1i...
.5 .
3").-

”am

 

1.3.. §
. . . f.

 

 

 

 

 

53:551. \
1E“ .

‘1 .
s

Wan. .
um

. 2233‘!
it flu.
, tn», 3‘

£135

‘cpfiflfil itii. sagflqfihfi

v73~tyauax\r.§i
v :5;
a.“ .11
uaxv.:
- h i I.
1...... x a
5.3.3.915. .
s... 3"}!

015: III;

.li kg:

.- ...
1.... Fifi...

3. I..... D. I \1
(v! 111-.»
I! ’11:,

it.

- wr.vkl(lauu_

t r...‘
6137113334‘2‘1‘:
39...!)11.’ ost-a

c.3911 3.1! I."

.,.p>..!x 1“

 

LIBRARY
Michigan State
University

 

 

 

This is to certify that the
dissertation entitled

NEW DISCOVERIES INVOLVING PLATELETS:
BIOENERGETICS AND BIOTECHNOLOGY

presented by

Chia-Jui Ku

has been accepted towards fulfillment
of the requirements for the

 

 

Ph.D. degree in Chemistry

 

 

Date

MSU is an Affirmative Action/Equal Opportunity Employer

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

DATE DUE I DATE DUE DATE DUE

 

 

Justnofig 2014

'I.

 

I, '4

 

 

 

 

 

 

 

 

 

 

 

5/08 K:IProj/Aoc&Pres/ClRC/DateDm.indd

 

 

 

 

 

NEW DISCOVERIES INVOLVING PLATELETS:
BIOENERGETICS AND BIOTECHNOLOGY

By

Chia-Jui Kn

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY
Chemistry

2009

ABSTRACT

NEW DISCOVERIES INVOLVING PLATELETS:
BIOENERGETICS AND BIOTECHNOLOGY

By
Chia-Jui Ku

The circulation is a complex network system comprised of arterioles and
capillaries, with a main function of moving oxygen to and from tissues and cells in the
body. It is also a complex mixture that includes, but not limited to, cells, macrophages,
proteins, and metabolites. Although white cells (leukocytes), red cells (erythrocytes) and
platelets have well-defined roles in the bloodstream, there also exist reports suggesting
that cells that flow through the circulation may actually participate in other processes in
blood vessels.

In this work, it is hypothesized that cells in the blood stream are communicating
through NO, which is mediated by the RBC’s ability to release ATP. NO is known as the
endothelium-derived relaxing factor (EDRF) that not only helps vasodilation, but also
regulates platelet activity. In 2006, our group proposed a mechanism to establish a
relationship between RBC deformability, REC-derived ATP, and subsequent
endothelium-derived NO production. Although numerous studies have been performed to
describe the relationship between RBC-derived ATP and endothelium-derived NO,

reports of the synergy between ATP released from RBCs and the ability of platelets to

produce NO are lacking.

Work in this thesis is divided into three concepts; first, to develop a method to
measure platelet NO production and release upon stimulation and activation. Secondly,
based on the ability of platelets to produce NO, communication between RBCs and
platelets were investigated in a capillary flow system. Finally, this newly obtained
knowledge was integrated into a microfluidic device developed as a tool to investigate
circulation using an in vitro format.

The ability to quantitatively determine platelet. NO production and release using
fluorescence probes provides a useful tool for further biochemical/medical application.
Data presented in this work also provides evidence suggesting that the relationship
between RBCs and platelets is ATP mediated. Moreover, physiological interactions
(adhesion) between cell types could be observed utilizing a microfluidic device with an
immobilized endothelium in channels while RBCs and platelets were pumped through.
Collectively, communication between three cell types was established in this work, and
might be helpful to explain conditions of certain diseases involving hyperactive platelets
with either low (e.g., diabetes, hypertension and cystic fibrosis) or high (e.g., sickle cell

disease and multiple sclerosis) REC-derived ATP release.

- My déarfamilj' anJC/iun-Juz'

I woufif not 6e wfiene I am now
wit/iout at? of your [ave and support

are iifiéfié ~ it; ~ %v1&1’&2§:

ACKNOWLEDGMENTS

First and foremost, I would like express my thankfulness to my advisor and
mentor, Dr. Dana Spence. I can not imagine how I will be with any other advisor in my
graduate career. Spence is not only a professor or an advisor to me, he is also everyone’s
friend. Not only he teaches us stuff, but also learns with us. I learned a lot from Spence
even though I might not be a satisfied student to him, he still patiently takes the time to
guide and encourage me. The past few years have been an unforgettable memory to me. I
always felt fortunate to have the chance joining Spence group.

Another deep gratitude is to all Spence group members (Luiza, Madushi, Teresa,
Wasanthi, Ajith, Andrea, Jen, Nicole, Wathsala, Adam. Steve, Kari, Paul and Suzanne)
who definitely make my graduate life more enjoyable. Especially to those start the
graduate school at the same year as mine, how lucky I am to be friends with you, work at
the same lab and graduate at the same year. All of you will always be remembered.

I would like to thank my family and Chun-Jui for always supporting and
encouraging me. Because of your love, I am who I am today; I am able to be strong
through the times alone. Even I never say, even we are so far away, you know I do love

you, miss you, and care about you all.

Also thanks to my committee members (Dr. Gary Blanchard, Dr. Robert
Maleczka and Dr. David Weliky) and Dr. Scott Martin those have assisted me through

the graduate career. You guidance and help have been always appreciated.

vi

TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................ x
LIST OF FIGURES ......................................................................................................... xi

CHAPTER 1 INTRODUCTION

1.1 DISSERTATION INTRODUCTION ........................................................................ l
1.2 THE PLATELET ....................................................................................................... 2
1.2.1 Platelet production ......................................................................................... 3
1.2.2 Platelet function ............................................................................................. 7
1.2.2.1 Platelet activation ................................................................................ 7
1.2.2.2 Platelet adhesion ................................................................................. 9
1.2.2.3 Shape change .................................................................................... 13
1.2.2.4 Secretion ........................................................................................... 13
1.2.2.5 Platelet aggregation ........................................................................... 15
1.2.3 Pathways of platelet regulation .................................................................... 16
1.2.3.1 Activator reactions ............................................................................ 16
1.2.3.2 Inhibitor reactions ............................................................................. 17
1.2.4 Role of platelets in thrombus formation ...................................................... 19
1.3 NITRIC OXIDE ....................................................................................................... 23
1.3.1 Nitric oxide biosynthesis .............................................................................. 23
1.3.2 Role of NO in the circulation ....................................................................... 29
1.3.3 NO and platelets ........................................................................................... 34
1.3.3.1 NOS in platelets ................................................................................ 34
1.3.3.2 Physiological regulation of platelet function by NO ........................ 35

1.4 PATHOLOGICAL ROLE OF NO IN VASCULAR DISORDERS
ASSOCIATED WITH PLATELET DYSFUNCTION ........................................... 39
1.4.1 Atherosclerosis, thrombosis and hypertension ............................................ 40
1.4.2 Diabetes mellitus .......................................................................................... 44
1.4.3 Cancer .......................................................................................................... 45
1.5 PROJECT OBJECTIVE .......................................................................................... 46
LIST OF REFERENCES .................................................................................................. 48

vii

CHAPTER2 FLUORESCENCE DETERMINATION OF NITRIC OXIDE
PRODUCTION IN PLATELETS

2.1 KNOWLEDGE OF NITRIC OXIDE DETECTION ............................................... 65
2.2 NO PRODUCTION AND RELEASE FROM STIMULATED AND ACTIVATED
PLATELETS ............................................................................................................ 70
2.3 EXPERIMENTAL METHODS ............................................................................... 74
Isolation and purification of platelets
Reagent preparation
Fluorescence determination
2.4 RESULTS AND DISCUSSION .............................................................................. 77
N0 in platelets

Optimization of DAF -F M DA concentration

Incubation time optimization

NO production and platelet concentration

Discussion of N0 in platelets

N0 released from platelets
2.5 CONCLUSIONS ...................................................................................................... 90
LIST OF REFERENCES .................................................................................................. 92

CHAPTER3 PLATELET-DERIVED NITRIC OXIDE IS AFFECTED BY

RBC-DERIVED ATP
3.1 INTRODUCTION TO CIRCULATION ................................................................. 98
3.1.1 General infonnation about the red blood cell .............................................. 99
3.1.2 REC-derived ATP related vasodilation ..................................................... 101
3.2 ATP-MEDIATED NO PRODUCTION IN PLATELETS .................................... 104
3.3 EXPERIMENTAL METHODS ............................................................................. 106
Isolation and purification of RBCs
Reagent preparation

Measurement of A TPfiom activated platelets
Fluorescence determination of platelet NO in a static system
Fluorescence determination of platelet N0 in a flow system
3. 4 RESULTS AND DISCUSSION ........................................................................... 112
Self-stimulated NO production in platelets
Measurement of platelet N0 in a microflow system
RBC-stimulated NO production in platelets
Increased RBC -stimulated platelet N0 with diabetic rats
3.5 CONCLUSIONS .................................................................................................... 127
LIST OF REFERENCES ................................................................................................ 128

viii

CHAPTER4 A CIRCULATORY MIMIC IN A MICROFLUIDIC DEVICE
INCLUDING MUTLIPLE CELL TYPES

4.1 INTRODUCTION TO MICROFLUIDICS ........................................................... 133
4.1.1 Materials for microfluidic devices ............................................................. 134
4.1.2 Photolithography ........................................................................................ 138
4.1.3 Soft lithography ......................................................................................... 140
4.1.4 Rapid prototyping and replica molding ..................................................... 141

4.2 CIRCULATORY MIMIC WITHIN A MICRODEVICE ..................................... 142

4.3 EXPERIMENTAL METHODS ............................................................................. 147
Preparation of a microfluidic device
Cell culture

Cell immobilization
Isolation and purification of platelets and RBCs
Reagent preparation
Fluorescence labeling of platelets
4.4 RESULTS AND DISCUSSION ............................................................................ 154
The eflect of NO on platelet adhesion
Applications for drug discovery
The eflect of A TP on platelet adhesion in a multiple cell types system
4.5 CONCLUSIONS .................................................................................................... 165
LIST OF REFERENCES ................................................................................................ 167

CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS

5.1 CONCLUSIONS .................................................................................................... 175
5.2 FUTURE DIRECTIONS ....................................................................................... 178
LIST OF REFERENCES ................................................................................................ 183

ix

Table 1.1

Table 1.2

Table 1.3

Table 1.4

Table 2.1

LIST OF TABLES

Naturally occurring and artificial activators of platelets ............................. 10
Selected platelet receptors ........................................................................... 12
Platelet a- and dense granule secretions ..................................................... 14
Properties of NOS isoforms ........................................................................ 27

Practical methods for the detection of NO in biological samples employing
common analytical techniques .................................................................... 66

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4

Figure 1.5

Figure 1.6

LIST OF FIGURES

Overall scheme of platelet production sequence. Pathways leading to
platelet production are indicated by solid arrows, other pathways are
indicated by dashed arrows. RBC, red blood cell; Gran, granulocyte; Mo,
monocyte ....................................................................................................... 4

Overview of megakaryocyte production of platelets. (a) megakaryocytes; (b)
cells first undergo nuclear endomitosis, organelle synthesis, and dramatic
cytoplasmic maturation and expansion, while a microtubule array,
emanating from centrosomes, is established; (c) centrosomes disassemble
and microtubules translocate to the cell cortex. Proplatelet formation
commences with the development of thick pseudopods; (d) sliding of
overlapping microtubules drives proplatelet elongation as organelles are
tracked into proplatelet ends, where nascent platelets assemble; (e) the
entire megakaryocyte cytoplasm is converted into a mass of proplatelets,
which are released from the cell. The nucleus is eventually extruded from
the mass of proplatelets, and individual platelets are released from
proplatelet ends ............................................................................................. 6

A simplified scheme of the hemostatic response .......................................... 8

(a) Structures of ADP and TXAz (b) The activator pathways that amplify
platelet aggregation ..................................................................................... 18

(a) The structure of prostacyclin. (b) The prostacyclin-thromboxane balance
in regulation of platelet aggregation. + denotes stimulation; - denotes

inhibition ..................................................................................................... 20

Biosynthetic pathway of nitric oxide from L-arginine ................................ 24

xi

Figure 1.7

Figure 1.8

Figure 1.9

Generation of NO form L-arginine. NOS catalyses a multi-electron
oxidation to form N-hydroxy-L-arginine (ArgOH), citrulline and NO. The
first step involves the binding of L—arginine followed by the reduction of
the ferric iron by an electron supplied by NADPH to form ArgOH.
Incorporation of an additional oxygen molecule forms an iron-dioxy species,
which abstracts a proton from ArgOH producing an iron-peroxy species and
ArgOH radical. The final stage progresses through a tetrahedral
intermediate between the iron-peroxy and ArgOH radical results in the
production of citrulline, NO and the regeneration of the ferric iron ........... 26

Schematic of NOS enzyme and participating co-factors. Increased Ca2+
levels enhances Ca2+/CaM binding results in the reduction of NOS, passing
electron to heme group and subsequent NO production ............................. 28

Schematic representation of (a) constitutive NO release and (b) induced NO
release and their signal transudation pathways ........................................... 30

Figure 1.10 NO induced smooth muscle relaxation ....................................................... 32

Figure 1.11

Figure 1.12

Figure 2.1

Figure 2.2

Overview of the role of NO in platelet function. NO generated from
L-arginine (L-Arg) by the endothelial cells and platelets activates the
soluble gunaylate cyclase (36C) to increase the levels of cGMP that control
the intracellular enzymes including protein kinase G (PKG),
cGMP-inhibited CAMP phosphodiesterase (PDE), and the function of ion
channel regulating calcium influx. NO can also react with superoxide anion
(02') to form peroxynitn'te (ONOO') .......................................................... 37

Overview actions of NO in myocardial infarction ...................................... 43

The reaction scheme of DAF-FM DA for the detection of intracellular NO
production. DAF-FM forms a fluorescent benzotriazole derivative that has
an excitation at 495 nm and emission at 515nm ......................................... 69

(a) Emission profiles for platelets in the presence of DAF-FM DA
molecular probe for nitric oxide (middle trace), the platelets in the presence
of the probe and 10 uM ATP (top trace), and the platelets in the presence of
the probe after incubation in the NOS inhibitor L-NAME (bottom trace) (b)
Quantitative data obtained from spectra, control consisted of platelets in the
absence probe. Error bars represent SEM. p < 0.05 (n = 5) ........................ 78

xii

Figure 2.3

Figure 2.4

Figure 2.5

Figure 2.6

Figure 2.7

Fluorescence intensities using various concentrations of the DAF-FM DA
probe. Emission intensities are shown for DAF-FM DA probe in the
absence of platelets (black bar), the probe in the presence of the platelets
(light gray bar), and in the presence of the probe, platelets, and ATP
stimulus (dark gray bar). Error bars represent SEM. p < 0.05 (n = 4) ........ 81

Measurement of the change in the fluorescence intensity as a function of
time. The lower two traces are for DAF-FM DA (circles) and DAF-FM DA
incubated with ATP (diamonds) in the absence of platelets. The top two
traces are for platelets incubated with DAF-FM DA for the times specified
on the time axis in the absence (squares) and presence (triangles) of ATP.
For each data point shown in the top two traces, the fluorescence intensity
is significantly higher for those platelets that were stimulated with ATP.
Error bars represent SEM. (n = 4) ............................................................... 83

Signal intensities as a function of platelet number for platelets in the
presence and absence of DAF-FM DA, in the presence of the probe with
ATP, and in the presence of the probe and L-NAME. Error bars represent
SEM. (n = 4) ............................................................................................... 85

Monitoring the production of nitric oxide as a function of concentration of
ATP and ADP. Relative to unstimulated platelets incubated with the
DAF-FM DA probe, the increase in fluorescence emissions were 39.1% :t
6.2% and 52.3% d: 8.2% for platelets stimulated with ATP and activated
with ADP, respectively. Error bars represent SEM. (n = 4) ........................ 87

(a) A calibration curve was prepared using the method of multiple standard
additions. The fluorescence intensity was measured after increments of NO
were added to aliquots of platelets containing DAF-FM. (b) Quantitative
determinations of NO released by the platelets are smnmarized in the
accompanying bar graph. The concentration of extracellular NO in the
presence of platelets alone (Plt) is 9.9 :l: 2.2 x 10'18 moles NO/platelet. The
extracellular NO levels increase to 2.0 :l: 0.1 x 10'17 and 2.8 i 0.3 x 10’17
moles NO/platelet in the presence of ATP (+ATP) and ADP (+ADP),
respectively. In the presence of a NOS inhibitor (+L-NAME), the
concentration of extracellular NO decreased to 3.1 :t 0.9 x 10'18 moles
NO/platelet. Error bars represent SEM. (n = 4) .......................................... 88

xiii

Figure 3.1

Figure 3.2

Figure 3.3

Figure 3.4

Illustration of the deformation-induced ATP release pathway. Activation of
the G-protein (Gs) coupled receptor (GPCR) by mechanical deformation
leads to the conversion of ATP to cyclic adenosine monophosphate (cAMP)
by adenylyl cyclase (AC), which results in phosphorylation of the cystic
fibrosis transmembrane regulator (CFTR) by protein kinase A (PKA), upon
which, stimulates ATP release from the cell. ATP further binds to sz
receptor on the endothelium results in NO production eventually leads to
vasodilation ............................................................................................... 103

Chemiluminescence assays were performed as a function of ADP
concentration in each sample to quantitatively detect ATP release from
activated platelets. ATP that is released from the activated platelets

. decreased in the presence of apyrase (1.0 units/mL as final concentration).

Error bars represent SEM. p < 0.05 (n = 5) .............................................. 114

Evidence that NO production in platelets is due to ATP stimulation of the
P2x receptor. Platelets NO was measured in the absence and presence of a
stimulus of NO (ATP) or activators of ATP release (ADP and thrombin)
which lead to NO production. As shown, NO production increases in each
case. However, identical measurements that were performed in the presence
of NF449, a reagent that blocks the ATP receptor on platelets resulted in a
decrease in NO production. This data suggested that NO production in
platelets is largely dependent upon ATP binding to the platelet receptor.
L-NAME, a NOS inhibitor decreased NO production with and without the
P2x blocker, verifying the measured signal in each case was due to NO.
Error bars represent SEM. p < 0.05 (except L-NAME measurements)
(it = 4) ........................................................................................................ 115

Increased NO production from platelets in the presence of 0.1 uM ATP. (a)
The traces represent the measured fluorescence intensities from platelets
only (lower trace), platelets in the presence of DAF-FM DA (middle trace),
and platelets with DAF-FM DA in the presence of ATP (upper trace). (b)
Bars represent the average of normalized results. Error bars represent SEM.
p<0.05 (n=3) ......................................................................................... 117

xiv

Figure 3.5

Figure 3.6

Figure 3.7

Figure 3.8

NO production of platelets incubated with supernatant from rabbit RBCs
incubated with zinc-activated C-peptide (+RBCs+10P10Zn) and in the
absence of zinc (+RBCs+10P) or absence of C-peptide (+RBCs+1-OZn) at 5
hours (black bars). Platelets NO remain unchanged when platelets were
pretreated with NF449 (light gray bars) and L-NAME (dark gray bars).
Error bars represent SEM. p < 0.05 (n = 3) .............................................. 119

The percent change in fluorescence due to platelet NO production
stimulated by RBCs in the presence and absence of stimulators and
inhibitors of ATP release. The percent changes were reported relative to
RBCs flowing with the DAF-FM DA-loaded platelets alone. In (a), RBCs
incubated with pentoxyfilline prior to flowing with platelets in microbore
tubing resulted in a 15.5% :I: 0.8 increase in emission intensity; in (b), the
RBCs were incubated with pentoxyfilline and diamide, resulting in a 36.9%
:I: 1.1% decrease in platelet NO; RBCs were treated with glybenclamide and
pentoxyfilline in (c) and the platelet NO decreased by 25.3% i 0.9%; in (d),
RBCs were incubated with iloprost, resulting in an increase in platelet NO
production of 10.0% :t 1.1%; the iloprost-induced increase in NO
production was reduced in (e) where RBCs treated with glybenclamide and
iloprost resulted in a decrease in platelet N0 of 50.9% :t 0.9%. Error bars
represent SEM. p < 0.05 (n = 3) ................................................................ 121

Control experiments of platelet NO production. Platelets were incubated
with zinc-activated C-peptide (Plt+10PlOZn) and in the absence of zinc
(Plt+10P) or absence of C-peptide (Plt+102n) at 5 hours. Each bar here was
statistically insignificant, suggesting that C-peptide has no direct effect on
platelet NO production. Error bars represent SEM. (n = 3) ...................... 124

The effect of metal-activated C-peptide on the NO production by platelets
from type 2 (BB/ZDB) and control rats. Black bars represent the
fluorescence intensity from platelet NO production incubated with
supernatant from rat RBCs and gray bars represent the fluorescence
intensity from platelet NO production incubated with supernatant from rat
RBCs incubated with metal-activated C-peptide at 5 hours. Platelet NO
production from type 2 rats increased 26.1% i 8.4% when RBCs were
incubated with metal-activated C-peptide. Error bars represent SEM.
p<0.05 (n=3) ......................................................................................... 125

XV

Figure 3.9

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

The effect of metal-activated C-peptide on the platelet NO production from
type 1 and control rats. Black bars showed the platelet NO production
incubated with supernatant from rat RBCs and gray bars showed the
platelet NO production incubated with supernatant from rat RBCs
incubated with metal-activated C-peptide at 5 hours. Error bars represent
SEM. p < 0.05 (n = 5) ............................................................................... 126

Two type of photoresist used result in different relief structure on wafer.
The portion of the negative photosist exposed to the radiation is insoluble
thereby create raised features corresponding to the mask, while positive
photoresist generate negative relef structures after development ............. 139

The process of photolithography for the master fabrication. (a) Spin-coat,
results in approximately 100 pm thick photoresist on the 4” silicon wafer.
(b) Bake, to evaporate solvent and compress the film. (c) Transparency
mask alignment. (d)UV exposure, cross-link the exposed portion. (e)
Development, remove the unpolymerized photoresist and obtain the master
with desired raised features ....................................................................... 143

Illustration of processes going on in the blood stream. ATP released from
RBCs results in platelet and endothelium NO production which leads to
inhibition of platelet recruitment and vasodilation ................................... 146

(a) Cross section of microfluidic array, each channel has dimensions of 100
um width and depth. (b) PDMS array with inlet and exit holes for
addressing flow to the system. (c) A confluent layer of bPAECs in a
microfluidic device for mimicing of resistance vessels ............................ 151

Reactions of CMFDA reagent. CMFDA is colorless and nonfluorescent

until cytosolic easterases cleave off acetates, releasing a fluorescent product
................................................................................................................... 153

xvi

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.10

Figure 4.11

The effect of ADP activation and L-NAME on platelets pumped over a
confluent channel of bPAECs. A stream of equilibrated medium was
pumped over the endothelium to ensure any nonadherent platelets were
removed before averaging the platelet count. Images in the left column
represent bright field images, while those on the right represent the
corresponding fluorescence images. (a) Untreated platelets, (b) platelets
incubated in 5 ,uM ADP, (c) untreated platelet adhering to a 10 mM
L-NAME treated endothelium, and (d) platelets activated with 5 uM ADP
adhered to a 10 mM L-NAME treated endothelium ................................. 156

Varying concentrations of ADP from 50 nM to 10 11M effects the number of
platelets adhered to an untreated and L-NAME treated bPAEC monolayer.
Non-activated platelets were also examined on similar untreated and treated
surfaces. Error bars represent SEM. p < 0.05 (n = 5) ............................... 157

In contrast to inhibition of endothelium NO, platelets were incubated with
10 mM L-NAME prior to flowing through the bPAEC layer channel. The
effect of exogenous NO was also examined shown as dark gray bars. Error
bars represent SEM. p < 0.05 (n = 4) ........................................................ 159

The effect of an anti-platelet drug, clopidogrel (Clop), on platelet adhesion
to a bPAEC monolayer. Platelets were incubated with clop for 30 minutes
before being pumped over the endothelium - (b), (d), and (f). In (d) and (f),
after clop incubation and prior to being pumped over the endothelium,
platelets were treated with 1.0 and 5.0 uM ADP. Error bars represent SEM.
p<0.05 (n=3) ......................................................................................... 160

(a) Platelet NO production as a function of concentration of ATP. As ATP
concentration was about 50 nM, platelet NO stopped increasing. Error bars
represent SEM. (n = 4) (b) Platelets were treated with various
concentrations of ATP (and were centrifuged to remove excess ATP) prior
to flowing through a channel coated with a confluent layer of bPAECs.
Error bars represent SEM. p < 0.05 (n = 5) .............................................. 162

Platelets were incubated with RBCs in the absence and presence of
glybenclamide (an ATP release inhibitor) or Zinc-activated C-peptide (an
ATP release stimulus) prior to flowing over the confluent bPAECs layer.
Error bars represent SEM. p < 0.05 (n = 6) .............................................. 164

xvii

Figure 5.1 A thrombus formation mimic in the microfluidic channel ........................ 181

xviii

CHAPTER 1 INTRODUCTION

1.1 DISSERTATION INTRODUCTION

The circulation is a complex system comprised of arterioles and capillaries
including cells, macrophages, proteins, and metabolites. An understanding of blood flow
maintance and cell communication is crucial to improving knowledge of certain diseases.
Recently, our group has been focusing on studying aspects of the blood stream, including
different cell types (e.g., red blood cells (RBCs), platelets and endothelial cells), and
successfully established a relationship between RBC deformability, RBC-derived ATP,

1, 2 . .
However, in this

and subsequent endothelium-derived nitric oxide (N 0) production.
dissertation, studies were focused on describing a relationship between platelets and NO,
using microfluidic technology as a circulation mimic. Therefore, in Chapter 1, the platelet
will be introduced from a biological point of view, highlighting aspects that are important
in our work. NO biochemistry will also be described later in Chapter 1, especially in
relation to platelets. In Chapter 2, a method to quantitatively measure platelet NO
production and release will be discussed. Furthermore, RBCs were introduced into the
same system with the platelets to establish a communication between RBCs and platelets

through ATP in Chapter 3. Finally, in Chapter 4, a microfludic device will be employed to

mimic the circulation. The development of a microfluidic device was first introduced in

the late 1970’s.3 However, within the past few years, a dramatic increase in microfluidic
research has taken place. Although early work in microfluidics ranged from sample
purification,4 amplifications’ 6 to high-throughput screening, applications such as
diagnostic testing7’ 8 and single molecule detectiong.ll have also been developed and
more information about microfluidics will be described in Chapter 4. A key feature of the
work here is emplying the microfluidic device as an in vitro platform to mimic in vivo

process.

1.2 THE PLATELET

Knowledge about the structure, biology, and function of platelets has evolved
considerably since 1882, when Giulio Bizzozero linked newly identified, discrete
particles in the blood, distinct from red and white blood cells, with the coagulation
processm'14 For more than 100 years, the dominant role of platelets in hemostasis and
thrombosis has been well documented.

Platelets are 2 — 4 pm, anucleate discoid circulating blood particles.15 They
circulate around the body in an inactive state and initiate hemostatic plug formation at a
site of vascular injury by promoting coagulation and subsequent wound healing. When

platelets adhere to the endothelial defect, they undergo processes such as shape change,

9

. 1
granule contents release, and adhesron to form aggregates.

Physiologically, these processes help to limit blood loss; however, inappropriate
or excessive platelet activation results in an acute obstruction of blood flow, for example,

18-20

in acute myocardial infarction (heart attack). However, activated platelets also

express and release species that stimulate a localized inflammatory response through the

. . 21-23
activation of leukocytes

and endothelial cells.24’ 25 It is now clear that platelet
fiinction is not only limited to the prevention of blood loss, but also has been implicated

in many pathological processes including host defense,26'28 inflammatory arthritis,29

. . 1 .
adult respiratory distress syndrome,” 3 and tumor growth and metastasrs.32

1.2.1 PLATELET PRODUCTION

The overall process of platelet production begins with the common hematopoietic
stem cell (HSC) (Figure 1.1).33 Two distinct types of blood cell lines are derived:
lymphoid, which includes all types of lymphocytes, and myeloid, which includes
granulocytes, monocytes, red blood cells and platelets. Polyploid megakaryocytes are the
immediate progenitors of platelets.

Megakaryocytes regenerate in human bone marrow34 at the rate about 108 cells
per day;33 and each megakaryocyte can generate more than 5,000 platelets, in turn, 10“
of platelets are replenished daily. The physiological number of platelets is 1.5 -— 4.0 x 108

per milliliter of blood.35 Production of such a number of cells, each with a relatively short

2.3180qu .22 ”Shoo—28%.. .520 Eco c003 won .Umm $32.8 8:93 .3 @88me one @3353 550
$38.8 23m .3 @8865 0.3 8:03on “Boga—m 3 @532 Ensign .oocoscom 8:03.05 6633 MO 0838 =Eo>O "A 2:me

 

      

. . oioobaxMMoE
M 0mm ,. 22.58
~81.in attire].
& EEO/w
first», fries... i... uoacomoa ,
.u Bantam
now—:owoa Neil...
oSoocoEBtnoo—zqfi0 WHEN w \
513131016». 1:11.“ ‘1
coamcomoa A. .50 a x \ 8:5on
8 a .8580 o ooba Mo
e. a Eb o fixer... 8%..on R a. 2
\1 EoEEQoSoobaxmon

:8 83m ouflonouafioa: now—comoa 929mm“
:oEEoo beam 91.; v@
. \ fin :00 3:050 cum
\1@ III—tmwxcouaowoa 3205095—

«mflnommnaaoam amaze—war.

life span (7 - 10 days), offers an advantage in terms of speed and adaptability to
hemostatic challenges.

The megakaryocyte undergoes a series of morphological changes during the 4 —
10 hour process of platelet production (Figure 1.2).36 Nuclear endomitosis (a process to
separate a cell nucleus into two identical nuclei) and organelle synthesis occur first, along
with expansion of the cytoplasm. An array of microtubules37 emerges from centrosomes
(the main microtubule organizing center in the cell for regulating cell-cycle procession),
which then migrate to the cell periphery.

Aided by sliding of the microtubules, the megakaryocyte’s cytoplasm then
develops multiple thick pseudopods in preparation for formation of 5 — 10 proplatelets.
Organelles and granules migrate along the microtubules to the developing, elongatinig
proplatelet ends, where new platelets will form. Proplatelets are 250 — 500 um38 long on
average and can produce 100 — 200 platelets each. Proplatelets are then released from the
cell into the vascular sinus (the cavity in vessel wall), often appearing paired in dumbbell
shape. The nucleus is ejected from the mass of platelets, and individual platelets are later
released or “budded off” from proplatelet ends. One third of platelets are normally stored
in the spleen as an interchangeable pool with circulating cells, and can be pushed into

general circulation in times of stress.

(a) (b)

(c
Centrosomal ., .., . Organelle

microtubules \e. .n.
. /Pseudopod \\1

format ion ‘3,"

"i...

  
   
 
     
 

  
 
 

   

Cytoplasmic . icrotubules
maturation

   
   
 
 

 

    
 

  

;. Trackingoforganelles and cell cortex

(d) .- s granules to nascentplatelets (e) PTO latelet
i . Microtubule sliding ‘ f Platelet
. . .0 power proplatelet .5 release

    

elongation ..

 
    

extrusion

Figure 1.2 Overview of megakaryocyte production of platelets. (a)
megakaryocytes; (b) cells first undergo nuclear endomitosis, organelle synthesis,
and dramatic cytoplasmic maturation and expansion, while a microtubule array,
emanating from centrosomes, is established; (c) centrosomes disassemble and
microtubules translocate to the cell cortex. Proplatelet formation commences with
the development of thick pseudopods; (d) sliding of overlapping microtubules
drives proplatelet elongation as organelles are tracked into proplatelet ends, where
nascent platelets assemble; (e) the entire megakaryocyte cytoplasm is converted
into a mass of proplatelets, which are released from the cell. The nucleus is
eventually extruded from the mass of proplatelets, and individual platelets are
released from proplatelet ends.36

1.2.2 PLATELET FUNCTION

Platelets control bleeding (hemostasis) when there is an injury to the blood vessel
(Figure 1.3), and the endothelial cell layer is disrupted exposing the underlying
extracellular matrix. Platelets are very reactive cells, and upon activation by suitable
triggers, such as exposure to subendothelial tissue, they are able to adhere at the site of
damage.39'41 Following adhesion, rapid signal transduction leads to platelet activation,
cytoskeletal changes associated with shape change, spreading and secretion, and
inside-out activation of integiins that support adhesion and aggregation. During these
processes, platelets also assist fibrin formation by providing a surface on which many of
the reactions of the coagulation cascade may occur. Many active substances are released:
growth factors that influence smooth muscle cells in the vessel wall or tumor growth,
serotonin that affects vascular integrity, vasoactive materials that modulate local blood
flow, and leukocyte chemoattractants. If these various functions go out of control, a

pathological bleeding or thrombotic state may develop.

1.2.2.1 PLATELET ACTIVATION
Platelet activation describes the process that converts the smooth, nonadherent

platelet into an adhesive speculated particle that releases and expresses biologically

. . . . . . . 39
active substances and acquires the ability to bind the plasma protein fibrinogen.

Normal intact
endothelium

Restoration of
ant ithromb otic
activity

 

 

    

    

  
   

Activation of fibrinoly sis
Clot dissolution
Lesion rep air

  

Exp osure of
subendothelium

 

Platelet adhesion,
recruitment and
aggregation

Clot formation
Blood arrest

 

     
   

Coagulation
cascade activation

Figure 1.3 A simplified scheme of the hemostatic response

Activation occurs rapidly following exposure to a variety of particulate and soluble
substances known as agonists (Table 1.1). Some of substances listed in Table 1.1 result in
physiological activation, while others occur in pathological states or are in vitro reagents.
Activation can also occur as a result of the physical stimulus of high fluid stress, such as
that found at the site of a critical arterial narrowing.41

On activation, platelets display four basic phenomena: adhesion, shape change,
secretion and aggregation. These may not necessarily occur in the same order, and some
can occur without others. The various activators elicit different types of response,
showing dose-dependence and sometimes synergism.

When the vascular endothelial cell lining becomes disrupted, the subendothelial
connective tissues are exposed and platelets rapidly adhere to them, spreading across the
site of damage, and changing shape. They then secrete their granular contents, which
recruit more platelets to the site, forming an aggregate of cells. Activated platelets also
provide a procoagulant surface supporting the reactions leading to thrombin generation

and ultimately producing fibrin, which adds mechanical strength to the platelet plug.

1.2.2.2 PLATELET ADHESION

One of the earliest events following blood vessel damage is the adhesion of

. . 40, 42 . .
platelets to areas where subendothelium 1S exposed. Platelet adhesron requires

 

Soluble activators

Insoluble activators

 

Adenosine diphosphate (ADP)
Adrenaline
Arachidonate
Epinephrine
Immune complexs
PAF-acether

PG endoperoxides
Proteolytic enzymes
Serotonin
Thrombin
Thromboxane A2
Vasopressin

Bacteria
Collagen
Glass

Kaolin

Latex particles
Viruses

 

Table 1.1 Naturally occurring and artificial activators of platelets

10

specific structural components of the subendothelium, plasma proteins and receptors
(Table 1.2) on the platelet membrane. A number of plasma proteins are candidates for
mediators of platelet adhesion to the endothelium, among them von Willebrand factor
(vWF),43 fibronectin, fibrinogen and thrombospondin, the so-called adhesive proteins.44
The best studied of this group is vWF and, interestingly, its contribution in
platelet adhesion has been studied in vitro using a flow chamber and exposed rabbit aorta,
and appears to be highly dependent on wall shear rate.45 At low shear rates, such as large
veins (200 s"), adhesion occurs independently of vWF. However, at high shear rates
comparable to those found at the arterial wall (500 -1000 s") and in small vessels (>1300
s'l), there is a significant adhesion defect in the absence of vWF. In the high fluid stress
environment of flowing arterial blood, initial adherence is mediated primarily by the
platelet membrane vWF receptor GP Ib-IX-Vfus-48 Circulating vWF binds to collagen
exposed in the subendothelium, allowing it to interact with GP Ib-IX-V.49 This interaction
is reversible and allows the adherent platelets to roll, eventually through clustering of the
receptors,50 results in platelet activation.51 Platelet number, viscosity and red blood cell
count have a linear relationship to adherence, which reflects the rheology of high shear
vessels where red blood cells occupy the central core position forcing the platelets to
migrate to the periphery of the blood vessel, thus increasing the platelet-vessel wall

contact.

11

 

 

Receptor Ligand
Adhesion
Integrins
GP Ia/IIa Collagen
GP Ic/IIa Laminin
GP Ic*/IIa F ibronectin
oLVIIIa Vitronectin, fibrinogen, vWF, thrombospondin
GP IIb/IIIa Fibrinogen, fibronectin, vWF, vitronectin
Others
P-selectin Selectin counter receptors
GP Ib vWF
GP IV Thrombospondin, collagen
Aggregation
GP IIb/IIIa Fibrinogen, fibronectin, vWF

 

Table 1.2 Selected platelet receptors

12

1.1.2.3 SHAPE CHANGE

Following adhesion to subendothelium, platelets spread, covering the exposed
connective tissue matrix, and in doing so change from the circulating discoid form to an
irregularly shaped elongated cell with cytoplasmic projections. Platelet pseudopod
formation appears to result from rearrangement of the cytoskeletal proteins (actin and
myosin) and results in contractile activity, which is analogous to activity seen in muscle

12, 52 . . . . .
Both microfilaments and microtubules are found in pseudopods and it 18

cells.
thought that the latter control recruitment and dissolution of microfilaments. In the early
stages of platelet activation, shape change is reversible, but strong stimuli result in the

centralization of organelles, degranulation, and release granule contents accompanied by

irreversible shape change and aggregation.

1.2.2.4 SECRETION

Platelets release a number of biologically active substances from granules upon
activation (Table 1.3). There are three types of granules: the alpha (or-) granule, the dense
granule, and lysosomes. or-granules contain platelet-specific proteins, such as
B-thromboglobulin and platelet factor 4,53.56 as well as some proteins which normally
circulate in the plasma at relatively high concentrations, e. g. fibrinogen. Dense granules

sequester a pool of nucleotides that are not interchangeable with those utilized in the

13

 

or-granule

dense granule

 

al-Antitrypsin
org-Macroglobulin
az-Antiplasmin
B-Thromboglobulin
Albumin

Coagulation factor V
F ibrinogen

F ibronectin
Platelet-derived grth factor
Platelet factor 4
P-selectin

von Willebrand factor

Adenosine diphosphate (ADP)

Adenosine triphosphate (ATP)
2+

Ca

Serotonin

 

Table 1.3 Platelet or- and dense granule secretions

14

general metabolism of the cell. The released ADP provides a feedback loop for further
platelet stimulation, and serotonin helps to maintain the integrity of the vascular
endothelium.57 The importance of these two types of granules is well illustrated by the
clinical syndromes associated with their deficiency or dysfunctionsg’ 59 Lysosomes
contain a variety of acid hydrolases such as lysozyme, acid phosphatase and elastase.60’
61 Unlike or- and dense granules, lysosomes contain enzymes that help digesting such as

particles, excess organelles, foreign microbes and do not release their contents while

platelet activation.

1.2.2.5 PLATELET AGGREGATION

The process of platelet aggregation describes the ability of platelets to co-adhere
with one another in a specific process requiring energy, intracellular processes and
initiators. A large number of platelet activators (Table 1.1) are able to cause aggregation.
Once tethered to the vessel wall, platelets form irreversible adhesion bonds through the
interaction of platelet receptors with specific subendothelial matrix proteins and plasma
proteins immobilized at the site of injury. The major platelet integrin oran3 (GP IIb/IIIa),
binds vWF and/or fibrinogen to facilitate the crosslinking (platelet aggregation) under

41,62

shear, and further activation of platelets, providing strength and stability to growing

the thrombus. The formation of a platelet plug stabilized by an insoluble fibrin network

15

serves to prevent firrther blood loss from a damaged vessel.

Although platelet adhesion and aggregation were historically viewed as distinct
processes in the formation of a thrombus, it is now clear that the fundamental mechanism
is similar,63 involving the interaction between platelet receptors GP IIb/IIIa and GP
Ib-IX-V complex with fibrinogen and vWF in flowing blood.64'66 For example, an
adherent platelet binds fibrinogen and vWF from the circulating blood, creating an ideal
surface for the recruitment of firrther platelets. However, additional receptors such as
collagen receptors are required for the stable attachment of platelets to subendothelium

structures while shear forces are generated on the platelet by the flowing blood.

1.2.3 PATHWAYS OF PLATELET REGULATION
1.2.3.1 ACTIVATOR REACTIONS

Research over the past 40 years has provided the biochemical rationale for these
dramatic alterations in basal behavior of platelets. It is now well established that the
formation of a platelet plug is supported by the activity of at least three platelet-derived
activator systems.

The release of ADP from platelet a-granules and the interactions of ADP with

purinergic receptors form the foundation of the first system. In vivo, a platelet-derived

pool of ADP may be supplemented with ADP released from red blood cells.67 The

16

discovery of the mechanism of action of aspirin and its analogs68m formed the
foundations for the discovery of the proaggregating metabolites of arachidonic acid in
platelets, namely, cyclic endoperoxides and thromboxane A2 (TXAz).72 TXA2 is
synthesized by the sequential action of platelet cyclooxygenase and thromboxane
synthase, and once formed, it acts on its receptors to amplify aggregation.73 74

Inhibition of the generation and the action of ADP (e.g., by ticlopidine or
clopidogrel) and thromboxane (e.g., by aspirin) is not sufficient to abolish platelet
aggregation stimulated by such potent agonists as thrombin. The discovery of matrix
metalloproteinase-2 (MMP-2), which is expressed in human platelets, revealed another
platelet-derived activator system. The release of MMP-2 is collagen or thrombin
stimulated and the subsequent platelet aggregation is in a non-thromboxane-,
non-ADP-dependet manner (Figure 1.4).75 Once released, MMP-2 can remodel platelet

. . 76 77
surface membranes Wthh enhances aggregation.

1.2.3.2 INHIBITOR REACTIONS

The vascular endothelium is a major contributor to the inhibitor reactions that
control platelet activation. Vane et al first discovered that endothelial cells generate
prostacyclin,78 a potent inhibitor of platelet aggregation and stimulator of platelet

deaggregation.79’ 80 Prostacyclin is a biological opponent of TXAZ on platelets

17

(a)

 

OH
Thromboxane A2

(b) Activator agents

Platelet release

I
I | I
ADP TXAZ MMP-Z

 

I»
Aggregation

Figure 1.4 (a) Structures of ADP and TXA2 (b) The activator pathways that amplify
platelet aggregation

l8

(Figure 1.5) and the vessel wall resulting in inhibition of platelet aggregation and

. . 73, 81, 82
vasodilation.

Prostacyclin binds to its specific receptors present on platelets that
are linked to adenylyl cyclase. Stimulation of prostacyclin receptor leads to increased

accumulation of intracellular CAMP and downregulation of all pathways involved in the

amplification of platelet aggregation.82 Prostacyclin exerts little influence on the process

of platelet adhesion to the subendothelial components of the vessel wa11.83’ 84

Prostacyclin acts as an indirect inhibitor of platelet activation. It is released close
to the endothelial surface in response to stimulation with various vasoactive mediators
including angiotensins and bradykinin.85 Platelets themselves lack the capacity to
synthesize prostacyclin; however, they may contribute to the endothelial synthesis of this
eicosanoid by generating and releasing arachidonic acid cyclic endoperoxides, which

9

may be taken up by the endothelial cells for prostacyclin synthesis.8

1.2.4 ROLE OF PLATELTS IN THROMBUS FORMATION
Hemostasis is the process that maintains the integrity of a closed, high-pressure
circulatory system after vascular damage. Vessel-wall injury and the loss of blood from
the circulation rapidly initiate events in the vessel wall and in blood that repair lesions.
Thrombi are complex structures that are composed not only of fibrin meshwork,

but also contain blood-home cellular elements like platelets, leukocytes and red blood

19

(a)

 

(b) Arachidonic acid

I

Cyclic endop eroxides

./ \

Prostacyclin Thromboxane A2

\ /

Aggregation

Figure 1.5 (a) The structure of prostacyclin. (b) The prostacyclin-thromboxane balance in
regulation of platelet aggregation. + denotes stimulation; - denotes inhibition

20

cells. Platelets play an essential role in the initial response to vascular injury as they
adhere to vessel wall components, become activated, aggregate and secrete mediators that
promote further platelet activation and also attract leukocytes. In addition to the plug
formation, which transiently stops bleeding, platelets provide a surface for the subsequent
steps of the coagulation cascade leading to fibrin formation. Blood coagulation
complexes function only in compartments and platelets serve the phospholipid surface for
these reactions.87 These events occur concomitantly, and under normal conditions,
regulatory mechanisms contain thrombus formation temporally and spatially.

There are two distinct pathways acting in parallel or separately that can activate

88, 89
In one of these pathways, exposure of

platelets for thrombus formation.
subendothelial collagen initiates platelet activation; in the other, thrombin, generated by
tissue factor derived from the vessel wall or present in flowing blood is the initiator.
Depending on the injury or the disease, one pathway or the other may predominate, but
the consequences of platelet activation triggered by these pathways are the same.

When the blood vessel is damaged, platelets are activated by two kinds of
interactions, one is the platelet GP VI (a collagen receptor on platelets) with the collagen
of the exposed vessel wall and the other is platelet GP Ib-V-IX (a cluster of adhesive

receptors for vWF binding on platelets) with collagen-bound vWF. Both interactions

result in adhesion of platelets to the site of injury. The relative importance of platelet

21

GP VI and GP Ib-V-IX in the initial tethering of platelets depends on the shear rate at the
vessel wall.90 However, the interaction of collagen with GP V1 is required, as is GP
Ib-V-IX with vWF.88’91’92

Tissue factor (factor III, a protein presents in platelets for initiating the thrombin
formation) triggers a second pathway that initiates platelet activation. Platelet activation
initiated by this pathway does not require disruption of the endothelium and is
independent of vWF23 and GP V1.88’ 93 Tissue factor forms a complex with factor VlIa,
initiating a proteolytic cascade that generates thrombin. Thrombin cleaves protease-
activated receptor 4 (Par4) (Parl in humans) on the platelet surface, thereby activating
platelets94 and causing them to release ADP, serotonin, and TXAz. In turn, these agonists
activate other platelets, and in doing so, amplify the signals for thrombus formation.

A developing thrombus recruits unstimulated platelets,93 and within the thrombus
activation occurs only in a subgroup of the recruited platelets. Others remain loosely
associated with the thrombus but do not undergo activation and may ultimately disengage
from the thrombus.93 In short, thrombus formation is a dynamic process in which some
platelets adhere to and others separate from the developing thrombus, and in which shear,

flow, turbulence, and the number of platelets in the circulation greatly influence the

architecture of the clot.

22

1.3 NITRIC OXIDE
Nitric oxide (NO) is a diffusible, short-lived, diatomic free radical ubiquitously
produced by mammalian cells as a biological mediator first identified as the

endothelium-deriver relaxing factor (EDRF),"B'97

The half-life of NO is typically on the
order of 1 - 2 hours in dilute aqueous solution and approximately 1 - 5 seconds in vivo
due to the formation of other NO-derived species such as nitrate and nitrite. Although NO
is a free radical, it is relatively stable, reacting predominantly with molecules that have
molecular orbitals with unpaired electrons such as oxygen, superoxide, and transition
metals such as heme iron. The biosynthesis of NO is achieved by sequential oxidation of
a terminal guanidino-nitrogen of L-arginine (L-Arg) yielding citrullinegs'100 (Figure 1.6).
NO plays a prominent role in controlling a variety of functions in the cardiovascular,

. . 101-104
immune, reproductive, and nervous systems.

1.3.1 NITRIC OXIDE BIOSYNTHESIS

NO is biosynthesized in mammals by the modified urea cycle,‘05 which has two

important functions: a secretory role to regenerate L-arginine for NO synthesis and an

excretory role to eliminate excess nitrogen created by cell metabolism. It is formed by a

. . . . . . . . . 106 .
series of oxrdation-reduction mechanisms from the ammo acrd L-arginine, (F igure 1.6)

which is normally present in high concentration in the plasma (80 11M) and at even higher

23

 

 

O
+
+H 3N H 3N o-
O'
NOS
N H > 1*
2x N / N H
O H
L-arginine (D
N -Hydroxyarginine
NH3 0
+H 3N it
0 ' N O
Nitric Oxide
N H
0 AN H 2
L-citrulline

Figure 1.6 Biosynthetic pathway of nitric oxide from L-arginine

24

concentrations intracellularly.

The reaction is catalyzed by the NO synthases (NOS), which all utilize reduced
nicotinamide adenine dinucleotide phosphate (NADPH) and 02 as cosubstrates
(Figure 1.7). Full activity of NOS requires the presence of four co-factors: flavin adenine
dinucleotide (FAD), flavin mononucleotide (F MN), tetrahydrobiopterin (H48) and a
heme group.107 Three NOS isoforms (Table 1.4) have evolved to function in animals, and
each gene is located on a different chromosomelos’ ‘09 Two of three NOS isoforms are
constitutively expressed in cells, and they synthesize NO in response to increased Ca2+ or
in response to Ca2+-independent stimuli such as shear stress.110 These particular NOS
firnction in signal transduction cascades by linking temporal changes in calcium level to
NO production. NO then serves as an activator of soluble guanylate cyclase (sGC).1 11

Important in these two types of NO syntheses is the presence of the calmodulin
(CaM) protein bound to the NOS enzyme. When increased levels of Ca2+ are present in
the environment, CaM will bind to the Ca2+ resulting in the reduction of NOS
(Figure 1.8). The constitutive enzymes are designated nNOS and eNOS (or NOS I and 111,
respectively), after the cell types in which they were originally discovered (rat neurons

and bovine endothelial cells). An inducible NOS (iNOS or NOS II) is constitutively

. . . . 112 . .
expressed only in select tissues, such as lung epithelium, and 18 more typically

. . . . . 113, 114
syntheSIZed in response to inflammatory or promflammatory mediators.

25

/ Enzyme / Enzyme / Enzyme

‘ From NADPH ‘
- - L-Arg § L-Ar
\ I / L-Arginme \ I / _\_ e' \ / g
1763+ Fe3+ Fe2+
/ \ / \ / \
Heme cofactor
l/OZ
/Enzyme /Enqyme /Enzyme
H o 2H+ ‘-
\ Iy/ ~,\ H 4__ \j 3+/ HEX“ N 2 \ § / L Arg
/Fe\/N\fi\/N\R / “C\ Y \R 6' /Ff}{
HO 0 - NH2

 

From NADPH . °
L-Arg ‘0‘

,O

N-hydroxy-L-Arginine

From
02 NADPH
e'
/Enzyme / Enzyme
From substrate \ I /

\

\? / \ {3 E H. FC3+ ‘,
\
R

flit-”i )0 ., .
gym ““2 “Rd” NH2
.0 . H

R + :N=O + Fe3+

L- citrulline Nitric oxide

 

Figure 1.7 Generation of NO form L-arginine. NOS catalyses a multi-electron oxidation
to form N-hydroxy-L-arginine (ArgOH), citrulline and NO. The first step involves the
binding of L-arginine followed by the reduction of the ferric iron by an electron supplied
by NADPH to form ArgOH. Incorporation of an additional oxygen molecule forms an
iron-dioxy species, which abstracts a proton from ArgOH producing an iron-peroxy
species and ArgOH radical. The final stage progresses through a tetrahedral intermediate
between the iron-peroxy and ArgOH radical results in the production of citrulline, NO
and the regeneration of the ferric iron

26

 

 

cNOS (constitutive) iNOS (inducible)
nNOS (NOS III) eNOS (NOS 1) NOS 11
Source Cardiovascular Central nervous Nonspecific immune
system system system
Calcium dependency Yes Yes NO
Function Regulatory Regulatory Host defense
Examples Relaxation of Neurotransmitter Kills bacteria and

smooth muscle

Regulates blood
flow and pressure

Inhibits platelet
activation

microorganisms

Seen in inflammatory
conditions

 

Table 1.4 Properties of NOS isoforms

27

N ADPH

Arginine
NADP+, H“

Citru llin e,
NO

 

Figure 1.8 Schematic of NOS enzyme and participating co-factors. Increased Ca2+ levels
enhances Caz+/CaM binding results in the reduction of NOS, passing electron to heme
group and subsequent NO production

28

Although expression of iNOS is beneficial in host defense or in modulating the immune
. . . . . . 113,115,116
response, its expressron 18 also linked to a number of inflammatory diseases.

NO as a signaling molecule is completely different from classical mediatorsm
Figure 1.9 illustrates the constitutive and induced NO release and its signal transduction
pathway. Unlike classical mediators, NO, a lipophilic, free radical gas, diffuses freely
through the plasma membrane and does not need vesicles secretion from signaling cells
or any cell surface receptors in the target cells in order to trigger a signal. It passes readily

103,104
The

to the underlying smooth muscle and stimulates vasorelaxation (Figure 1.9a).
molecular targets of NO in victim cells are Cu-Fe proteins, releasing free Cu2+ and F e2+

and generating 02' and highly toxic hydroxyl radicals, thus leading to large scale

oxidative injury (Figure 1.9b).

1.3.2 ROLE OF NO IN THE CIRCULATION

Small arteries play an important role in the regulation of peripheral vascular
resistance. The endothelium of resistance arteries regulates vascular function by way of
its barrier role, through interaction with circulating cells such as platelets, which may
release vasoactive or growth regulating agents, and by production of substances that
modulate vascular tone and smooth muscle cell growth. However, NO serves as an

important mediator in this regulation. The endothelium is an obvious target organ of

29

   

  

  
  
 

   
    
  

 

 

\

 

J

 

(a) Acetylcholine, ADP, bradykinin
Shear stress Ca2+
Ca2+
Cahnodulin
NADPH
L- ' ° nNOS
argmme jeNOS
L-citrulline
NO
f i
NO
GTP>1
cGMP sCG
K cGMP-PK —> Relaxation
(b) Endotoxin, cytokines

  

   

OZ'
Stimulation of gene
exp res sion of iNOS

I

   
      
   
  

Nuclear activating factor
L-arginine

>1 iNOS
L-citrulline
NO

 

 

/ .
- _ NO
02 '" """" * O2 Cu2+proteins
Fez“ proteins
Peroxynitrite
K Oxidative injury

 

J

 

Stimuli

Generator cells:

Neurons
Endothelial cells

Target cells:

Neurons
Smooth muscles
Platelets

Stimulus cells

Killer cells:

Macrophages
Kup ffer cells

Victim cells:

Cancer cells
Parasites

Figure 1.9 Schematic representation of (a) constitutive NO release and (b) induced NO
release and their signal transudation pathways

30

cardiovascular risk factors. Accordingly, functional alterations do occur with aging,
hypertension and hypercholesterolemia, all of which are associated with a decreased
basal and stimulated release of endothelium-derived NO.

Relaxations in response to the abluminal release of endothelium-derived NO are
associated with stimulation of sGC and in turn the formation of cyclic guanosine
3’,5’-monophosphate (cGMP) in vascular smooth muscle cells (Figure 1.9a). Briefly,
cGMP targets specific G-dependent protein kinases (PKG) that phosphorylate several key
target proteins, including ion channels, ion pumps, receptors and enzymes. Once
phosphorylated, these targets actively reduce the intracellular calcium concentration,
which decreases myosin light chain kinase (MLCK) activity resulting in smooth muscle

“8421 sGC, also present in platelets, is activated by the luminal

relaxation (Figure 1.10).
release of endothelium-derived NO,122 which limits adhesion and aggregation]23
Therefore, endothelium-derived NO is a determinant in both vasodilation and platelet
deactivation, and thereby represents an important antithrombotic feature of the
endothelium.

NO also plays a crucial role in the regulation of blood pressure.124 When infused
intravenously, inhibitors of NOS such as L-NG-monomethylarginine (L-NMMA) or
Nm-nitro-L-arginine methylester (L-NAME) have been shown to induce long-lasting

125, 126

increases in blood pressure and vascular resistance in the rabbit and human. This

31

Smooth muscle
relaxation

I

MLCKI

No\1 I

sGC [C32+]i l

/\ I

GTP cGMP > PKGT

 

Figure 1.10 NO induced smooth muscle relaxation

32

demonstrates that the resistance circulation is in a constant state of vasodilation due to
continuous basal release of picomolar quantities of NO by the vascular endothelium.
Furthermore, NO plays an important role in modulating vascular structure under
physiological and pathophysiological conditions. In hypertension, resistance arteries
adapt to the increased wall tension by changing their geometry. Accordingly, a reduced
lumen diameter, an increased wall thickness, or both, can normalize the excessive tension
applied on the vessel wall, which may protect the microcirculation against the blood
pressure rise. The alterations in vascular wall structure and composition, induced by
long-term changes in blood flow that lead to normalization of shear stress, are
multifactorial in etiology, as the endothelium can regulate cell proliferation and
extracellular matrix production through both NO-dependent and -independent
mechanismsnl129
Creation of an arteriovenous fistula (an artificial passageway between two vessels
that are not normally connected) in the rabbit carotid circulation leads to an increase in
carotid artery diameter and remodeling of the media that normalizes wall shear stress.
These adaptive changes are partially attenuated by eNOS inhibition.129 In marked
contrast to wild-type mice, remodeling of the carotid artery is prevented in knockout mice

with targeted disruption of eNOS.130 Instead, eNOS mutants display a paradoxical

hyperplastic increase in arterial wall thickness, suggesting that NO activity prevents

33

pathological changes in vessel wall morphology. 130

1.3.3 N0 AND PLATELETS

The appreciation of endogenous inhibitors for platelet activation was stimulated
by the discovery of prostacyclin,79 a major platelet-regulatory prostaglandin. However, it
became apparent that the generation and release of this eicosanoid can only account in
part for non-thrombogenic properties of vascular endothelium. Endothelial cells express
the antiaggregatory activity even under conditions of complete inhibition of prostaglandin
generation.83

Shortly after NO was recognized to be the EDRF with an important role in

vasomotor control through its actions on vascular muscle,96 it was also demonstrated that
NO is an inhibitor of platelet fimction and plays a physiological role in the reduction of

123, 131

platelet activation. This deactivation by NO is achieved as platelets being the

smallest of the blood cells, circulate closest to the endothelium, which is considered to be
the most important source of N0 in the vasculature. However, it was soon realized that

platelets themselves are capable of biosynthesizing NO when they are activated.106

1.3.3.1 NOS IN PLATELETS

In contrast to megakaryocytes, which contain large amounts of RNA and DNA,

34

platelets contain trace amounts of DNA and small amounts of RNA. Therefore, the
identification of DNA fragments coding NOS proteins requires application of polymerase
chain reaction (PCR). A number of researchers extracted platelet RNA and amplified

DNA fragments consistent with the expression of endothelial NOS but not inducible NOS

132-134

or neuronal NOS in platelets. Although the presence of eNOS in normal platelets

appears to be beyond dispute, the identification of iNOS has proved to be controversial.

However, it has been proven that both eNOS and iNOS are expressed in normal human

134-137

and porcine platelets. Irnportantly, platelet NOS, similar to endothelial NOS, is

associated with a particular fraction of the platelet and undergoes intracellular

. . . . . . 10 ,12 ,1 6
translocation and activation during platelet activation. 6 3 3

1.3.3.2 PHYSIOLOGICAL REGULATION OF PLATELET FUNCTION BY NO
NO available for platelet regulation is generated by both endothelium- and
platelet- NOS. Stimulation of platelet and endothelial function plays an important role in

the generation of NO. Tonic release of NO from the endothelial cells is mediated by shear

139,140

stress,138 while resting platelets generate small amounts of NO. Platelet adhesion

and aggregation stimulate platelet NOS, leading to release of NO.106’ 139’ 141’ 142

Both basal (shear stress dependent) and agonist stimulation release of NO have

been implicated in platelet regulation. Experiments have shown the coronary and

35

pulmonary vasculatures generate NO to inhibit platelet adhesion under constant flow

143,

conditions. 144 Similarly, bradykinin-stimulated endothelial cells release NO in

quantities sufficient to inhibit platelet adhesion.]45'147
Platelet aggregation induced by a variety of agonists, as well as by shear stress, is

84, 122, 146, 148-152

inhibited by NO released from endothelial cells. In addition to

inhibition of adhesion and aggregation, NO disaggregates preformed platelet
aggregates84 and inhibits platelet recruitment to the aggregate.153 Figure 1.11 summarizes
the role of NO in regulating platelet function.

Similar to other cell types and tissue systems, the effects of NO on platelets are
largely dependent on the stimulation of sGC and the resultant increase in the intraplatelet
cGMP levels, hence activation of cGMP-dependent protein kinase (PKG). This in turn
results in inhibition of platelet activation through various pathways. PKG promotes
sarcoplasmic reticulum ATPase (SERCA)-dependent refilling of intraplatelet Ca2+
stores,154 thereby inhibiting influx of Ca2+ and other cations and decreasing intracellular
Ca2+ levels. PKG also phosphorylates the TXA2 receptor, thereby inhibiting its function

resulting in decreased platelet aggregation.155

In addition, two other mechanisms have been identified whereby cGMP prevents

platelet activation. Firstly, CGMP indirectly increases intracellular CAMP through

inhibition of phosphodiesterase;l56 cGMP and CAMP act synergistically to inhibit platelet

36

T Platelet aggregation

T P-selectin I Platelet aggregation
I MMP activation i Platelet adhesion
Vascular dysfunction 1 Disaggregation

T Inhibition of recruitment

  
  
  
     

 

Stimuli \ ON 0 O‘ Platelet

T [Ca2+],

  

Activation of PKG
N—O§>*fl> cGMP cGMP-inhibited CAMP PDE
0 Reduction of cytosolic Ca2+

  
 

     

 

 

\ Endothelium /

Figure 1.11 Overview of the role of NO in platelet function. NO generated from
L-arginine (L-Arg) by the endothelial cells and platelets activates the soluble gunaylate
cyclase (sGC) to increase the levels of cGMP that control the intracellular enzymes
including protein kinase G (PKG), cGMP-inhibited CAMP phosphodiesterase (PDE), and
the function of ion Channel regulating calcium influx. NO can also react with superoxide

anion (02') to form peroxynitrite (ONOO')

37

146’ ‘57 Secondly, cGMP indirectly inhibits the activation of GP IIb/IIIa

aggregability.
. 158
fibrinogen receptors.

Apart from the cGMP-dependent pathways described above, there is evidence that
NO can also regulate platelet fiinction independently of cGMP. NO has been shown to
inhibit ATP-dependent Ca2+ uptake into platelet membrane vesicles in a manner which
cannot be attributed to cGMP, because cGMP itself only has a weak effect on this uptake
even at high concentrations.159 However, the inhibitory effect of adenosine on platelet
aggregation can be partially prevented by NOS inhibition,160 suggesting that, while
platelet-derived NO does not necessarily and consistently inhibit platelet aggregation in
response to proaggregants, it enhances the antiplatelet effects of antiaggregatory
mediators.

Platelet-derived NO inhibits recruitment of platelets to the growing

161-163
thrombus.

This process is initiated by activated platelets at the site of vascular
injury by secretion of ADP, serotonin, and TXAZ, and further promotes thrombin
deposition and thrombus formation on the platelet surface. In vitro, platelet-derived NO

inhibits aggregation between leukocytes and platelets, and in particular, between

monocytes and platelets. This is considered an early and robust marker of platelet

. . 164. . . . . . 165,166
activation implicated in the mechanism of atherogenesrs and thrombosrs.

Platelet-derived NO also modulates the rate of thrombus growth, through altering

38

platelet adhesion on to the surface, and is also sensitive to insulin or shear stress.l67’ ‘68

Collectively, these data support a physiological role of platelet-derived NO in the ‘

modulation of platelet function and hence thrombus formation.

1.4 PATHOLOGICAL ROLE OF NO IN VASCULAR DISORDERS ASSOCIATED
WITH PLATELET DYSFUNCTION ’

The vasodilator and platelet-regulatory functions of endothelium are impaired

during the course of vascular disorders including atherosclerosis, coronary artery disease,

169,170
A number of researchers correlated

essential hypertension and diabetes mellitus.
the changes in the endothelial function with the generation of NO. The endothelial
dysfunction was ascribed to both decreased and enhanced generation of NO. To explain

this discrepancy, it was proposed that these Changes in NO generation are often

accompanied by reduced bioactivity of NO.171 The metabolism of NO and the

interactions of NO with reactive oxygen species account for this reduced bioactivity of

qull72

A detrimental effect of superoxide ion generation on the NO-dependent cellular
signaling was first demonstrated by Gryglewski et 01.173 In 1990, Beckman et al reported

that the reaction of NO with superoxide could take place under physiological conditions

174,175

and lead to the formation of peroxynitrite (ONOO_), a highly reactive species that

can oxidize various biomolecules in the cellular microenvironment. In 1994, it was found

39

that ONOO— can decrease the vasodilator and platelet-inhibitory activity of NO and

176, 177

. 176 . .
prostacyclin. However, thiols and glucose attenuated these detrimental effects

of ONOOL The reaction of ONOO_ with thiols in cell membranes and glucose in the
extracellular fluid results in synthesis of NO donors that counteract the vasoconstrictor
and platelet-aggregatory activities of the parent oxidant.l76’ 178 Interestingly, there is now
evidence that small amounts of ONOO— may be generated during aggregation of normal
platelets.179 Thus, ONOO— generated by platelets, is rapidly detoxified and converted to
NO donors following reactions with platelet membrane thiols.178 The oxidizing stress

could decrease the efficiency of this regulating mechanism and precipitate platelet

dysfunction and damage.

1.4.1 ATHEROSCLEROSIS, THBOMBOSIS AND HYPERTENSION

Thrombosis appears to be a major determinant of the progression of
atherosclerosis. In early atherosclerosis, microthrombi present on the luminal surface of
vessellgo’ 18] can potentiate progression of atherosclerosis by exposing the vessel wall to
clot-associated mitogens. In later stages of atherosclerosis, mural thrombosis is associated
with the growth of atherosclerotic plaques and progressive luminal occlusion. Platelet
activation and participation in thrombotic responses to ruptures of atherosclerotic plaques

are critical determinants of the extent of thrombosis, increasing plaque growth, and the

40

2, 183
Increased adherence of platelets to vessel wall

development of occlusive thrombi.l8
manifesting early atherosclerotic changes and the release of growth factors from
a-granules can exacerbate the evolution of atherosclerosis.184

Atherogensis is associated with profound changes in the oxidative status of the

vascular wall. Oxidative modifications of low-density lipoproteins (LDL) play a key role

. . . 171 . .
in atherogenesrs, and a number of studies have examined the effects of native and

oxidized LDL on NO-mediated vascular fimctions. In most of these studies lipoproteins

decreased the bioactivity of NO.185’ 186

The decreased bioactivity of NO in
atherosclerosis could also result from Changes in the metabolism and the generation of
mod from superoxide and inducible No.174 In addition, LDL inhibit L-arginine
uptake into platelets and through this mechanism, decreases NOS activity and promotes
thrombosis.187 These effects are prevented by the administration of L-arginine in the

188, 189

diet In contrast to LDL, high-density lipoproteins (HDL) decreased platelet

. . . . . . . . 187
activation and thrombosrs by increasmg NOS acthlty in platelet. Moreover, human

apolipopeotein E, which mediates hepatic Clearance of lipoproteins, exerts a significant

inhibitory effect on platelets through stimulation of platelet NOS. 190
Lipid peroxidation also leads to free radical-catalyzed generation of prostaglandin

isomers from peroxidation of arachidonic acid. Interestingly, some of these isoprostanes

reduce the antiadhesive and antiaggregatory activity of NO on platelets.191 Thus, lipid

41

peroxidation contributes to the pathomechanism of impaired bioactivity of NO in the
cardiovascular system.

Ischemic heart disease and myocardial infarction are common manifestations of
coronary atherosclerosis. NO inhibited microthromboembolism in the ischemic heart,

protected myocardium against intracoronary thrombosis (Figure 1.12), and decreased

2, 193

. . . . 9
platelet deposrtlon ow1ng to carotid endarterectomy.] Moreover, decreased

generation of NO by platelets is predictive of the presence of acute coronary syndromes

in patients with coronary atherosclerosis.162 In addition, acetylcholine-induced release of
NO is impaired in patients with coronary artery disease, contributing to a reduction in the
endothelial capacity to regulate platelet activation.184 These observations Clearly show
that the alterations in the generation and action of NO are important for the pathogenesis
of atherogenesis and its ischemic complications.

Interestingly, an impaired NO generation or action also underlies the

. . . . . 194, 195
pathomechamsm of vasospastic and thrombotic changes of essential hypertenSion.

Camilletti et al found that platelet NO production is reduced in hypertensive patients.196

Platelet L-Arginine transport has also been reported to be reduced in hypertensives,

197, 198

attributable to downregulation of the membrane transport system. Asymmetric

dimethylarginine plasma levels are greater in hypertensive patients compared with

norrnotensive controls,199 and this gives rise to enhanced inhibition of platelet NOS in

42

Vascular endothelium

NO (-)

Atherosclerotic vascular dysfunction

 

 

 

 

Vasoconstriction
I Blood flow

 

 

Platelet
adhesion/aggregation

 

 

Leukocyte activation

 

 

 

 

Thrombus formation

Myocardial infarction

Figure 1.12 Overview actions of NO in myocardial infarction

43

 

hypertensives. Patients recently diagnosed, but not treated, with mild essential
hypertension have been found to exhibit impairment in stimulated platelet NOS activity;
in this study, although albuterol and collagen both increased platelet NOS activity in
normotensive subjects, they failed to generate such an increase in hypertensives.I63 As
these two agonists stimulate NOS through different pathways, it is likely that a
generalized defect exists in the ability of platelet NOS to undergo stimulation in the

context of hypertension.

1.4.2 DIABETES MELLITUS

There are indications that Changes in the bioactivity and metabolism of NO are
involved in the pathogenesis of vasculopathy in diabetes mellitus. Insulin, at
physiological conditions, inhibits platelet activation via stimulation of platelet NOS.200
Exposure of platelets to insulin decreases platelet aggregation in part by increasing
synthesis of NO that, in turn, increases intraplatelet concentrations of cyclic nucleotides,
cGMP and CAMP. Both of these cyclic nucleotides are known to inhibit activation of
platelets. Thus, an insulin-dependent increase in NO production exerts antiaggregatory
effects. In the context of diabetes, basal platelet NOS activity has been found to be

decreased in both type I and type II diabetes mellitus as compared with healthy

individuals.201 This suggests that insulin deficiency of type I diabetes and in advanced

44

stages of type II diabetes contribute to platelet hyperactivity and diabetic angiopathy202

by decreasing the inhibition of platelet reactivity induced by insulin.

1.4.3 CANCER

Platelets contribute to the cytotoxic cell effector system controlling neoplasia
(tumor formation) and a part of this cytotoxic mechanism of platelets could be NO
dependent.203

Platelets also play a role in the pathogenesis of tumor metastasis by increasing the
formation of tumor cell-platelet aggregates, thus facilitating cancer cell arrest in the
microvasculature. Tumor cell-induced platelet aggregation in vivo is modulated by the
ability of tumor cell to generate NO, and this correlates with their propensity for

204, 205

metastasis. Indeed, human colon carcinoma cells isolated from metastases

exhibited lower NO activity than cells isolated from primary tumor. Moreover, the
expression of iNOS by murine melanoma cells inversely correlated with their ability to
form metastases in vivo.206 These data suggest that a differential synthesis of NO
distinguishes between cells of low and high metastatic potential.

Another aspect of NO action on the metastatic cascade of events is its interactions
with matrix metalloproteinases (MMPs). MMPs represent a family of matrix-degrading

enzymes that play an important role in the growth, invasion, and metastasis of cancer

45

cells.207 Sawicki et al have found that MMP-2 plays a crucial role in tumor cell-induced

208’ 209 The release of MMP-2 was inhibited by NO donor agents,

platelet aggregation.
suggesting that NO interfere with cancer invasion and spread by reducing the release of

MMPs.

1.5 PROJECT OBJECTIVE

Hyperactive platelets and associated thrombosis have been related to a number of
. . . . . . 162, 210-212
cardiovascular diseases, and NO plays an important role in this physrology.

When NO is released by the endothelium it prevents platelet adhesion to the vessel wall.

However, when released by platelets, NO inhibits further recruitment of platelets to a
growing thrombus.153 Previous data from our research group showed that RBCs, upon

1 213

deformation or under the influence of different agonists, release ATP ’ that can further

stimulate NO production from both the endothelial cells214 and plateletszls’ 216 resulting
in vascular relaxation and inhibition of platelet activation.

In this work, it is hypothesized that these different cell types in the circulation are
communicating through NO, mediated by the RBCs’ ability to release ATP. Experiments
have been performed not only to investigate platelet NO production release upon
stimulation and activation using fluorescence spectroscopy, but also quantitatively

measured the amount of NO employing a fluorescence probe with intracellular and

46

extracellular components. Moreover, this work also demonstrates the ability to measure
NO production in platelets stimulated by RBC-derived ATP by employing a continuous
flow analysis system as an in vivo system mimic. More specifically, an in vitro platform
to immobilize endothelial cells in the channels of a microfluidic device to mimic in vivo
microcirculation was used to monitor cell communication at the molecular level. This
device is employed to monitor the physical interaction (adhesion) of platelets to an
immobilized endothelium in the presence of platelet activator, inhibitor and RBCs. This
approach is the first microfluidic device that allows multiple cell types to physically
interact in the channels and this work further demonstrates the potential of these devices

in the drug discovery process and drug efficacy studies.

47

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

LIST OF REFERENCE

Carroll, J .; Raththagala, M.; Subasinghe, W.; Baguzis, S.; Oblak, T. D. a.; Root, P.;
Spence, D. Mol. BioSyst. 2006, 2, 305-311.

D'Amico Oblak, T.; Root, P.; Spence Dana, M. Anal Chem 2006, 78, 3193-3197.

Terry, S. C.; Jerman, J. H.; Angell, J. B. IEEE Trans. Electron Devices 1979,
130-26, 1880-1886.

Li, P. C. H.; Harrison, D. J. Anal. Chem. 1997, 69, 1564-1568.

Kopp, M. U.; De Mello, A. J .; Manz, A. Science (Washington, D. C.) 1998, 280,
1046-1048.

Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J .; Foote, R. 8.;
Ramsey, J. M. Anal. Chem. 1998, 70, 5172-5176.

Kricka, L. J.; Wilding, P. Pure Appl. Chem. 1996, 68, 1831-1836.

Colyer, C. L.; Tang, T.; Chiem, N.; Harrison, D. J. Electrophoresis 1997, 18,
1733-1741.

Dorre, K.; Brakmann, S.; Brinkmeier, M.; Han, K.-T.; Riebeseel, K.; Schwille, P.;
Stephan, J.; Wetzel, T.; Lapczyna, M.; Stuke, M.; Bader, R.; Hinz, M.; Seliger, H.;
Holm, J.; Eigen, M.; Rigler, R. Bioimaging 1997, 5, 139-152.

Effenhauser, C. S.; Bruin, G. J. M.; Paulus, A.; Ehrat, M. Anal. Chem. 1997, 69,
3451-3457.

Chou, H.-P.; Spence, C.; Scherer, A.; Quake, S. Proc. Natl. Acad. Sci. U. S. A.
1999, 96,11-13.

48

(12) Wurzinger, L. J. Adv Anat Embryo] Cell Biol 1990, 120, 1-96.

(13) Brewer Douglas, B. Br J Haematol 2006, 133, 251-258.

(14) Kleiman, N. S.; Freedman, J. E.; Tracy, P. B.; Furie, B. C.; Bray, P. F.; Rao, S. V.;
Phillips, D. R.; Storey, R. F.; Rusconi, C. E; French, P. A.; Steinhubl, S. R.;
Becker, R. C. Platelets 2008, 19, 239-251.

(15) Rao, G. H. R. Handb. Platelet Physiol. Pharmacol. 1999, 1-20.

(16) Caen, J. P.; Michel, H. Nature 1972, 240, 148-149.

(17) Gear, A. R. L. Can. J. Physiol. Pharmacol. 1994, 72, 285-294.

(18) Oliva, P. B.; Breckinridge, J. C. Circulation 1977, 56, 366-374.

(19) Heptinstall, S.; Mulley, G. P.; Taylor, P. M.; Mitchell, J. R. A. Br. Med. J. 1980,
280, 80-81.

(20) Levine, S. P.; Towell, B. L.; Suarez, A. M.; Knieriem, L. K.; Harris, M. M.;
George, J. N. Circulation 1985, 71, 1129-1134.

(21) Bouchard, B. A.; Tracy, P. B. Curr Opin Hematol 2001, 8, 263-269.

(22) Wagner, D. D.; Burger, P. C. Arterioscler, Thromb, Vasc. Biol. 2003, 23,
2131-2137.

(23) Weyrich, A. S.; Zimmerman, G. A. Trends Immunol. 2004, 25, 489-495.

(24) Heller, R.; Bevers, E. M. Handb. Exp. Pharmacol. 1997, 126, 585-618.

(25) Diacovo, T. G.; Puri, K. D.; Wamock, R. A.; Springer, T. A.; von Andrian, U. H.
Science (Washington, D. C.) 1996, 273, 252-255.

49

(26)

(27)

(23)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38)

(39)

von Hundelshausen, P.; Weber, C. Circ. Res. 2007, 100, 27-40.

Weyrich, A.; Cipollone, F.; Mezzetti, A.; Zimmerman, G. Curr. Pharm. Des. 2007,
13,1685-1691.

MCNicol, A.; Israels, S. J. Cardiovasc. Hemato]. Disord: Drug Targets 2008, 8,
99-117.

Zhang, Q.; Lu, Y.; Proulx, S. T.; Guo, R.; Yao, Z.; Schwarz, E. M.; Boyce, B. F.;
Xing, L. Arthritis Res. T her. 2007, 9, No pp given.

Bone, R. C.; Francis, P. 8.; Pierce, A. K. Am J Med 1976, 61, 585-589.

Bozza, F. A.; Shah, A. M.; Weyrich, A. S.; Zimmerman, G. A. Am. J. Respir. Cell
Mol. Biol. 2009, 40, 123-134.

GasiC, G. J. Cancer Metastasis Rev 1984, 3, 99-114.

Deutsch, V. R.; Tomer, A. Br: J. Haematol. 2006, 134, 453-466.

Ogawa, M. Blood 1993, 81 , 2844-2853.

Burstein, S. A.; Harker, L. A. Clin Haematol 1983, 12, 3-22.

Patel, S. R.; Hartwig, J. H.; Italiano, J. E., Jr. J. Clin. Invest. 2005, 115,
3348-3354.

Italiano, J. E., Jr.; Lecine, P.; Shivdasani, R. A.; Hartwig, J. H. J. Cell Biol. 1999,
147,1299-1312.

Italiano, J. E., Jr.; Hartwig, J. H. Platelets 2002, 21-35.

Baumgartner, H. R.; Muggli, R.; Tschopp, T. B.; Turito, V. T. Thromb.

50

(40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(48)

(49)

(50)

(51)

Haemostasis 1976, 35, 124-138.

Mustard, J. F.; Packham, M. A. Inflammation, Immun. Hypersensitivity (2nd Ed.)
1979, 557-664.

Kroll, M. H.; Hellums, J. D.; McIntire, L. V.; Schafer, A. I.; Moake, J. L. Blood
1996, 88, 1525-1541.

Sixma, J. J. Recent Adv. Blood Coagulation 1981, 3, 175-192.

Ruggeri, Z. M. J. Thromb. Haemostasis 2003, 1, 1335-1342.

Packham, M. A.; Mustard, J. F. Prog. Hemostasis Thromb. 1984, 7, 211-288.

Turitto, V. T.; Weiss, H. J.; Baumgartner, H. R. J Lab Clin Med 1983, 102,
551-564.

Savage, 8.; Saldivar, E.; Ruggeri, Z. M. Cell (Cambridge, Mass.) 1996, 84,
289-297.

Savage, B.; Almus-Jacobs, F.; Ruggeri, Z. M. Cell (Cambridge, Mass.) 1998, 94,
657-666.

Andrews, R. K.; Shen, Y.; Gardiner, E. E.; Dong, J .-F.; Lopez, .J. A.; Bemdt, M. C.
Thromb. Haemostasis 1999, 82, 357-364.

Ruggeri, Z. M. Thromb. Haemostasis 1997, 78, 611-616.

Kasirer-Friede, A.; Wares, J.; Leng, L.; Marchese, P.; Ruggeri, Z. M.; Shattil, S. J.
J. Biol. Chem. 2002, 277, 11949-11956.

Zaffran, Y.; Meyer, S. C.; Negrescu, E.; Reddy, K. B.; Fox, J. E. B. J. Biol. Chem.
2000, 275, 16779-16787.

51

(52)

(53)

(54)

(55)

(56)

(57)

(53)

(59)

(60)

(61)

(62)

(63)

(64)

(65)

Holmsen, H. Eur J Clin Invest 1994, 24 Supp] 1, 3-8.

Kaplan, K. L.; Owen, J. Methods Enzymol. 1982, 84, 93-102.

Kaplan, K. L.; Owen, J. Methods Enzymol. 1982, 84, 83-92.

Kaplan, K. L.; Owen, J. Methods Hematol. 1983, 8, 115-125.

Walz, D. A.; Hung, G. L. Semin. Thromb. Hemostasis 1985, 11, 40-47.

Dale, G. L.; Friese, P.; Bater, P.; Hamilton, S. F.; Reed, G. L.; Jackson, K. W.;
Clemetson, K. J .; Alberlo, L. Nature (London, U. K.) 2002, 415, 175-179.

Holmsen, H.; Weiss, H. J. Brit. J. Haematol. 1970, 19, 643-649.

Gerrard, J. M.; Phillips, D. R.; Rao, G. H.; Plow, E. F.; Walz, D. A.; Ross, R.;
Harker, L. A.; White, J. G. J Clin Invest 1980, 66, 102-109.

Holmsen, H. Platelets: Cell. Response Mech. Their Biol. Signif, Proc. EMBO
Workshop 1980, 249—263.

Niewiarowski, 8.; Paul, D. Res. Monogr. Cell Tissue Physiol. 1981, 5, 91-106.

Xiong, J.-P.; Stehle, T.; Goodman, S. L.; Arnaout, M. A. Blood 2003, 102,
1155-1159.

Ruggeri, Z. M. J. Clin. Invest. 2000, 105, 699-701.
Ruggeri, Z. M.; Dent, J. A.; Saldivar, E. Blood 1999, 94, 172-178.

Tsuji, S.; Sugimoto, M.; Miyata, S.; Kuwahara, M.; Kinoshita, S.; Yoshioka, A.
Blood 1999, 94, 968-975.

52

(66)

(67)

(68)

(69)

(70)

(71)

(72)

(73)

(74)

(75)

(76)

(77)

(78)

Wu, Y.-P.; Vink, T.; Schiphorst, M.; Van Zanten, G. H.; Ijsseldijk, M. J. W.; De
Groot, P. G.; Sixma, J. J. Arterioscler, Thromb., Vasc. Biol. 2000, 20, 1661-1667.

Radomski, M. W.; Radomski, A. S. Handb. Platelet Physiol. Pharmacol. 1999,
458-477.

Vane, J. R. Nature (London), New Biol. 1971, 231, 232-235.

Vane, J. R.; Flower, R. J .; Botting, R. M. Stroke (Dallas) 1990, 21, IV-12-IV-23.
Vane, J. R.; Botting, R. M. Thromb. Res. 2003, 110, 255-258.

Wang, C.; Ainiwaer Zhongguo Xueye Liubianxue Zazhi 2007, 1 7, 167-169.

Hamberg, M.; Samuelsson, B. Proc. Natl. Acad. Sci. U. S. A. 1974, 71,
3400-3404.

Moncada, S. Cardiovasc. Pharmacol. Prostaglandins, [Meet] 1982, 101-114.

Moncada, S.; Higgs, E. A.; Vane, J. R. Adv. Pharmacol. Ther., Proc. Int. Congr,
8th 1982, 6, 39-47.

Sawicki, G.; Salas, E.; Murat, J.; Miszta-Lane, H.; Radomski, M. W. Nature
(London) 1997, 386, 616-619.

Sawicki, G.; Sanders, E. J .; Salas, E.; Wozniak, M.; Rodrigo, J.; Radomski, M. W.
Thromb. Haemostasis 1998, 80, 836-839.

Abou-Saleh, H.; Theoret, J .-F.; Yacoub, D.; Merhi, Y. Thromb. Haemostasis 2005,
94, 1230-1235.

Vane, J. R. Brit. J. Pharmacol. 1969, 35, 209-242.

53

(79)

(80)

(81)

(82)

(83)

(84)

(35)

(86)

(37)

(38)

(89)

(90)

Moncada, S.; Gryglewski, R.; Bunting, S.; Vane, J. R. Nature (London) 1976, 263,
663-665.

Moncada, S.; Gryglewski, R. J .; Bunting, S.; Vane, J. R. Prostaglandins 1976, 12,
715-737.

Moncada, S. Arteriosclerosis (Dallas) 1982, 2, 193-207.
Moncada, S. Br. J. Pharmacol. 1982, 76, 3-31.

Radomski, M. W.; Palmer, R. M. J.; Moncada, S. Br. J. Pharmacol. 1987, 92,
181-187.

Radomski, M. W.; Palmer, R. M. J.; Moncada, S. Br. J. Pharmacol. 1987, 92,
639-646.

Nowak, J.; Radomski, M.; Kaijser, L.; Gryglewski, R. J. Acta Physiol. Scand.
1981, 112, 405-411.

Dusting, G. J .; Moncada, S.; Vane, J. R. Adv. Prostaglandin, Thromboxane,
Leukotriene Res. 1982, 10, 59-106.

Sachs, U. J. H.; Nieswandt, B. Circ. Res. 2007, 100, 979-991.

Dubois, C.; Panicot-Dubois, L.; Merrill-Skoloff, G.; Furie, B.; Furie, B. C. Blood
2006, 107, 3902-3906.

Mangin, P.; Yap Cindy, L.; Nonne, C.; Sturgeon Sharelle, A.; Goncalves, 1.; Yuan,
Y.; Schoenwaelder Simone, M.; Wright Christine, E.; Lanza, R; Jackson Shaun, P.
Blood 2006, 107, 4346-4353.

Ruggeri, Z. M. Ann. Med. (Helsinki, F in]. ) 2000, 32, 2-9.

54

(91) Massberg, S.; Gawaz, M.; Gruner, S.; Schulte, V.; Konrad, 1.; Zohlnhofer, D.;
Heinzmann, U.; Nieswandt, B. J. Exp. Med. 2003, 197, 41-49.

(92) Bergmeier, W.; Piflam, C. L.; Goerge, T.; Cifuni, S. M.; Ruggeri, Z. M.; Ware, J .;
Wagner, D. D. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 16900-16905.

(93) Dubois, C.; Panicot-Dubois, L.; Gainor, J. F.; Furie, B. C.; F urie, B. .1. Clin. Invest.
2007, 11 7, 953-960.

(94) Vu, T. K. H.; Hung, D. T.; Wheaton, V. 1.; Coughlin, S. R. Cell (Cambridge, Mass. )
1991, 64, 1057-1068.

(95) Furchgott, R. F.; Zawadzki, J. V. Nature (London) 1980, 288, 373-376.

(96) Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature (London) 1987, 327,
524-526.

(97) Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Hypertension (Dallas) 1988, 12,
365-372.

(98) Marletta, M. A. Cell 1994, 78, 927-930.

(99) Kerwin, J. F., Jr.; Lancaster, J. R.; Feldman, P. L. J. Med. Chem. 1995, 38,
4343-4362.

(100) Griffith, O. W.; Stuehr, D. J. Annu. Rev. Physiol. 1995, 57, 707-736.

(101) Knowles, R. G.; Palacios, M.; Palmer, R. M. J .; Moncada, S. Proc. Natl. Acad. Sci.
U. S. A. 1989, 86, 5159-5162.

(102) Bredt, D. S.; Hwang, P. M.; Snyder, S. H. Nature (London) 1990, 347, 768-770.

(103) Moncada, S.; Higgs, E. A. Eur. J. Clin. Invest. 1991, 21, 361-374.

55

(104)

(105)

(106)

(107)

(108)

(109)

(110)

(111)

(112)

(113)

(114)

(115)

(116)

(117)

Moncada, S.; Palmer, R. M. J .; Higgs, E. A. Pharmacol. Rev. 1991, 43, 109-142.

Nathan, C. Faseb J. 1992, 6, 3051-3064.

Radomski, M. W.; Palmer, R. M. J.; Moncada, S. Br. J. Pharmacol. 1990, 101,
325-328.

Hevel, J. M.; Marletta, M. A. Methods Enzymol. 1994, 233, 250-258.

Nathan, C.; Xie, Q.-W. Cell (Cambridge, Mass.) 1994, 78, 915-918.

Nathan, C.; Xie, Q. W. J. Biol. Chem. 1994, 269, 13725-13728.

Fleming, 1.; Bauersachs, J.; Fisslthaler, B.; Busse, R. Circ. Res. 1998, 82,
686-695.

Ignarro, 1.; Murad, F.; Editors Nitric Oxide: Biochemistry, Molecular Biology, and
Therapeutic Implications. [Ins Adv. Pharmacol. (San Diego), 1995; 34], 1995.

Dweik, R. A.; Laskowski, D.; Abu-Soud, H. M.; Kaneko, F. T.; Hutte, R.; Stuehr,
D. J.; Erzurum, S. C. J. Clin. Invest. 1998, 101, 660-666.

Kroncke, K.-D.; Fehsel, K.; Kolb-Bachofen, V. Biol. Chem. Hoppe-Seyler 1995,
376, 327-343.

Hierholzer, C.; Menezes, J.; Kalff, J. C.; Villavicencio, R. T.; Harbrecht, B.;
Billiar, T. R.; Bauer, A. J .; Tweardy, D. J. Surg. Forum 1998, 49, 78-80.

Nathan, C. J. Clin. Invest. 1997, 100, 2417-2423.

MacMicking, J .; Xie, Q.; Nathan, C. Annu. Rev. Immunol. 1997, 15, 323-350.

Burnett, A. L.; Lowenstein, C. J.; Bredt, D. M.; Chang, T. S. K.; Synder, S. H.

56

(118)

(119)

(120)

(121)

(122)

(123)

(124)

(125)

(126)

(127)

(128)

Science (Washington, D. C., 1883-) 1992, 257, 401-403.

Rapoport, R. M.; Draznin, M. B.; Murad, F. Trans. Assoc. Am. Physicians 1983,
96, 19-30.

Rapoport, R. M.; Murad, F. J. Cyclic Nucleotide Protein Phosphorylation Res.
1983, 9, 281-296.

Ignarro, L. J. J. Physiol. Pharmacol. 2002, 53, 503-514.

Ignarro, L. J.; Sisodia, M.; Trinh, K.; Bedrood, 8.; Wu, G.; Wei, L. H.; Buga, G.
M. Nitric Oxide 2002, 7, 83-90.

Busse, R.; Lueckhoff, A.; Bassenge, E. Naunyn-Schmiedeberg's Arch. Pharmacol.
1987, 336, 566-571.

Radomski, M. W.; Palmer, R. M. J .; Moncada, S. Proc. Natl. Acad. Sci. U. S. A.
1990, 87, 5193-5197.

Gardiner, S. M.; Compton, A. M.; Bennett, T.; Palmer, R. M. J.; Moncada, S.
Hypertension (Dallas) 1990, 15, 486-492.

Haynes, W. G.; Noon, J. E; Walker, B. R.; Webb, D. J. J. Hypertens. 1993, 11,
1375-1380.

Haynes, W. G.; Noon, J. P.; Walker, B. R.; Webb, D. J. Lancet 1993, 342,
931-932.

Garg, U. C.; Hassid, A. J. Clin. Invest. 1989, 83, 1774-1777.

De Mey, J. G. R.; Dijkstra, E. H.; Vrijdag, M. J. J. F. Am. J. Physiol. 1991, 260,
H1128-H1134.

57

(129)

(130)

(131)

(132)

(133)

(134)

(135)

(136)

(137)

(138)

(139)

(140)

(141)

Tronc, F.; Wassef, M.; Esposito, B.; Henrion, D.; Glagov, S.; Tedgui, A.
Arterioscler, Thromb., Vasc. Biol. 1996, 16, 1256-1262.

Rudic, R. D.; Shesely, E. G.; Maeda, N.; Smithies, 0.; Sega], S. S.; Sessa, W. C. J.
Clin. Invest. 1998, 101, 731-736.

Radomski, M. W. Acta Physio] Pol 1989, 40 Suppl 33, 97-109.

Sase, K.; Michel, T. Life Sci. 1995, 5 7, 2049-2055.

Mehta, J. L.; Chen, L. Y.; Kone, B. C.; Mehta, R; Turner, P. J. Lab. Clin. Med.
1995, 125, 370-377.

Wallerath, T.; gath, 1.; Aulitzky, W. E.; Pollock, J. S.; Kleinert, H.; Foerstermann,
U. Thromb. Haemostasis 1997, 77, 163-167.

Chen, L. Y.; Mehta, J. L. J. Cardiovasc. Pharmacol. 1996, 27, 154-158.

Berkels, R.; Bertsch, A.; Zuther, T.; Dhein, S.; Stockklauser, K.; Roesen, P.;
Roesen, R. Eur. J. Haematol. 1997, 58, 307-313.

Berkels, R.; Stockklauser, K.; Roesen, P.; Roesen, R. T hromb. Res. 1997, 87,
51-55.

Busse, R.; Fleming, 1. J. Vasc. Res. 1998, 35, 73-84.

Malinski, T.; Radomski, M. W.; Taha, Z.; Moncada, S. Biochem. Biophys. Res.
Commun. 1993, 194, 960-965.

Zhou, Q.; Hellerrnann, G. R.; Solomonson, L. P. Thromb. Res. 1995, 77, 87-96.

Lantoine, F.; Brunet, A.; Bedioui, F.; Devynck, J.; Devynck, M.-A. Biochem.
Biophys. Res. Commun. 1995, 215, 842-848.

58

(142)

(143)

(144)

(145)

(146)

(147)

(148)

(149)

(150)

(151)

(152)

(153)

(154)

(155)

Polanowska-Grabowska, R.; Gear, A. R. Blood 1994, 83, 2508-2515.

Venturini, C. M.; Fenton, J. W., 11; Minnear, F. L.; Kaplan, J. E. Thromb.
Haemostasis 1989, 62, 1006-1010.

Pohl, U.; Busse, R. Circ. Res. 1989, 65, 1798-1803.

Radomski, M. W.; Palmer, R. M.; Moncada, S. Lancet 1987, 2, 1057-1058.

Radomski, M. W.; Palmer, R. M. J.; Moncada, S. Biochem. Biophys. Res.

_ Commun. 1987, 148, 1482-1489.

Sneddon, J. M.; Vane, J. R. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 2800-2804.

Furlong, B.; Henderson, A. H.; Lewis, M. J .; Smith, J. A. Br. J. Pharmacol. 1987,
90, 687-692.

Macdonald, P. 8.; Read, M. A.; Dusting, G. J. Thromb. Res. 1988, 49, 43 7-449.

Alheid, U.; Reichwehr, 1.; Foerstermann, U. Eur. J. Pharmacol. 1989, 164,
103-110.

Houston, D. S.; Robinson, P.; Gerrard, J. M. Blood 1990, 76, 953-958.

Broekman, M. J .; Eiroa, A. M.; Marcus, A. J. Blood 1991, 78, 1033-1040.

Freedman, J. E.; Loscalzo, J.; Barnard, M. R.; Alpert, C.; Keaney, J. F., Jr.;
Michelson, A. D. J. Clin. Invest. 1997, 100, 350-356.

Trepakova, E. S.; Cohen, R. A.; Bolotina, V. M. C irc. Res. 1999, 84, 201-209.

Wang, G.-R.; Zhu, Y.; Halushka, P. V.; Lincoln, T. M.; Mendelsohn, M. B. Proc.
Natl. Acad. Sci. U. S. A. 1998, 95, 4888-4893.

59

(156)

(157)

(158)

(159)

(160)

(161)

(162)

(163)

(164)

(165)

(166)

(167)

(168)

Maurice, D. H.; Haslam, R. J. Mol. Pharmacol. 1990, 37, 671-681.
Bowen, R.; Haslam, R. J. J. Cardiovasc. Pharmacol. 1991, I 7, 424-433.

Zhang, J.; Zhang, J .; Shattil, S. J .; Cunningham, M. C.; Rittenhouse, S. E. J. Biol.
Chem. 1996, 271 , 6265-6272.

Pernollet, M.-G.; Lantoine, F.; Devynck, M.-A. Biochem. Biophys. Res. Commun.
1996, 222, 780-785.

Anfossi, G.; Russo, 1.; Massucco, P.; Mattiello, L.; Cavalot, F.; Balbo, A.; Trovati,
M. Thromb. Res. 2002, 105, 71-78.

Storey, R. F.; Heptinstall, S. Clin Lab Haematol 1999, 21, 317-329.

Freedman, J. E.; Ting, B.; Hankin, B.; Loscalzo, J.; Keaney, J. F., Jr.; Vita, J. A.
Circulation 1998, 98, 1481-1486.

Gkaliagkousi, E.; Ritter, J .; Ferro, A. Circ. Res. 2007, 101, 654-662.

Michelson, A. D.; Barnard, M. R.; Krueger, L. A.; Valeri, C. R.; Furman, M. I.
Circulation 2001, 104, 1533-1537.

Celi, A.; Pellegrini, G.; Lorenzet, R.; De Blasi, A.; Ready, N.; Furie, B. C.; Furie,
B. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 8767-8771.

Radomski, M. W.; Salas, E. Nitric Oxide 1995, 43-74.
Williams, R. H.; Nollert, M. U. Thromb. J. 2004, 2, No pp given.

Gambaryan, S.; Geiger, J .; Renne, T.; Walter, U. J. Thromb. Haemostasis 2009, 7,
371-373.

60

(169)

(170)

(171)

(172)

(173)

(174)

(175)

(176)

(177)

(178)

(179)

(180)

(181)

de Belder, A. J .; Radomski, M. W. J Hypertens 1994, 12, 617-624.

Cooke, J..P. Cardiovasc Clin 1992, 22, 3-17.

Radomski, M. W.; Salas, E. Atherosclerosis (Shannon, Irel.) 1995, 118, 869-880.
Radomski, M. W. Top. Mol. Med. 1995, I, 151-167.

Gryglewski, R. J .; Palmer, R. M. J .; Moncada, S. Nature (London) 1986, 320,
454-456.

Beckman, J. S.; Chen, J .; Ischiropoulos, H.; Crow, J. P. Methods Enzymol. 1994,
233, 229-240.

Liu, S.; Beckman, J. S.; Ku, D. D. J. Pharmacol. Exp. Ther. 1994, 268,
1114-1121.

Moro, M. A.; Darley-Usmar, V. M.; Goodwin, D. A.; Read, N. G.; Zamora-Pino,
R.; Fellisch, M.; Radomski, M. W.; Moncada, S. Proc. Nat]. Acad. Sci. U. S. A.
1994, 91, 6702-6706.

Moro, M. A.; Darley-Usmar, V. M.; Lizasoain, 1.; Su, Y.; Knowles, R. G.;
Radomski, M. W.; Moncada, S. Br. J. Pharmacol. 1995, 116, 1999-2004.

Brown, A. S.; Moro, M. A.; Masse, E. M.; Cramer, E. M.; Radomski, M.;
Darley-Usmar, V. Cardiovasc. Res. 1998, 40, 380-3 88.

Naseem, K. M.; Low, S. Y.; Sabetkar, M.; Bradley, N. J.; Khan, J.; Jacobs, M.;
Bruckdorfer, K. R. FEBS Lett. 2000, 473, 119-122.

Velican, C.; Velican, D. Med Interne 1980, 18, 181-187.

Spurlock, B. 0.; Chandler, A. B. Scanning Microsc 1987, 1, 1359-1365.

61

(182)

(183)

(184)

(185)

(186)

(187)

(188)

(189)

(190)

(191)

(192)

(193)

Fuster, V. Scand. J. Haematol, Supp]. 1981, 38, 1-37.

Willerson, J. T.; Buja, L. M. Am. J Cardiol. 1984, 54, 16-20.

Diodati, J. G.; Dakak, N.; Gilligan, D. M.; Quyyumi, A. A. Circulation 1998, 98,
17-24.

Cooke, J. P.; Singer, A. H.; Tsao, P.; Zera, P.; Rowan, R. A.; Billingham, M. E. J.
Clin. Invest. 1992, 90, 1168-1172.

Luscher, T. F. Baillieres Clin Haematol 1993, 6, 609-627.

Chen, L. Y.; Mehta, J. L. Life Sci. 1994, 55, 1815-1821.

Tsao, P. S.; McEvoy, L. M.; Drexler, H.; Butcher, E. C.; Cooke, J. P. Circulation
1994, 89, 2176-2182.

Tsao, P. S.; Theilmeier, G.; Singer, A. H.; Leung, L. L. K.; Cooke, J. P.
Arterioscler. T hromb. 1994, 14, 1529-1533.

Riddell, D. R.; Graham, A.; Owen, J. S. J. Biol. Chem. 1997, 272, 89-95.

Minuz, P.; Andrioli, G.; Degan, M.; Gaino, S.; Ortolani, R.; Tommasoli, R.;
Zuliani, V.; Lechi, A.; Lechi, C. Arterioscler., Thromb., Vasc. Biol. 1998, 18,
1248-1256.

Komamura, K.; Kitakaze, M.; Nishida, K.; Naka, M.; Tamai, J.; Uematsu, M.;
Koretsune, Y.; Nanto, S.; Hori, M.; Inoue, M.; et al. J Am Coll Cardiol 1994, 24,
370-377.

Olsen, S. B.; Tang, D. B.; Jackson, M. R.; Gomez, E. R.; Ayala, B.; Alving, B. M.
Circulation 1996, 93, 327-332.

62

(194)

(195)

(196)

(197)

(198)

(199)

(200)

(201)

(202)

(203)

(204)

(205)

Calver, A.; Collier, J.; Moncada, S.; Vallence, P. Bio]. Nitric Oxide, Proc. Int.
Meet, 2nd 1992, I, 322-325.

Cadwgan, T. M.; Benjamin, N. J Hypertens 1993, 11, 417-420.

Camilletti, A.; Moretti, N.; Giacchetti, G.; Faloia, E.; Martarelli, D.; Mantero, F.;
Mazzanti, L. Am. J. Hypertens. 2001, I 4, 382-386.

Moss, M. B.; Brunini, T. M. C.; Soares de Moura, R.; Malagris, L. E. N.; Roberts,
N. B.; Ellory, J. C.; Mann, G. E.; Ribeiro, A. C. M. Clin. Sci. 2004, 107, 391-397.

Schlaich, M. P.; Parnell, M. M.; Ahlers, B. A.; Finch, 8.; Marshall, T.; Zhang,
W.-Z.; Kaye, D. M. Circulation 2004, 110, 3680-3686.

Brunini, T. M. C.; Moss, M. B.; Siqueira, M. A. S.; Meirelles, L. R.; Rozentul, A.
L.; Mann, G. E.; Ellory, J. C.; Soares de Moura, R.; Mendes-Ribeiro, A. C. Clin.
Exp. Pharmacol. Physiol. 2004, 31, 738-740.

Trovati, M.; Anfossi, G.; Massucco, P.; Mattiello, L.; Costamagna, C.; Piretto, V.;
Mularoni, E.; Cavalot, F.; Bosia, A.; Ghigo, D. Diabetes 1997, 46, 742-749.

Martina, V.; Bruno, G. A.; Trucco, F.; Zumpano, E.; Tagliabue, M.; Di Bisceglie,
C.; Pescarmona, G. Thromb. Haemostasis 1998, 79, 520-522.

Queen, L. R.; Ji, Y.; Goubareva, 1.; Ferro, A. Diabetologia 2003, 46, 1474-1482.

Okada, M.; Sagawa, T.; Tominaga, A.; Kodama, T.; Hitsumoto, Y. Immunology
1996, 89, 158-164.

Radomski, M. W.; Jenkins, D. C.; Holmes, L.; Moncada, S. Cancer Res. 1991, 51,
6073-6078.

Radomski, M. W.; Moncada, S. Dev. Cardiovasc. Med. 1991, 126, 27-48.

63

(206)

(207)

(208)

(209)

(210)

(211)

(212)

(213)

(214)

(215)

(216)

Dong, Z.; Staroselsky, A. H.; Qi, X.; Xie, K.; Fidler, I. J. Cancer Res. 1994, 54,
789-793.

Stetler-Stevenson, W. G. Am. J. Pathol. 1996, 148, 1345-1350.

Brooks, P. C.; Stromblad, S.; Sanders, L. G; von Schalscha, T. L.; Aimes, R. T.;
Stetler-Stevenson, W. G.; Quigley, J. P.; Cheresh, D. A. Cell (Cambridge, Mass.)
1996, 85, 683-693.

Jurasz, P.; Sawicki, G.; Duszyk, M.; Sawicka, J.; Miranda, C.; Mayers, 1.;
Radomski, M. W. Cancer Res. 2001, 61, 376-382.

Sobol, A. B.; Watala, C. Diabetes Res. Clin. Pract. 2000, 50, 1-16.

Freedman, J. E.; Loscalzo, J. J. Thromb. Haemostasis 2003, 1, 1183-1188.

Tziros, C.; Freedman, J. E. Curr. Drug Targets 2006, 7, 1243-1251.

Sprague, R. S.; Olearczyk, J. J .; Spence, D. M.; Stephenson, A. H.; Sprung, R. W.;
Lonigro, A. J. Am. J. Physiol. 2003, 285, H693-H700.

Oblak, T. D. A.; Root, P.; Spence, D. M. Anal. Chem. 2006, 78, 3193-3197.

Carroll, J. S.; Ku, C.-J.; Karunarathne, W.; Spence, D. M. Anal. Chem.
(Washington, DC, U. S.) 2007, 79, 5133-5138.

Ku, C.-J.; Karunarathne, W.; Kenyon, S.; Root, P.; Spence, D. Anal. Chem. 2007,
79, 2421-2426.

64

CHAPTER 2 FLUORESCENCE DETERMINATION OF NITRIC
OXIDE PRODUCTION IN PLATELETS

2.1 KNOWLEDGE OF NITRIC OXIDE DETECTION

NO concentrations existing physiologically is essential for developing a
quantitative understanding of NO signalling, for performing in vitro experiments with
NO, and for measuring NO concentrations in disease states. Moreover, NO is involved in
a wide range of biological systems in the body, such as the cardiovascular, nervous,
reproductive, and immune systems.“4 Therefore, most research involving NO has
primarily focused on the detection and quantitative determination of NO within these
biological systems. Detection of NO in situ is often difficult due to its short half-life and
low concentrations. However, a series of practical methods to detect NO (Table 2.1) has
been developed using analytical methods including absorbance, chemiluminescence,
amperometric, and fluorescence techniques.

Briefly, the first method utilizes horseradish peroxidase, a commercially available
heme protein with ferric iron, to form a stable NO-ferric complex that induces large
spectral changes at 396.5 and 420.0 nm in an absorbance spectrophotometer with a
detection limit of 10 nmol/L.5

Chemiluminescence, more specifically the luminol/peroxide system, has also been

used to detect NO in biological samples as low as 100 fmol/L.6.9 The reaction of NO and

65

 

Method Species detected

Detection scheme

Detection limit

 

Horseradish NO __ HRP complex
peroxide (HRP)
Griess reaction -
N02
Luminal reaction -
ONOO
Nafion coated NO
carbon electrod
DAF-FM (DA) NO

Absorbance

Absorbance

Chemiluminescence

Amperometry

Fluorescence

10 nmol/L

0.1 umol/L

100 fmol/L

10 umol/L

3 nmol/L

 

Table 2.1 Practical methods for the detection of NO in biological samples employing

common analytical techniques

66

hydrogen peroxide generates peroxynitrite, a stronger oxidizing agent than hydrogen
peroxide itself, which can then react with luminol to produce a detectable
chemiluminescent product.

Another technique utilizes the Griess reaction, a method that measures the
conversion of nitrite, an oxidation product of NO, to the diazonium ion that is then
coupled to N-(l-naphtyl)ethylenediamine to form an azo derivative that is chromophoric.
The limit of detection for the Griess reaction is about 0.1 umol/L.10

Amperometry has also proven to be a useful tool in the detection of NO. By

coating a carbon ink electrode with Nafion, a modification used to block nitrite from the

electrode, NO was detected at concentrations as low as 10 nmol/L.ll A decade ago, a

12-16

value of about 1 uM seemed reasonable based on early electrode measurements and

a provisional estimate of the potency of NO for its guanylyl cyclase-coupled receptors,

which mediate physiological NO signal transduction.”19 Since then, numerous efforts to

measure NO concentrations directly using electrodes in cells and tissues have yielded an

14-16, 20-23

irreconcilably large spread of values. In compensation, data from several

alternative approaches have now converged to provide a more coherent picture. These
approaches include the quantitative analysis of NO-activated guanylyl cyclase, computer
modeling based on the type, activity and amount of NO synthase enzyme contained in

18, 24-26

cells the use of novel biosensors to monitor NO release from single endothelial

67

cells and neurones, and the use of guanylyl cyclase as an endogenous NO biosensor in
tissue subjected to a variety of challenges.27’ 28

The bulk of results reported here have focused on utilizing fluorescence based
techniques for the detection of NO. Accordingly, of recent interest is the family of
diaminofluorescein (DAF) fluorogenic indicators developed by Kojima et al,29 but more
specifically 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM DA).
Membrane permeable, this probe is deacylated by intracellular esterases to
4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). The diacetate probe is
essentially nonfluorescent until it reacts with byproducts of NO oxidation to form
fluorescent heterocycles. Upon nitrosylation, the probe becomes trapped within the
cytoplasm as shown in Figure 2.1. Importantly, the detection limit of NO with DAF-FM
is approximately 3 nmol/L, which is 1.4 times lower than that of DAF-2, a probe similar
to DAF-FM. DAF-FM is also known to be stable above pH 5.8 and because of the
specificity for NO, DAF-FM will not react (in neutral solution) with nitrate, nitrite or any

oxygen reactive species. DAF-FM is also not sensitive to ascorbic acid, a common

species in medium and buffer system.

68

Sam g m we nommmmfio can 8: now an. 8:865 SW mm: :2: margarine Boummboflnfi
E8385: m was“ SE¢<Q .530:on 0 Z Hui—Eombfi no 830326 05 no.“ <Q Saddam mo 25:8 .8388 23. mm 8:me

9588026 058225 3x826 0:885 9:8 ufiomeozmcozv
gumzbw 2o~m5o§0m 2nd/ad 285808 :00 88236 SEAZQ

 

 

 

 

69

2.2 NO PRODUCTION AND RELEASE FROM STIMULATED AND
ACTIVATED PLATELETS

The circulation is a complex mixture that includes, but is not limited to, cells,
macrophages, proteins, and metabolites. The white blood cells (leukocytes), red blood
cells (erythrocytes) and platelets have well-defined roles in the bloodstream. For example,
it is well-established that leukocytes play a major role in the immune system. Red blood
cells (RBCs) are recognized as oxygen carriers in vivo, while platelets are generally
considered major determinants in the blood clotting process. While each of the
aforementioned roles of these cell types are understood and accepted, there also reports
suggesting that the cells that flow through the circulation may actually participate in other
processes in blood vessels.

RBCs have been shown to be determinants in the control of vascular caliber in the

pulmonary and systemic circulation by releasing nanomolar to micromolar amounts of

adenosine triphosphate (ATP) when these cells are deformed“)-35 or subjected to brief

9

periods of hypoxia.3 37 Certain pharmacological agents are also capable of stimulating

the release of ATP from RBCs.38 The importance of this release of ATP in vivo is that

ATP is a recognized stimulus of nitric oxide synthase (NOS) in endothelial cells”.42 NO

. . . . . . . . 43-46 . .
IS a multitasking molecule in vzvo. It IS a recognized dilator of blood vessels, It 1S an
51, 52

. . . . . . 47-50 . . .
1nh1b1tor of platelet activatlon and aggregat1on, and it 18 also a neurotransm1tter.

Although a relationship between ATP released from RBCs and endothelium-derived NO

70

has been established, there have been no reports of the synergy between ATP derived
from RBCs and the ability of platelets to produce NO.

The relationship between ATP and platelet-derived NO may be profound.
Sprague et a] postulated that ATP was released from the RBC in response to mechanical
deformation, as would occur in association with vasoconstriction or increased velocity of
blood flow.30 This RBC-derived ATP can then act on the endothelial cell to stimulate
endogenous NO synthesis and enable the RBC to participate in local regulation of

3

vascular caliber.3 34 In accordance with the availability of NO along the vascular wall,

we have recently found that platelets have the ability to release ATP and produce their
own NO (also has been shown to inhibit platelet adhesion to endothelial cells). These
findings suggest that the RBC may be a determinant of platelet adhesion in vivo.

Platelets normally circulate without adhering to undisturbed vascular endothelium.
Upon vascular insult, subendothelial collagen is exposed that acts as the primary stimulus
for platelet activation. However, several other endogenous agonists also exist such as
thrombin, ADP, thromboxane A2, serotonin and epinephrine, which also promote platelet
activation.53 Platelet activation initiates a change in the platelet shape, thus promoting
adhesion to the vascular walls and the subsequent recruitment of additional platelets. NO

has been widely shown to mediate this process by activating soluble guanylate cyclase

(sGC) which initiates a protein kinase G (PKG) dependant pathway.54 In this construct,

71

NO becomes an important determinant in platelet adhesion in vivo.50 If uncontrolled,
these adhered platelets become a major constituent of thrombus formation and
subsequent vessel blockage.

Recently, platelets have been reported to have unique properties in disease states
other than cardiovascular problems. For example, a procedure used in multiple sclerosis,
plasma replacement therapy, involves removing such formed elements as RBCs and
leukocytes from the patient’s plasma and then replenishing new plasma with the
previously removed cells. The plasma contains the platelets so, in addition to obtaining
new plasma, the patient has also receives new platelets. Interestingly, the platelets of
people with multiple sclerosis have been shown to be more susceptible to hyperactivity,
often aggregating more easily than the platelets of healthy people that do not have
multiple sclerosis.55 The importance of this platelet activity is that platelets are known to
produce NO upon activation56 and people with multiple sclerosis have been shown to
have high levels of NO and NO metabolites in their cerebral spinal fluid and urine.57’ 58
This trait of platelets (prone to activation and aggregation) is also known to exist for
patients that have cystic fibrosis.59 Finally, patients with diabetes also have platelets that
are known to be activated more readily than healthy, non-diabetic controls.6O

Exemplifying the importance and complexity of the relationship between platelets and

NO is that NO has the ability to inhibit platelet activation.50

72

Interestingly, platelets have the ability to create bioavailable NO in the
bloodstream in multiple ways other than by activation. For example, NO can be
synthesized inside of the platelet by one of multiple isoforms of NOS61’62 or from the
denitrosation of S-nitrosothiols (S-nitrosoglutathione or S-nitrosoalbumin)
enzyrnatically63 or by copper-containing proteins.64 In each of these scenarios, the most
common method that has been employed to date for measuring NO derived from cells is
amperometry with either carbon or platinum electrodes.“’ 65 Advantages of such
measurement schemes is the ability to measure the released NO without having to add
any molecular probes to the system or performing any sort of derivatization chemistry to
create a fluorophore or chromophore. Moreover, amperometric methods also enable the
analyst to add some type of selectivity to their measurement when a modified electrode
surface is employed during the measurement.“’ 65 However, the degree of selectivity

, 23
and

depends on the number of coatings of the electrode surface and its integrity,”
day-to-day electrode behavior can change dramatically.22

Here, we have employed diaminofluorofluorescein (DAF) probes66 which,
depending on the form, are rather specific for NO in the absence of certain interferences
(such as ascorbate).67'69 Specifically, we have used DAF -F M DA to perform intracellular

measurements of NO when produced via platelet eNOS. However, to date there have

been no reports of measuring platelet-derived NO using fluorescence spectrophotometry.

73

The DAF family of probes has been employed to determine NO production in, or
NO release from, numerous cell types. However, none of the DAF probes have been
employed to measure NO production in platelets. In the work reported here, the optimum
conditions for measuring NO production in platelets, including probe concentration and
incubation time, is determined. In addition, data is reported that demonstrates the ability
to measure NO production in platelets using DAF-FM DA. When combined with an
extracellular measurement (using DAF-FM) prior to the stimulation of NO production
with either ATP or ADP, the ability to quantitatively determine the amount of NO

released from the platelets is possible.

2.3 EXPERIMENTAL METHODS

Isolation and purification of platelets. Rabbits were anesthetized with ketamine
(100 mg/mL, 0.12 mI/kg, i.m.) and xylazine (20mg/mL, 0.08 mL/kg, i.m.) followed by
pentobarbital sodium (3 mg/mL, 3.2 mL/kg, i.v.). A cannula was placed in the trachea
and the animals were ventilated with room air. A catheter was then placed into a carotid
artery for administration of heparin (1000 units/mL, 0.5 mL/kg) and for phlebotomy.
After heparin, animals were exsanguinated and the whole blood collected in a 50 mL
centrifuge tube. Generally, 70 - 90 mL of blood was collected from the animal. Blood

was centrifuged at 500 x g at 37°C for 10 minutes. The platelet rich plasma (PRP) was

74

decanted for the subsequent isolation of platelets. Platelets were isolated from the PRP by
adding 1 mL of acid citrate dextrose (ACD, 41.6 mM citric acid anhydrous, 76.7 mM
sodium citric acid anhydrous, 122.1 mM D-glucose) to 9 mL of the PRP and centrifuging
at 1500 x g at 37°C for 10 minutes. The harvested platelets were then washed twice by
centrifugation in a mixture containing Hank’s Balanced Salt solution (HBSS, H1387
from Hanks’ Balanced Salt, Sigma) and ACD (6:1 v/v). The washed platelets were then
resuspended in HBSS as higher concentrated platelet stock solution.

In all experiments, platelet count was measured on the hemacytometer then
adjusted to a platelet count of 3.0 x 108 platelets mL'l as final concentration with HBSS.
Platelets were generally harvested and prepared on the day of use. Our group has found
that, if kept in the PRP, the platelets can be purified within 1-2 days after the surgical
procedure and still produce reliable data. However, the platelets are generally consumed
through experimental procedures on the day of harvesting them from the rabbit. All
studies involving animal use were approved by the Animal Investigation Committee at
Michigan State University.

Reagent preparation. All reagents were purchased from Sigma Chemical Co.
(St. Louis, MO) and used as received without further purification unless otherwise noted.

L-arginine (the substrate of NO production, pH was adjusted before experiments),

N‘D-Nitro-L-arginine methylester hydrochloride (L-NAME, a selective eNOS/nNOS

75

inhibitor), and NG-monomethyl-L-arginine (L-NMMA, a non-selective NOS inhibitor)
were prepared as 10 mM stock solutions in HBSS. Adenosine 5'-triphosphate (ATP) and
adenosine 5'-diphosphate (ADP) were prepared as 100 11M stock solutions in distilled and
deionized water (DDW, 18.2 MQ-cm). The DAF-FM/DAF-FM DA was prepared as a 5
mM stock solution in dimethyl sulfoxide (DMSO). In all studies, the total volume of
platelets, DAF-FM/DAF-FM DA, inhibitor/agonist, and buffer were equal to 1.0 mL.
Nitric oxide was prepared as a 38mM‘ stock solution from spennine NONOate (Cayman
Chemical, Ann Arbor, MI) by dissolving 10mg of the sperrnine NONOate solid in 1 mL
of 0.01M sodium hydroxide (NaOH) solution. Working solutions were prepared by
dilution of the alkaline NONOate solution in 0.1 M phosphate buffer (pH 7.4).
Fluorescence determination. Aliquots of DAF-FM/DAF-FM DA were added
into each vial and incubated with the platelets for 30 minutes before each fluorescence
measurement was taken. For those studies involving stimulation/activation with
ATP/ADP, the agonist was added and equilibrated with platelets for another 30 minutes
before measurements of NO production. In the contrast, studies involving inhibition with
L-NAME/L-NMMA, the inhibitor was added with platelets for 30 minutes prior to
fluorescent probe incubation. Fluorescence measurements were performed on an
RF-5301PC spectrofluorometer (Shirnadzu, Columbia, MD) at room temperature.

Fluorescence emission spectra were obtained in a quartz cuvette with excitation

76

wavelength at 495 nm and emission wavelength at 515 nm. Both excitation and emission
slit widths of 3 nm were used in all experiments. The sampling interval was 0.2 nm with
a data acquisition rate of 50 Hz. All error bars in the figure represent standard error of the
mean (SEM) for at least 11 = 4 different rabbits exsanguinated on different days and each

bar was statistically significant at minimum p < 0.05.

2.4 RESULTS AND DISCUSSION

NO in platelets. Platelets are known to contain NOS, which upon stimulation, will
produce and subsequent release NO.6I’ 62 In order to determine if basal levels of NO
could be measured using the DAF-FM DA probe, 100 uL of a suspension containing 3 x
109 platelets mL.1 were added to 100 1.1L of the DAF-FM DA probe (10 uM) and, after
the solution was brought to 1.0 mL in total volume with HBSS, allowed to incubate for
30 minutes; all experiments were performed at room temperature. This time enabled the
DAF-FM DA probe to cross the platelet membrane bilayer; once inside the platelet, the
probe is able to detect NO after the diacetate groups are cleaved from the parent
DAF-FM DA molecule. The incubation period enables this cleaving process to occur. A
typical fluorescence spectrum from the platelets loaded .with DAF-FM DA is shown in
Figure 2.2a (middle trace). Also shown in this figure is a spectrum (t0p trace) obtained

after DAF-FM DA loaded platelets were stimulated with 100 uL of 100 uM ATP, a

77

 

_, — Platelets
10‘. - +ATP
‘ ‘ +L-NAME

 

 

 

Fluorescence Intensity (an)
as

 

 

 

 

460 480 500 520 540 560 580 600
Wavelength (nm)

 

b.)

F luorascence intensity (a.u.)
'— N

 

 

 

Figure 2.2 (a) Emission profiles for platelets in the presence of DAF-FM DA molecular
probe for nitric oxide (middle trace), the platelets in the presence of the probe and 10 11M
ATP (top trace), and the platelets in the presence of the probe after incubation in the
NOS inhibitor L-NAME (bottom trace) (b) Quantitative data obtained from spectra,
control consisted of platelets in the absence probe. Error bars represent SEM. p < 0.05
(n = 5)

78

recognized stimulus of NOS.39 The increase in fluorescence emission is due to the
increase in NO production inside of the platelets upon NOS activation by ATP. In order
to provide evidence that this increase in the measured fluorescence emission was due to
an increase of NO production (via NOS activation) in the presence of the ATP stimulant,
platelets were incubated with L-NAME, a competitive inhibitor of NOS. As shown in
Figure 2.2a, a 50.4% i 2.3% decrease in fluorescence emission is measured from those
platelets incubated with L-NAME (bottom trace). Figure 2.2b contains summarized data
obtained from the spectra. The control consisted of platelets alone in the absence of
DAF-FM DA, and demonstrated that no NO production could be measured unless
DAF-FM DA is present. Upon stimulation or activation, the NO production in platelets
was increased 22.3% :1: 4.4% and 36.8% :1: 5.1%, respectively compared to normal
platelets incubated with DAF-FM DA. Since there is no nNOS in platelets, the inhibition
of NOS activity by L-NAME or L-NMMA resulted in approximately equal decreases in
NO production: 50.4% :1: 6.0% and 44.5% i 3.5%, respectively.

Optimization of DAF-FM DA concentration. Key to the success in measuring
NO production in any cell type is the optimization of those variables affecting the signal
intensity. An important variable in the studies reported here is the Optimal concentration
of the DAF-FM DA probe. Typically, most users of DAF-type probes employ

concentrations of 10 uM. However, fluorescein-based probes are somewhat notorious for

79

resulting in large background signals, thus affecting the overall S/N ratio in a
measurement scheme. Therefore, in order to optimize the resultant emission from NO
production, various DAF-FM DA concentrations were loaded into a suspension of
platelets (3 x 108 platelets mL'1 as final concentration), and allowed to incubate for 30
minutes before the fluorescence intensity was measured. Next, a mixture containing 100
11L of 10 mM L-arginine and 100 uL of 100 1.1M ATP was added to the platelet solution
and, after 30 minutes, the emission resulting from ATP-induced NOS activation was
measured. The data in Figure 2.3 show that platelets incubated with 0.1 and 0.5 uM
DAF-FM DA displayed a change in fluorescence intensity upon the addition of DAF-F M
DA to the platelet suspension. However, upon addition of the ATP stimulus, there was no
statistically significant change in the fluorescence emission. There were significant
changes in the fluorescence emission for concentrations of DAF -F M DA between 1 and
10 11M. In addition, results obtained from the studies involving the 1-10 1.1M
concentrations indicate the background emission (DAF-FM DA alone represented by
black bars in Figure 2.3) was lowest for the 1 uM trial. The signal from stimulated
platelets (expressed as a percent change from the emission from platelets loaded with
DAF-F M DA that were not stimulated with ATP) was highest for the 5 11M and 10 11M
trials where an increase in emission intensity of ~40% was measured. Although a percent

change of 37% was measured for the 1 uM DAF-FM DA concentration, the SIN ratio did

80

r—I
O\

 

        

 

 

 

 

 

 

1 — DAF-FM DA
14 4 [:3 +Platelet
2 j — +ATP
g 12 a
g 10 -j
5 . 13
.§ 8:
“g :
o 6: ‘
5: 45 +
u- 1
2 5
O E 7x IX \ \ x '
$9 a“? ‘5‘ij $2.8“ ‘5‘?“ 0,9

Figure 2.3 Fluorescence intensities using various concentrations of the DAF-F M DA
probe. Emission intensities are shown for DAF-FM DA probe in the absence of platelets
(black bar), the probe in the presence of the platelets (light gray bar), and in the presence
of the probe, platelets, and ATP stimulus (dark gray bar). Error bars represent SEM.
p < 0.05 (n = 4)

81

not statistically increase from 1 11M to 10 uM DAF-FM DA. Therefore, for the remaining
studies, a 10 11M DAF-FM DA stock was diluted to a working concentration of 1 uM
DAF -F M DA for all NO measurements.

Incubation time optimization. In addition to the concentration of the DAF-FM
DA probe employed for monitoring the NO production in platelets, it is also important to
investigate the time allotted for the DAF-FM DA to enter the cell and for the cleavage of
diacetate from the parent probe molecule. Figure 2.4 contains data showing results from
subjecting 100 uL of a 3 x 109 platelets mL'l solution to 100 uL of a 10 11M DAF—FM
DA solution (the final volume was adjusted to 1.0 mL with HBSS) for a specified period
of time and monitoring the increase in fluorescence emission. Controls consisting of
buffer alone with DAF-FM DA or buffer with L-arginine, ATP, and DAF-FM DA in the
absence of platelets were measured to demonstrate that no significant increase in
emission intensity is measured unless platelets are included in the reaction mixture.
However, in the presence of platelets, a significant increase in fluorescence emission is
measurable, even after 2.5 minutes of incubation time. This increase in fluorescence is
even more pronounced in the presence of ATP. Interestingly, the highest ratio of
fluorescence intensity in the presence of ATP to that when no ATP is added occurs
within 2 - 3 minutes. In other words, although longer incubation periods for the DAF-FM

DA will increase the overall emission intensity, the difference in fluorescence emission

82

7-
' _._ DAF-FMDA

6 _ Platelets + DAF-FM DA
/; +ATP
g 5 A Platelets + ATP + DAF-FM DA
5: . 1 E 1 1
84-
.§ 1 i E ,6 § 1 E
2‘33- § JE— 6 BE
8 : § 1 E
9213 l ‘3
a . .

1 E

 

_,_:_1_|_|._J_n . A;
i-

q

4

O

 

O
p—t
O
N
O

30 40 50 60
Time (minute)

Figure 2.4 Measurement of the change in the fluorescence intensity as a function of time.
The lower two traces are for DAF-FM DA (circles) and DAF-FM DA incubated with
ATP (diamonds) in the absence of platelets. The top two traces are for platelets incubated
with DAF-FM DA for the times specified on the time axis in the absence (squares) and
presence (triangles) of ATP. For each data point shown in the top two traces, the
fluorescence intensity is significantly higher for those platelets that were stimulated with
ATP. Error bars represent SEM. (n = 4)

83

between basal levels of NO and those levels resulting from stimulation can be optimally
measured within a minute or two of subjecting the platelets to the NO stimulus. The
reason that the signal does not stabilize with time could be due to the ability of platelets
to continue producing NO. Therefore, a fixed-time method is used to perform all
experiments here.

NO production and platelet concentration. As mentioned above, the volume of
platelets obtained from the whole blood of the rabbit is limited. Thus, in order to optimize
the number of experiments that can be performed with a single harvesting of platelets
from a mammalian subject, studies were performed to investigate the effect of platelet
number on the measured emission signal. Volumes of a platelet solution containing 3 x
109 platelets mL'1 were incubated in 1.0 uM DAF-FM DA for 30 minutes prior to
measuring the fluorescence emission from the probe-NO product. Data in Figure 2.5
indicate that the signal intensity increases as a fimction of the number of platelets in the
reaction mixture. However, the overall ratio of the emission intensity between platelets
with DAF-FM DA and those platelets incubated with the probe and ATP stimulus was
statistically unchanged even up to 200 uL of platelets.

Discussion of N0 in platelets. Figures 2.2 -— 2.5 demonstrate that the DAF-FM
DA probe can be employed for the intracellular determination of NO, not only in the

presence of external agonists, ATP and ADP, but also after the inhibition by L-NAME

84

4 ‘ + Platelets
: + Platelets+ L-NAME
, -O— Platelets + DAF-FM DA
+ Platelets + ATP + DAF-F M DA

‘19
1 I 1

I 4 LL 1 J l I

Fluorescence intensity (a.u.)
N

 

 

 

 

0 ,3: fit F A
0 50 100 150 200
Platelet volume (uL)

Figure 2.5 Signal intensities as a function of platelet number for platelets in the presence
and absence of DAF -F M DA, in the presence of the probe with ATP, and in the presence
of the probe and L-NAME. Error bars represent SEM. (n = 4)

85

and L-NMMA. The ATP stimulates the production of NO in platelets by binding to
purinergic receptors on the platelet membrane. Therefore, although the ATP is able to
stimulate NO production in the platelets, it does not result in the activation or aggregation
of platelets. It is known that agonists of platelet activation and subsequent aggregation,
such as ADP and thrombin, are able to stimulate NO production. However, this type of
stimulation of NO (through platelet activation) is important because many features of
platelet function are related to their ability to activate and aggregate. The data in
Figure 2.6 show that the maximum NO produced in platelets upon activation with the
ADP agonist increased by 52.3% d: 8.2% while ATP stimulation resulted in a maximum
increase in fluorescence emission of 39.1% i 6.2%. Thus, the use of the DAF-FM DA
probe can be employed to measure NO production even in the presence of activated
platelets. Importantly, these results also suggest that a greater amount of NO is produced
in platelets that are activated by ADP as compared to stimulation with ATP.

N0 released from platelets. A quantitative determination of the amount NO was
also performed. The NO donor, spennine NONOate, was used as a standard in the
method of multiple standard additions (Figure 2.7a). A 3.8 uM NONOate solution was
incubated in HBSS at 37°C for 15 minutes, while at the same time, 100 11L of platelet
solution (3 x 109 platelets mL'l) and 100 uL of 10 uM DAF-FM DA were mixed and

allowed to incubate for 15 minutes. Various amount of NONOate solution was added into

86

 

 

 

4.0:
3.8“: 1 t
:i I '
3; 3.6 ‘ I]
b .
'5', 3.4: ,
8 « a -
H 4 ' - g
.S 32 — l a
d) .1 '3
8 3.0 . I
o
H 4 ..
g 2.8:
pt. .
2.6% —0— Platelets+ADP
1 + Platelets+ATP
2,4-.fififfi

 

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00
concetration ( 1.1M)

Figure 2.6 Monitoring the production of nitric oxide as a function of concentration of
ATP and ADP. Relative to unstimulated platelets incubated with the DAF-FM DA probe,
the increase in fluorescence emissions were 39.1% :t 6.2% and 52.3% i 8.2% for
platelets stimulated with ATP and activated with ADP, respectively. Error bars represent
SEM. (n = 4)

87

(a) 3.0
2.5

2.0

Fluorescence intensity (a.u.)
UI

 

1.0
0.5
0 40 80 120 160
NONOate volume (uL)

 

i?

V

.N s» u

U! C M
n A I

NO concentration (x1 0' 1 7 mole NO/plt)
N
'o

0.5 j

 

 

 

.9
o

Plt +ATP +ADP +L-NAME

Figure 2.7 (a) A calibration curve was prepared using the method of multiple
stande additions. The fluorescence intensity was measured after increments of NO
were added to aliquots of platelets containing DAF-FM. (b) Quantitative
determinations of NO released by the platelets are summarized in the accompanying
bar graph. The concentration of extracellular NO in the presence of platelets alone
(Plt) is 9.9 i 2.2 x 10'18 moles NO/platelet. The extracellular NO levels increase to
2.0 :1: 0.1 x 10‘17 and 2.8 i 0.3 x 10'17 moles NO/platelet in the presence of ATP
(+ATP) and ADP (+ADP), respectively. In the presence of a NOS inhibitor
(+L-NAME), the concentration of extracellular NO decreased to 3.1 i 0.9 x 10'18
moles NO/platelet. Error bars represent SEM. (n = 4)

88

DAF-FM DA loaded platelet solution and allowed to incubate for another 15 minutes.
The solution volume in each vial was adjusted to 1.0 mL with HBSS. The results showed
that the amount of intracellular NO in platelets is (2.7 i 0.3) x 10'16 moles of NO/platelet
(data not shown) as basal level of NO. Employing the method previously discussed with
the addition of the DAF-FM probe (for extracellular NO determinations), Figure 2.7b
indicates that a quantitative determination of NO released from platelets upon stimulation
by ATP or activation by ADP is feasible. The data in Figure 2.7 revealed that the
concentration of extracellular NO in the presence of platelets alone (i.e., no stimulus or
activator) is (9.9 :1: 2.2) x 10'18 moles of NO/platelet. The extracellular NO levels
increase to (2.0 d: 0.1) x 10'17 and (2.8 i 0.3) x 10'17 moles of NO/platelet in the presence
of ATP and ADP, respectively. To verify that the fluorescence signal was due to NO, the
experiment was also performed in the presence of L-NAME. In the presence of the
inhibitor, the concentration of extracellular NO decreased to (3.1 i 0.9) x 1048 mole of
NO/platelet. Importantly, Freedman et a] demonstrated that, upon activation, platelets
released about 5 x 10717 moles of NO/platelet.5O Here, we reported a value approximately
3 x 10'17 moles of NO/platelet,7o which is less than that reported using amperometry, but

still in good agreement with previous results.

89

2.5 CONCLUSIONS

The spectrofluorometric monitoring of NO production in platelets has been
determined using DAF -F M DA as a molecular probe. The NO that was measured existed
either as a basal level of NO found in the platelets, NO that was stimulated by ATP, or
NO stimulated by activating the platelets with ADP. There have been some reports
describing the use of DAF-2 DA, another member of the DAF family of molecular
probes, for fluorescent monitoring of intracellular NO production. However, these reports
were lacking since none of these previous studies investigated the parameters affecting
the overall emission signal from the DAF-FM/NO product such as probe concentration,
reaction time, and platelet density. Another important feature of the results presented here
is that NO production in platelets can be determined upon activation with an agonist
known to initiate platelet aggregation. That is, upon addition of ADP, platelets. will
activate and eventually aggregate. This activation has been shown to lead to the
production and release of NO from platelets. The ability to monitor this NO production
using a fluorescence probe may be advantageous over other forms of measuring NO
production from platelets (such as amperometry) because, once activated, a platelet’s
ability to adhere to surfaces is dramatically increased. In fact, in vivo, it is the ability of
the platelet to adhere to surfaces that leads to repair of lesions or clotting of blood. These

same adhesion properties are also known to be determinants in the formation of a

90

thrombus on the inner walls of the vascular system, thus leading to thrombosis and,
potentially, atherosclerosis and vessel blockage. The ability to measure NO produced in
platelets using fluorescence spectrophotometry will enable those researchers studying the
role of platelets in other biologically or medically related fields (diabetes, multiple
sclerosis, cystic fibrosis) to monitor this important molecule using instrumentation found

in most laboratories and microliter volumes of sample.

91

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

LIST OF REFERENCES

Ignarro, L. J.; Buga, G. M.; Byms, R. E.; Wood, K. S.; Chaudhuri, G. J.
Pharmacol. Exp. T her. 1988, 246, 218-226.

Toda, N.; Okamura, T. Pharmacol. Rev. 2003, 55, 271-324.

Daniel, H.; Hemart, N.; Jaillard, D.; Crepel, F. Eur J Neurosci 1993, 5,
1079-1082.

Cook, H. T.; Cattell, V. Clin. Sci. 1996, 91, 375-384.

Kikuchi, K.; Nagano, T.; Hirobe, M. Biol. Pharm. Bull. 1996, 19, 649-651.

Kikuchi, K.; Nagano, T.; Hayakawa, H.; Hirata, Y.; Hirobe, M. J. Biol. Chem.
1993, 268, 23106-23110.

Kikuchi, K.; Nagano, T.; Hirobe, M. Kassei Sanso, Furi Rajikaru 1993, 4,
487-496.

Kojima, H.; Kikuchi, K.; Hirobe, M.; Nagano, T. Neurosci. Lett. 1997, 233,
157-159.

Kurumatani, H.; Kikuchi, K.; Nagano, T.; Hirobe, M.; Yamazaki, J.; Nagao, T.
Biol. Pharm. Bull. 1998, 21, 1286-1289.

Nims, R. W.; Cook, J. C.; Krishna, M. C.; Christodoulou, D.; Poore, C. M. B.;
Miles, A. M.; Grisharn, M. B.; Wink, D. A. Methods Enzymol. 1996, 268, 93-105.

Spence, D. M.; Torrence, N. J.; Kovarik, M. L.; Martin, R. S. Analyst (Cambridge,
U. K.) 2004, 129, 995-1000.

92

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

Malinski, T.; Radomski, M. W.; Taha, Z.; Moncada, S. Biochem. Biophys. Res.
Commun. 1993, 194, 960-965.

Malinski, T.; Taha, Z.; Grunfeld, S.; Burewicz, A.; Tomboulian, P.; Kiechle, F.
Anal. Chim. Acta 1993, 279, 135-140.

Wang, Y.; Lin, S.-Z.; Chiou, A.-L.; Williams, L. R.; Hoffer, B. J. J. Neurosci.
1997, I 7, 4341-4348.

Kalinowski, L.; Dobrucki, I. T.; Malinski, T. J. Cardiovasc. Pharmacol. 2001, 37,
713-724.

Mochizuki, S.; Miyasaka, T.; Goto, M.; Ogasawara, Y.; Yada, T.; Akiyama, M.;
Neishi, Y.; Toyoda, T.; Tomita, J .; Koyama, Y.; Tsujioka, K.; Kajiya, F.; Akasaka,
T.; Yoshida, K. Biochem. Biophys. Res. Commun. 2003, 306, 505-508.

Besse, J. C.; F urchgott, R. F. J. Pharmacol. Exp. Ther. 1976, 197, 66-78.

Zhao, Y.; Brandish, P. E.; Ballou, D. P.; Marletta, M. A. Proc. Nat]. Acad Sci. U.
S. A. 1999, 96, 14753-14758.

Griffiths, C.; Wykes, V.; Bellamy, T. C.; Garthwaite, J. Mo]. Pharmacol. 2003, 64,
1349-1356.

Malinski, T.; Bailey, F .; Zhang, Z. G.; Chopp, M. J. Cereb. Blood Flow Metab.
1993, 13, 355-358.

Wang, Y.; Li, Q.; Hu, S. Bioelectrochemistry 2005, 65, 135-142.

Wadsworth, R.; Stankevicius, E; Simonsen, U. J. Vasc. Res. 2006, 43, 70-85.

Santos, R. M.; Lourenco, C. F.; Piedade, A. P.; Andrews, R.; Pomerleau, F.;
Huettl, P.; Gerhardt, G. A.; Laranjinha, J.; Barbosa, R. M. Biosens. Bioelectron.
2008, 24, 704-709.

93

(24)

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

Mo, E.; Amin, H.; Bianco, I. H.; Garthwaite, J. J. Biol. Chem. 2004, 279,
26149-26158.

Roy, B.; Garthwaite, J. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 12185-12190.

Roy, B.; Halvey, E. J.; Garthwaite, J. J. Biol. Chem. 2008, 283, 18841—18851.

Sato, M.; Hida, N.; Umezawa, Y. Proc. Natl. Acad. Sci. U. S. A. 2005, 102,
14515-14520.

Sato, M.; Nakajima, T.; Goto, M.; Umezawa, Y. Anal. Chem. 2006, 78,
8175-8182.

Kojima, H.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Hirata, Y.; Nagano, T. Angew.
Chem, Int. Ed. 1999, 38, 3209-3212.

Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J. Am. J. Physiol.
1996, 271, H2717-H2722.

Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Kleinhenz, M. E.; Lonigro,
A. J. Am. J. Physiol. 1998, 275, H1726-H1732.

Edwards, J.; Sprung, R.; Spence, D.; Sprague, R. Analyst (Cambridge, U. K.)
2001, 126, 1257-1260.

Sprung, R.; Sprague, R.; Spence, D. Anal. Chem. 2002, 74, 2274-2278.

Sprague, R. S.; Olearczyk, J. J.; Spence, D. M.; Stephenson, A. H.; Sprung, R. W.;

Lonigro, A. J. Am. J. Physiol. 2003, 285, H693-H700.

Price, A. K.; Fischer, D. J.; Martin, R. S.; Spence, D. M. Anal. Chem. 2004, 76,
4849-4855.

94

(36)

(3 7)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(43)

Bergfeld, G. R.; Forrester, T. Cardiovasc. Res. 1992, 26, 40-47.

Ellsworth, M. L. Acta Physiol. Scand. 2000, 168, 551-559.

Olearczyk, J. J .; Stephenson, A. H.; Lonigro, A. J .; Sprague, R. S. Am. .I. Physio].
2004, 286, H940-H945.

Bogle, R. G.; Coade, S. B.; Moncada, 8.; Pearson, J. D.; Mann, G. E. Biochem.
Biophys. Res. Commun. 1991, 180, 926-932.

Hassessian, H.; Burnstock, G. Br. J. Pharmacol. 1995, 114, 846-850.

Kotsis, D. H.; Spence, D. M. Anal. Chem. 2003, 75, 145-151.

Carroll, J .; Raththagala, M.; Subasinghe, W.; Baguzis, S.; Oblak, T. D. a.; Root,
P.; Spence, D. Mo]. BioSyst. 2006, 2, 305-311.

Furchgott, R. F.; Zawadzki, J. V. Nature (London) 1980, 288, 373-3 76.

Ignarro, L. J .; Buga, G. M.; Chaudhuri, G. Eur. J. Pharmacol. 1988, 149, 79-88.

Palmer, R. M. J.; Ferrige, A. G.; Moncada, S. Nature (London) 1987, 327,
524-526.

Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Biochem. Pharmacol. 1989, 38,
1709-1715.

Radomski, M. W.; Palmer, R. M.; Moncada, S. Lancet 1987, 2, 1057-1058.

Wang, G.-R.; Zhu, Y.; Halushka, P. V.; Lincoln, T. M.; Mendelsohn, M. B. Proc.
Natl. Acad. Sci. U. S. A. 1998, 95, 4888-4893.

95

(49)

(50)

(51)

(52)

(53)

(54)

(55)

(56)

(5 7)

(58)

(59)

(60)

Sogo, N.; Magid, K. 8.; Shaw, C. A.; Webb, D. J.; Megson, 1. L. Biochem.
Biophys. Res. Commun. 2000, 279, 412-419.

Freedman, J. E.; Loscalzo, J.; Barnard, M. R.; Alpert, C.; Keaney, J. F., Jr.;
Michelson, A. D. J. Clin. Invest. 1997, 100, 350-356.

Martin, W.; Gibson, 1. F.; Gillespie, J. S.; Liu, X. Aspects Synaptic Transm. 1991,
258-279.

Bicker, G.; Stern, M. Neuroforum 2009, 15, 4-12.

Packham, M. A. Can. J. Physiol. Pharmacol. 1994, 72, 278-284.

Morrell, C. N.; Matsushita, K.; Chiles, K.; Scharpf, R. B.; Yamakuchi, M.; Mason,
R. J. A.; Bergmeier, W.; Mankowski, J. L.; Baldwin, W. M., 111; Faraday, N.;
Lowenstein, C. J. Proc. Nat]. Acad. Sci. U. S. A. 2005, 102, 3782-3787.

Neu, I. S.; Prosiegel, M.; Pfaffenrath, V. Acta Neurol Scand 1982, 66, 497-504.

Radomski, M. W.; Palmer, R. M. J .; Moncada, S. Proc. Natl. Acad Sci. U S. A.
1990, 87, 5193-5197.

Giovannoni, G.; Heales, S. J. R.; Land, J. M.; Thompson, E. J. Mult. Scler. 1998,
4, 212-216.

Smith, K. J .; Lassmann, H. Lancet Neurol. 2002, I, 232-241.

O'Sullivan, B. P.; Linden, M. D.; Frelinger, A. L., 111; Barnard, M. R.;
Spencer-Manzon, M.; Morris, J. E.; Salem, R. 0.; Laposata, M.; Michelson, A. D.
Blood 2005, 105, 463 5-4641.

Coppola, L.; Verrazzo, G.; La Marca, C.; Ziccardi, P.; Grassia, A.; Tirelli, A.;
Giugliano, D. Diabet Med 1997, 14, 959-963.

96

(61)

(62)

(63)

(64)

(65)

(66)

(67)

(63)

(69)

(70)

Muruganandam, A.; Mutus, B. Biochim. Biophys. Acta, Gen. Subj. 1994, 1200,
1-6.

Sase, K.; Michel, T. Life Sci. 1995, 5 7, 2049-2055.

Root, P.; Sliskovic, 1.; Mutus, B. Biochem. J. 2004, 382, 575-580.

Gordge, M. P.; Hothersall, J. S.; Neild, G. H.; Dutra, A. A. N. Br. J. Pharmacol.
1996, 119, 533-538.

Lee, Y.; Yang, J.; Rudich, S. M.; Schreiner, R. J.; Meyerhoff, M. E. Anal. Chem.
2004, 76, 545-551.

Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.;
Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-2453.

Zhang, X.; Kim, W.-S.; Hatcher, N.; Potgieter, K.; Moroz, L. L.; Gillette, R.;
Sweedler, J. V. J. Biol. Chem. 2002, 277, 48472-48478.

Ye, X.; Kim, W.-S.; Rubakhin, S. S.; Sweedler, J. V. Analyst (Cambridge, U. K.)
2004, 129, 1200-1205.

Kim, W.-S.; Ye, X.; Rubakhin, S. S.; Sweedler, J. V. Anal. Chem. 2006, 78,
1859-1865.

Ku, C.-J.; Karunarathne, W.; Kenyon, S.; Root, P.; Spence, D. Anal. Chem. 2007,
79, 2421-2426.

97

CHAPTER3 PLATELET-DERIVED NITRIC OXIDE IS
AFFECTED BY RBC-DERIVED ATP

3.1 INTRODUCTION TO CIRCULATION

The main function of the circulatory system is to move nutrients, gases, and
wastes to and from tissues and cells, as well as stabilize body temperature and pH to
maintain homeostasis. Distinguished by types, it is considered that the circulatory system
consists of the cardiovascular and lymphatic systems.

The main components of cardiovascular system are the heart and blood vessels.
The blood vessels are comprised of arteries, arterioles, capillaries, veins and venules and
the heart itself can be considered as a specialized type of blood vessel that has become
adapted for pumping blood throughout the cardiovascular system.

Separate from the cardiovascular system is the lymphatic circulatory system,
which contains capillaries and large vessels. This system is responsible for the transport
of tissue fluid, or lymph, back to the circulatory system.

Distinguished by route, the circulation is composed of two systems, the
pulmonary and systemic circulations. The vessels that supply blood to and from the lungs
from the heart are referred to as the pulmonary circulation. Blood vessels serving the
remainder of the body make up the systemic circulation. More specifically, the

pulmonary circulation carries oxygen depleted blood away from the heart and to the

98

lungs where it is oxygenated; it then returns the oxygenated blood to the heart, while the
systemic circulation carries oxygenated blood away from the heart and to the body

returning deoxygenated blood back to the heart.

3.1.1 GENERAL INFORMATION ABOUT THE RED BLOOD CELL

Blood is responsible for a variety of processes in the body; it transports nutrients
from the small intestine to the liver and from the liver and adipose tissue to other organs;
it also transports waste products from the tissues to the kidneys. The bloodstream is
responsible for carrying hormonal signals from one tissue to another and the transfer of
oxygen from the lungs to tissues, as well as transport of carbon dioxide generated from
tissue respiration to the lungs.

On average, most adults have 5 - 6 liters of blood circulating in the body. Over
half of this volume is occupied by three types of cells: leukocytes, that are responsible for
aiding the body in fighting infection; platelets, an essential component of the blood
clotting process; and erythrocytes, or red blood cells (RBCs), that are responsible for
carrying oxygen in the bloodstream. There are approximately 5 x 106 RBCs, 4 — 10 x 103
leukocytes, and 1.5 - 4.0 x 105 platelets in each microliter of blood in an average human.

The remaining volume of blood is comprised of plasma that contains proteins,

99

lipoproteins, nutrients, waste, inorganic salts, and hormones; it is also responsible for
carrying carbon dioxide back to the lungs.

Approximately 8 — 10 pm in diameter, RBCs are unlike other cell types because
they are anuclueate when mature, which means they contain no nucleus and no DNA.
RBCs are primarily composed of hemoglobin, a complex meta110protein containing heme
groups. The iron center of heme is responsible for the temporary linking of oxygen to
RBCs in the circulation. The color associated with RBC is also due to the heme
constituents on hemoglobin; oxyhemoglobin appears scarlet in color while
deoxyhemoglobin is darker; it appears blueish in color in the vessels and through skin.
RBCs are described as circular, biconcave disks. Importantly, this shape is optimal as it
allows for the exchange of oxygen with surrounding tissues and organs.

In general, RBCs mature from stem cells in 7 days and circulate through the body
for an average of 120 days. Human RBCs are produced in bone marrow by a process
known as erythropoiesis. Once aged, an erythrocyte will undergo changes in its plasma
membrane making it more susceptible to phagocytes and subsequent phagocytosis in the
spleen. The majority of the RBC breakdown is recycled throughout the body including

hemoglobin.

100

3.1.2 RBC-DERIVED ATP RELATED VASODILATION

Vasodilation is the widening of the blood vessels resulting from a relaxation of
the smooth muscle wall of the vessels. This relaxation relies on removing the stimulus for
contraction, which is primarily dependent on calcium and the phosphorlyation of myosin
light chain. Therefore, vasodilation works by decreasing the intracellular concentration of
calcium or a dephosphorylation of the myosin light chain." 2 There are a number of
substances that can dilate blood vessels including acetylcholine, bradykinin, as well as
ATP.”

ATP, commonly known as a “universal energy carrier” or “molecular currency” is
a multifunctional nucleotide generated and consumed within the body in a multitude of
anabolic and catabolic reactions. ATP is mainly generated during respiration in
non-photosynthetic organisms and during photosynthesis in photosynthetic organisms,

6,

and is readily consumed by many anabolic and catabolic enzymes. 7 It is involved in

signal transduction pathways and acts as a substrate for kinases, a group of enzymes that
catalyze the transfer of a phosphate group fiom high energy donors, to specific target

8, 9
molecules.

Interestingly, ATP is known to be present in millimolar amounts within

10,11
8

RBC and upon being subjected to a stimuli such as mechanical deformation (as

RBCs traverse through the microcirculation), 02 tension (hypoxia) or low pH, these

101

5

RBCs release nanomolar to low micromolar amounts of ATPIZ'l into the lumen of the

12, 14, 16, 17

vessel. This is significant because it has previously been reported that

endogenous NO production in the circulation is dependent on the presence of RBCs in
the perfusate flow through an isolated rabbit lung.18 It has also been determined that the
production of NO was dependent on the RBCs’ ability to release ATP in response to
mechanical deformation.19 The production of NO has been linked to changes in the
endothelial lining of blood vessels as the result of stimulation from substances such as
ATP20 as well as shear stress.21’ 22 The binding of ATP to the sz receptor located on the
endotheliurn23'27 results in the synthesis of N028’ 29 eventually lead to smooth muscle
relaxation and an increase in blood flow to the target tissue. ‘4

Collectively, these reports suggest a mechanism for control of vascular caliber in
the circulation. In response to the RBC being continually deformed during flow through a
vessel, it releases ATP thereby stimulating the synthesis of endothelial NO resulting in
subsequent relaxation of the smooth muscle cells and this is illustrated in Figure 3.1.

Accordingly, multiple groups have proposed a relationship between RBC-derived ATP

, 19, 29 28, 30-32

. . 18 . . .
release and vascular resrstance in the pulmonary and systemic Circulation.

102

88:38? 3
86.2 3:388 88:85 OZ 5 338: 8:22:88 2: :o 5382 AN: 8 8:5 85.5.: A.;. .=8 05 :8: 8:22 9:4 moan—:88
.835 :3: Afinc < 89:: 588: 3 DELUV :82:on 05582:me ammofic 0:98 05 .«o :ouflboammonm E 838: :02?
AU<V 8298 31:8: 3 332$ ofifimonaogfi 038:8: 0:93 9 ER mo 888280 ufi 8 £82 8:88:80: 32582: 3
20mg 8580: 83:8 A69 £8290 05 mo 83:38.34. 5:358 883: PC: wooéfléoumgomuu 05 mo 8:882: mm oBME

103

 

 

 

:ofinEouoc
3252—32

3.2 ATP-MEDIATED NO PRODUCTION IN PLATELETS

Not only is RBC-derived ATP a stimulus of NO production in endothelial cell,
resulting in relaxation of smooth muscle and subsequent vascular dilation, but also in
platelets.33'35 In question concerning the production and release of platelet-derived NO is
the mechanism by which the NO production is stimulated. For example, ATP is a
recognized stimulus of NOS and subsequent platelet NO production as previously
discussed in Chapter 2; moreover, it is also known that ATP is released from platelets
upon activation.36 However, it has not been established if ATP released from activated
platelets has the ability to stimulate the production of NO in platelets. Also, previous
reports concerning RBC-derived ATP and ATP-stimulated NO production suggests that,
in vivo, the ability of RBCs to release ATP may act as a stimulus for NO production in
platelets. This is an extension of the RBCs role to stimulate endothelium NO.

To determine if ATP released from platelets affect platelet NO production and, to
establish that communication between RBCs and platelets exists, experiments were
performed to examine ATP-mediated platelet NO production and measure platelet NO in
the presence of stimuli for RBC-derived ATP. These measurements were performed
using a microflow system in conjunction with fluorescence detection.

Three stimuli (iloprost, pentoxifylline and metal-activated C-peptide) known to

increase the release of RBC-derived ATP37’39 were used in experiments. Iloprost has

104

already been shown to increase ATP release from RBCs due to the stimulation of IP

receptor (prostacyclin receptor) and the increased RBC deformability.40’ 41

Pentoxifylline (Trental), which has been reported to have beneficial effects in the

9, 42, 43 . . ,
3 has also been recently 1nvest1gated as a stimulus of ATP release

diabetic kidney,
by our group. Previous reports have shown that a pentoxifylline derivative may act as an
anti-inflammatory agent by suppressing oxygen radical production or behave as a
scavenger of reactive oxygen species. More recently, reports have suggested that adding
pentoxyfilline to a diabetic patient’s daily medicinal intake improved some of the
complications associated with the disease, perhaps by reducing the amount of lipid
peroxidation in the cell.43 However, several reports have also demonstrated that
pentoxyfilline acts specifically on improving RBC deformability, ultimately reducing
fibrinogen concentration, platelet adhesion, and improving blood viscosity.

C-peptide, a byproduct of insulin synthesis, was initially believed to be a

biologically inactive peptide.44 45 It is a 31 amino acid peptide that is released in
equimolar amounts with insulin from the pancreatic beta cells. It is known that C-peptide

facilitates the folding of insulin prior to cleavage from the proinsulin C-peptide hormone.

However, our group has recently shown that metal (Fe2+, Cr3+ and Zn2+) —activated

C-peptide can promote ATP release from RBCs by increasing glycolysis.39’ 46

105

In this chapter, data are presented suggesting that pentoxyfilline and iloprost, two
drugs known to improve blood flow in vivo, have the ability to indirectly increase NO
production in platelets due to the ability of increasing RBC-derived ATP that are
subjected to deformation while flowing through microbore tubing. Moreover, patients
with type 1 and type 2 diabetes are known to have hyperactive platelets,47’ 48 which is
defined as platelets that are more adhesive or are easier to active; and interestingly reports
by separate groups have demonstrated that RBCs obtained from type 2 diabetic patients

15, 39, 49

release less ATP than those obtained from healthy controls. These results led us

to speculate that increased platelet activity reported in diabetes is due to decreased
stimulation of NO production secondary to reduced RBC-derived ATP. Here, data
provide evidence suggesting that platelet NO is significantly increased when platelets
were exposed to RBC supernatant that has been incubated with physiological
concentrations of metal-activated C-peptide, not only in healthy control rats but also in

type 1 and type 2 diabetic rats.

3.3 EXPERIMENTAL METHODS
Isolation and purification of RBCs. Whole blood was obtained from
exsanguinated rabbits, and platelets were isolated and purified as previously described in

Chapter 2. After the first centrifugation of whole blood and removing the platelet rich

106

plasma (PRP) and buffy coat, packed RBCs were resuspended and centrifuged three
times in physiological salt solution (PSS; 4.7 mM KCl, 2.0 mM CaClz, 1.2 mM MgSO4,
140.5 mM NaCl, 21.0 mM tris(hydroxymethyl)aminomethane, and 11.1 mM dextrose
with 5% bovine serum albumin in 18.2 MW-cm deionozed, distilled water (DDW), pH
adjusted to 7.4) at 500 x g at 37°C for 10 min. Typically, 65 — 75% hematocrit RBC was
obtained as stock solution.

In addition, for experiments involving in rats, whole blood was obtained from
4-month type 1 diabetic BB/Wor-rats, and age-matched control rats. Blood was obtained
by cardiac puncture after anesthesia with isoflurane in a closed gas chamber. Typically,
6-8 ml of blood were collected into heparinized tubes from each rat. Platelets and RBCs
were further washed in the same manner as mentioned in Chapter 2. All surgical
procedures involving animals used in this study were performed under protocols
approved by the Animal Investigation Committee.

Reagent preparation. All reagents were purchased from Sigma Chemical (St.
Louis, MO) without further purification unless otherwise noted. The preparation of
reagents (ATP, ADP, L-NAME, and DAF-FM DA) was described in Chapter 2.
Thrombin (a platelet activator) was prepared at 1.2 units/mL in HBSS as stock solution.
Platelets prepared for use with NF449 (an inhibitor of the P2x receptor on platelets)

(4',4",4"'- carbonylbis [imino-5,1,3-benzenet1iyl bis(carbonyl-imino)] tetrakis(benzene-

107

1,3-disulfonic acid) octasodium salt) studies, were prepared by washing three times in an
HBSS/ACD solution. The first two washes contained lunit/mL apyrase (A6410, Apyrase
Grade VI, Sigma, this grade has an exceptionally high ATPase/ADPase ratio to convert
ATP in the solution to ADP/AMP). The third wash contained HBSS/ACD solution in the
absent of apyrase.

For inhibitor studies of ATP release, glybenclamide (Sigma) was prepared by
adding 49 mg to 2 mL of 0.1 M NaOH and 7.94 mL of a dextrose solution (1 g dextrose
in 20 mL of DDW). The solution was carefully heated to 52°C until dissolved, resulting
in a final stock concentration of 0.01 M. This stock was then diluted in the PSS to create
a buffered solution of glybenclamide, to which washed RBCs were added (final
concentration in 1% hematocrit RBCs was 100 MM).

For studies involving diamide, 0.0018 g of diamide was dissolved in 50 mL of
DDW, creating a 200 pM stock solution. The stock solution was then diluted three times
(1:10) in PSS, resulting a 0.2 uM diamide working solution (final concentration in 1%
hematocrit RBCs was 40 nM). 500 uL of 54 uM solution of pentoxyfilline (0.0015 g
dissolved in 100 mL of DDW) was prepared in PSS prior to the addition of 1.0 mL of 5%
hematocrit RBCs (final concentration in 1% hematocrit RBCs was 5.4 nM). For studies
involving iloprost, a procedure identical to that involving pentoxyfilline was followed.

0.5 g of iloprost was dissolved in 100 mL of DDW, creating a 13.8 mM stock solution.

108

50 uL of 100 uM solution of iloprost were prepared in PSS prior to adding 1.0 mL of 5%
hematocrit RBCs (final concentration in 1% hematocrit RBCs was 1.0 uM).

Human C-peptide (American Peptide, Sunnyvale, CA, USA), 0.25 mg (MW 3020
g/mol) was dissolved in 10 ml of DDW to yield a concentration of 8.3 pM. The solution
of Cry/Zn2+ (derived from chromic chloride hexahydrate and zinc chloride) was
prepared in DDW. The metal solution was then added in equimolar amounts to the
C-peptide solution through a series of dilutions. Before adding to the RBCs, the
metal—C-peptide mixture was diluted in PSS to avoid cell lysis that may occur upon
direct contact of RBC with a non-buffered aqueous solution. All RBC solution in
experiments had a final hematocrit of 7%.

Measurement of A T P from activated platelets. The experiment was performed
using microbore tubing (Polymicro Technologies, Phoenix, AZ, USA) as a
microcirculation mimic.” 50 A syringe pump (Harvard Apparatus, Holliston, MA, USA)
was used to propel two solutions; one solution contained luciferase with 2 mg of luciferin
added per 5 mL of solution to enhance the chemiluminescence sensitivity. The other
syringe contained mixture of 300 uL of various concentrations of ADP and 200 uL
aliquot of the platelets. After allowing 5 minutes for the ADP/platelet mixture to incubate,

the two solutions were pushed through microbore tubing having an inside diameter of

50 um and combined at a mixing tee at a flow rate of 6.7 uL min'l. The resultant mixed

109

flow that eluted from this tee then traveled through a third section of tubing. This last
section of tubing was placed over a photomultiplier tube (Hamamatsu, Iwata City, Japan)
in order to measure the chemiluminescence at 540 nm resulting from the mixing of the
luciferase solution with ATP that was released from activated platelets while flowing
through the microbore tubing. The amount of ATP released was averaged from the
activated platelets obtained from the whole blood of n = 5 exsanguinated rabbits. The
system was calibrated each day with authentic ATP standards having concentrations from
0 to 1.5 uM. The correlation coefficient for this calibration is generally > 0.99, thus
establishing a linear relationship between the emission and ATP concentration.
Fluorescence determination of platelet N0 in a static system. Fluorescence
emission spectra were obtained using 1 uM DAF-FM DA in a quartz cuvette with
excitation wavelength at 495 nm and emission wavelength at 515 nm. This system for
measuring platelet NO with DAF-FM DA is described in detail in Chapter 2. For studies
involving the inhibition of the P2x receptor, a 100 uM stock solution of NF449 was
prepared by adding 1 mg of the NF449 to 6.6 mL of HBSS. NF449 is a selective P2X
receptor antagonist which inhibits ATP binding to the receptor. When using the inhibitor,
30 uL of the 100 “M stock solution was added to 3 mL of an apyrase-washed stock
platelet suspension that contained 3 x 109 platelets/mL (DAF-FM DA loaded), the final

concentration of NF449 used in all studies was 0.1uM. This solution was allowed to

110

incubate for 30 minutes prior to addition of a NOS stimulus (ATP) or a platelet activator
(ADP or thrombin). After addition of these final reagents, the solution was allowed to
incubate for an additional 30 minutes prior to obtaining the emission at 515 nm. For
studies involving metal-activated C-peptide stimulated RBC-derived ATP, RBCs
suspension was incubated with C-peptide in the manner as previously described for 5
hours prior to obtaining the supernatant. The supernatant from 7% hematocrit RBC
suspension was obtained by centrifugation at 500 x g at 37°C for 10 minutes, then added
and equilibrated with platelets for an additional 30 minutes prior to measuring the
fluorescence.

Fluorescence determination of platelet N0 in a flow system. Platelets were
passed through microbore tubing in a manner similar to that described previously for
measuring ATP from activated platelets. The system consists of 3 major components,
namely a dual syringe pump, the sections of microbore tubing, and a flow-through
fluorescence detector (Jasco) housed with a capillary flow cell. DAF-FM DA-loaded
platelets and RBCs were mixed prior to being pumped through a section of microbore
tubing using a syringe pump to propel the solution at a flow rate of 6.7 uL min]. The
pump was a conventional syringe pump where the syringe was easily accessible. For
studies involving stimulation with RBC-derived ATP in a flow system, 350 pL of 1%

hematcroit RBC suspension was added and equilibrated with 150 uL of 3 x 108 platelets

111

mL'l solution for 5 minutes prior to being pumped through for fluorescence detection.
Previous experience with this type of syringe has proven that platelet sedimentation
during the measurement portion of the analysis is not problematic because the platelets
are usually in solution as a suspension. The fluorescent signal that is produced in the
platelets due to NO production was determined using the aforementioned flow-through
fluorescence detector and monitored with a program written in house with LabView

(National Instruments).

3.4 RESULTS AND DISCUSSION

Self-stimulated NO production in platelets. Previous work by other groups
provide evidence that platelets have the ability to release ATP upon activation with ADP
and other agonists that result in activation of the platelets.36 In addition, the data
previously described in Chapter 2 shows that these platelets can also produce their own
NO (intracellularly through NOS) via stimulation with ATP.51 Such a scheme suggests
that platelets can self-stimulate their own NO; that is, upon activation, the platelets
release ATP which may, in turn, stimulate NOS and subsequent NO production. To date,

there has been one example of self-stimulated production of NO in platelets.52 This

measurement was performed using a carbon electrode and showed a transient of NO

112

production upon activation with ADP. Moreover, that same measurement was not clear
on whether the activation of the NOS was due to the ADP or the released ATP.
Substances such as collagen, thrombin, fibrinogen, and ADP all activate platelets
and induce an ATP release. It was hypothesized that ATP release, and not the activators
themselves, stimulate the NO production in platelets. To provide evidence for this theory,
experiments were performed in which platelets were stimulated with various of ADP
concentration to induce ATP release as shown in Figure 3.2 (black bars). Measurements
of this ATP release, via the chemiluminescence assay for ATP described above, upon
activation were verified. However, after 3 minutes of incubation with ADP, the platelet
mixture was combined with 10 uL of a form of apyrase (50 units/mL) that breaks down
ATP. After allowing the apyrase to react with this mixture for 2 additional minutes
(which now contains ATP that was released from the activated platelets), the
chemiluminescence from any remaining ATP was measured as shown in Figure 3.2 (gray
bars). The ATP that was released from the activated platelets decreased in the presence of
apyrase. Moreover, agents were shown to increase the production of NO by platelets as
shown in Figure 3.3. However, when the ATP receptor on platelets (the P2x receptor) was
inhibited, the NO production decreased for every activator studied (gray bars in Figure
3.3). Collectively, the importance of this finding is that only ATP is able to stimulate NO

production in platelets; the platelet activators (which results in a shape change of the

113

 

 

 

    

 

 

 

 

 

 

5
- Normalplt
+Apyrase
4 ..
”g: .
v 3 -
(D
g .
2 ..
9?. 2 *
fl .
< .
1;
0* 5% LI] ,3;
10 20 30

[ADP] (HM)

Figure 3.2 Chemiluminescence assays were performed as a function of ADP
concentration in each sample to quantitatively detect ATP release from activated platelets.
ATP that is released from the activated platelets decreased in the presence of apyrase (1.0
units/mL as final concentration). Error bars represent SEM. p < 0.05 (n = 5)

114

 

q — Normal Plt
m NF449 treated plt

 

0.4 {

Fluorescence intensity (a.u.)
o
ox
l__l_ _l L LL;

.9
N
l l 1 A

1 j

 

 

 

F3
O
l A 1

 

 

Figure 3.3 Evidence that NO production in platelets is due to ATP stimulation of the P2x
receptor. Platelets NO was measured in the absence and presence of a stimulus of NO
(ATP) or activators of ATP release (ADP and thrombin) which lead to NO production.
As shown, NO production increases in each case. However, identical measurements that
were performed in the presence of NF449, a reagent that blocks the ATP receptor on
platelets resulted in a decrease in NO production. This data suggested that NO production
in platelets is largely dependent upon ATP binding to the platelet receptor. L-NAME, a
NOS inhibitor decreased NO production with and without the P2x blocker, verifying the
measured signal in each case was due to NO. Error bars represent SEM. p < 0.05 (except
L-NAME measurements) (11 = 4)

115

platelets) do result in NO production, but perhaps through an ATP-mediated mechanism.
This is potentially very important because it is known that some patient groups known to
have low NO production or complications involving blood flow are also known to have
RBCs that release lower than normal amounts of ATP upon pharmacological activation
or physical deformation of the erythrocyte. Patients with primary pulmonary
hypertension,53 cystic fibrosis,54 and diabetes”’ 49 are example patient groups with these
lower ATP release values.

Measurement of platelet N0 in a microflow system. In chapter 2, we
demonstrated that platelet NO, in either extracellular or intracellular form, could be
quantitatively determined using variations of the DAF family of probes.” However,
those measurements were performed in a static system that did not involve flow. In vivo,
platelets are part of the flowing blood stream, typically occupying the space closest to the
vessel wall due to the non-Newtonian characteristics of whole blood (i.e., the platelets are
displaced from the center of the flow by the larger RBCs). Therefore, to more closely
mimic the physical conditions that the platelet would be subjected to in vivo, and to
prepare for subsequent studies involving the RBC, it was imperative that the NO
production by platelets be measured in a flowing stream.

The data shown in Figure 3.4 demonstrated the ability to measure both basal

levels of NO in platelets and the increase in NO production when these platelets are

116

 

(a) 0.018 ,
0.016
0.014
0.012

0.010

[111.111

0.008

Fluorescence intensity (a. u.)

0.006 {

 

 

 

0.004'........................
0 20 40 60 80 100

Time period (second)

 

(b) 0.016 ,
0.014
0.012
0.010
0.008

0.006

lLlALJAlAAA‘lAA

Fluorescence intensity (a.u.)

0.004

0.002 -

 

 

Inn:

 

0.000

Plt Plt+DAF Plt+DAF+ATP

Figure 3.4 Increased NO production from platelets in the presence of 0.1 uM ATP.
(a) The traces represent the measured fluorescence intensities from platelets only (lower
trace), platelets in the presence of DAF-FM DA (middle trace), and platelets with
DAF-FM DA in the presence of ATP (upper trace). (b) Bars represent the average of
normalized results. Error bars represent SEM. p < 0.05 (n = 3)

117

stimulated with 0.1 11M ATP. The bottom trace in Figure 3.4a is essentially the
background due to platelets flowing through the microflow system described above.
The middle trace is the emission resulting from platelets incubated in 1 uM DAF-F M DA
for 30 minutes prior to being pumped through the system. The upper trace is the
fluorescence intensity of another aliquot of the DAF-FM DA-loaded platelets moving
through the system; however, these platelets were stimulated with 0.1 uM ATP. As
evident by the increase in the traces, the system enables the detection of basal and
stimulated levels of NO production in platelets in a flowing stream.

RBC-stimulated NO production in platelets. Collectively, in Figures 3.2 and 3.3,
the data suggested that the increase in NO production via platelet activation is due to an
ATP stimulus alone. Moreover, the data shown in Figure 3.4 demonstrated that platelet
NO can be measured via fluorescence spectrophotometry in tubing having a diameter that
approximates an arteriole in vivo. These results suggested a possible unique relationship
between RBCs and platelets in the circulation thus render the RBC a possible determinant
of platelet physiology in vivo. Specifically, the RBC may be able to communicate with
the platelet through RBC-derived ATP due to stimulus,39 mechanical deformationls’ 49
50 or pharmacologically,19 resulting in NO production in platelets. Here, we determined
that ATP derived from RBCs that had been incubated with zinc-activated C-peptide was

able to increase the NO production by platelets by 67.5% is 16.4% (Figure 3.5) compared

118

 

1.0 2

0 9 _2 _ Normal plt
' I 2...... NF449 treated plt *

0.8 g — L-NAME treated plt
0.7 3

 

0.5
0.4 3
0.3 g
0.2

Fluorescence intensity (a.u.)

 

 

 

   

0.0 5
6‘?

\
Q
xe

 

33’ C?

X

Figure 3.5 NO production of platelets incubated with supernatant from rabbit RBCs
incubated with zinc-activated C-peptide (+RBCs+10PIOZn) and in the absence of zinc
(+RBCs+10P) or absence of C-peptide (+RBCs+IOZn) at 5 hours (black bars). Platelets
NO remain unchanged when platelets were pretreated with NF449 (light gray bars) and
L-NAME (dark gray bars). Error bars represent SEM. p < 0.05 (n = 3)

119

to platelets alone. Importantly, when platelets were incubated without zinc or in the
presence of only zinc (no C-peptide) the increase in NO production was insignificant. To .
demonstrate that the increase in NO production was due to the ATP from the RBCs, the
platelets were incubated with NF449, an inhibitor of the P2x ATP receptor on the surface
of the platelet. As shown in Figure 3.5 (light gray bars), the NF449 reduced the NO
production from platelets that had been incubated with supernatant from RBCs incubated
with zinc-activated C-peptide to levels that were statistically equivalent to that of
platelets alone. After establishing that the increase in NO production was due to ATP
binding to the P2x receptor on the platelet, the platelets were incubated with L-NAME
prior to the addition of supernatant from RBCs that had been incubated with
zinc-activated C-peptide. As expected, the fluorescence intensity was reduced to levels
equivalent to platelets alone (dark gray bars in Figure 3.5) indicating that it was NO
production that was being measured by the DAF-FM DA probe.

More specifically, data verifying the relationship between RBCs and platelets in
the circulation was summarized in Figure 3.6. Here, the ability to measure
platelet-derived NO upon stimulation with ATP secreted from deformed RBCs in a
microflow system is demonstrated. The percent increase or decrease in platelet-derived
NO (measured spectrofluorometrically with the DAF-FM DA probe) is presented for

RBCs in the presence of either pentoxyfilline or iloprost. Pentoxyfilline is thought to

120

 

Fluorescence percent change

 

 

 

 

Figure 3.6 The percent change in fluorescence due to platelet NO production stimulated
by RBCs in the presence and absence of stimulators and inhibitors of ATP release. The
percent changes were reported relative to RBCs flowing with the DAF-FM DA-loaded
platelets alone. In (a), RBCs incubated with pentoxyfilline prior to flowing with platelets
in microbore tubing resulted in a 15.5% :t 0.8 increase in emission intensity; in (b), the
RBCs were incubated with pentoxyfilline and diamide, resulting in a 36.9% :1: 1.1%
decrease in platelet NO; RBCs were treated with glybenclamide and pentoxyfilline in (c)
and the platelet NO decreased by 25.3% :t 0.9%; in (d), RBCs were incubated with
iloprost, resulting in an increase in platelet NO production of 10.0% d: 1.1%; the
iloprost-induced increase in NO production was reduced in (e) where RBCs treated with
glybenclamide and iloprost resulted in a decrease in platelet N0 of 50.9% i 0.9%. Error
bars represent SEM. p < 0.05 (n = 3)

121

improve blood flow via its ability to make the RBC more deformable through a radical

. . 5 56 .
scavenging mechamsm. Therefore, due to flow-induced shear, RBCs become more

deformed in the presence of pentoxyfilline, and an increase in deformability would then
be anticipated to lead to an increase in deformation-induced release of ATP from the
RBC. In the presence of pentoxyfilline, the fluorescence from the platelet NO increased
by 15.5% :t 0.8% due to the increased RBC-derived ATP release. However, this increase
in platelet NO due to pentoxyfilline was reduced by 36.9% :1: 1.0% and 25.3% i: 0.9% in
the presence of diamide or glybenclamide, respectively. Diamide is a recognized oxidant
that results in a stiffened membrane and reduced RBC deformability.57 Glybenclamide is
an anion transport inhibitor that will inhibit ATP release from RBCs. There have been
reports of other pharmaceutical agents having the ability to stimulate ATP release from
RBCs. Specifically, iloprost, a stable analogue of prostacyclin, has been reported to
increase the release of RBC-derived ATP independent of flow.13 In the presence of
iloprost, a 10.0% :1: 1.1% increase in platelet NO production, and a value that was
reduced by 50.9% d: 0.9% in the presence of glybenclamide. Moreover, neither
pentoxyfilline nor iloprost stimulated NO production in platelets in the absence of the
RBC.

Increased RBC-stimulated platelet N0 with diabetic rats. People with diabetes

often suffer cardiovascular complications as a result of poor circulation of the blood.

122

Interestingly, RBCs from people with diabetes release less ATP than healthy
indivuals.15’ 49 Because ATP is a primary stimulus for NO production in platelets, then
recent reports involving ATP release from RBCs of patients with diabetes becomes
important to platelet physiology. The significant decrease in concentrations of
RBC-derived ATP from diabetic RBCs may contribute to decreased levels of NO
production by platelets. Previously, it was shown that C-peptide had no direct effect on
platelet behavior,58 and studies performed in our laboratory have agreed with this prior
report (even when the C-peptide was activated with zinc) (Figure 3.7). However,
although there was no direct effect on the platelets due to treatment with C-peptide, there
was an indirect effect due to C-peptide’s ability to stimulate ATP release from RBC,
which subsequently affected platelet behavior. As shown in Figure 3.8, the NO
production from platelets obtained from type 2 rat models increased 26.1% i 8.4%, while
the NO production from control platelets was 43.8% suggesting a slight resistance to the
effects of metal-activated C-peptide. Type 1 rat models, however, demonstrated an NO
production increase of 27.8% i 11.1% compared to platelets alone with the control
increased similarly (31.1% i 3.2%) as shown in Figure 3.9. This data suggested that ATP
derived from zinc-activated C-peptide treated RBCs may play an important role in

platelet hyperactivity and aggregation.

123

 

0.5
0.4 3
0.3

0.2 3

Fluorescence intensity (a.u.)

0.1

 

 

IJLLJLJ I I .

 

0.0

Q‘“ \§ 6‘? 6‘?
$3} ‘25 QQN
«1‘ xx
42)"

Figure 3.7 Control experiments of platelet NO production. Platelets were incubated with
zinc-activated C-peptide (Plt+10PlOZn) and in the absence of zinc (Plt+10P) or absence
of C-pepu'de (Plt+IOZn) at 5 hours. Each bar here was statistically insignificant,
suggesting that C-peptide has no direct effect on platelet NO production. Error bars
represent SEM. (n = 3)

124

 

: — +RBCS
1.4 - [:2] +RBCs+metal-C-pep

Fluorescence Intensity (a.u.)

 

 

 

Control Type 2

Figure 3.8 The effect of metal-activated C-peptide on the NO production by platelets
from type 2 (BB/ZDB) and control rats. Black bars represent the fluorescence intensity
from platelet NO production incubated with supernatant from rat RBCs and gray bars
represent the fluorescence intensity from platelet NO production incubated with
supernatant from rat RBCs incubated with metal-activated C-peptide at 5 hours. Platelet
NO production from type 2 rats increased 26.1% :t 8.4% when RBCs were incubated
with metal-activated C-peptide. Error bars represent SEM. p < 0.05 (n = 3)

125

 

— +RBCS
1_4 - E2123 +RBCs+metal-C—pep

*

Fluorescence Intensity (a.u.)

 

 

 

Control Type 1

Figure 3.9 The effect of metal-activated C-peptide on the platelet NO production from
type 1 and control rats. Black bars showed the platelet NO production incubated with
supernatant from rat RBCs and gray bars showed the platelet NO production incubated
with supernatant from rat RBCs incubated with metal-activated C-peptide at 5 hours.
Error bars represent SEM. p < 0.05 (n = 5)

126

3.5 CONCLUSIONS

Once produced by the platelet, NO acts as a platelet inhibitor, reducing the
activation and aggregation of platelets. The over-aggregation of platelets results in a
thrombus formation, which if broken off, can become lodged in a small arteriole or
capillary resulting in an emboliSm. As previously mentioned, ATP can stimulate platelet
NO, here, the data provided evidence that RBC-derived ATP actually plays an important
factor of platelet function in the circulation, especially considering the results from
Figure 3.3 that suggest platelet NO is stimulated through ATP. Results shown here
demonstrated that the ATP released by RBCs is able to stimulate NO production in
platelets. The decreased NO production in platelets decreases substantially when RBCs
are in the presence of a cell stiffening agent (diamide) or an inhibitor of ATP release
(glybenclamide) or in the case of RBCs obtain from diabetic rat models. It should also be
noted that neither of these agents have an effect on platelet NO production in the absence
of RBCs. While in vivo studies will be necessary to truly verify any conclusions drawn
here from in vitro studies, however, the data presented suggested that RBCs are able to
stimulate NO production in platelets via their ability to release ATP and more in vivo
circulation mimic experiments will be performed in next chapter to have a closer

investigation of communications between these different cell types.

127

LIST OF REFERENCES

(1) Faraci, F. M.; Sobey, c. G. Brain Res. 1999, 821, 368-373.

(2) Lehen'kyi, V. V.; Zelensky, S. N.; Stefanov, A. V. Nitric Oxide 2005, 12,
105-113.

(3) Amezcua, J. L.; Dusting, G. J .; Palmer, R. M. J .; Moncada, S. Br. J. Pharmacol.
1988, 95, 830-834.

(4) Marshall, J. J .; Wei, E. P.; Kontos, H. A. Am. J. Physio]. 1988, 255, H847-H854.

(5) Ascuitto, R. J.; Ross-Ascuitto, N. T.; Ramage, D.; McDonough, K. H.; Kadowitz,
P. J. Pediatr Res 1992, 32, 236-242.

(6) Conover, T. E. Curr. Top. Bioenerg. 1967, 2, 235-267.

(7) Funk, G. D.; Huxtable, A. G.; Lorier, A. R. Respir. Physio]. Neurobiol. 2008, 164,
131-142.

(8) Dubyak, G. R. Am. J. Respir. Cell Mol. Biol. 1991, 4, 295-300.

(9) Lippton, H. L.; Hao, Q.; Hauth, T.; Hyman, A. Am. .1. Physio]. 1992, 262,
H926-H929.

(10) Bergfeld, G. R.; Forrester, T. Cardiovasc. Res. 1992, 26, 40-47.

(11) Miseta, A.; Bogner, P.; Berenyi, E.; Kellermayer, M.; Galambos, C.; Wheatley, D.
N.; Cameron, I. L. Biochim. Biophys. Acta, Mo]. Cell Res. 1993, 1175, 133-139.

(12) Dietrich, H. H.; Ellsworth, M. L.; Sprague, R. S.; Dacey, R. G., Jr. Am. J. Physiol.
2000, 278, H1294-H1298.

128

(13)

(14)

(15)

(16)

(17)

(13)

(19)

(20)

(21)

(22)

(23)

(24)

Olearczyk, J. J .; Stephenson, A. H.; Lonigro, A. J .; Sprague, R. S. Med. Sci. Monit.
2001, 7, 669-674.

Price, A. K.; Fischer, D. J.; Martin, R. S.; Spence, D. M. Anal. Chem. 2004, 76,
4849-4855.

Carroll, J .; Raththagala, M.; Subasinghe, W.; Baguzis, S.; Oblak, T. D. a.; Root,
P.; Spence, D. Mo]. BioSyst. 2006, 2, 305-311.

Edwards, J.; Spnmg, R.; Spence, D.; Sprague, R. Analyst (Cambridge, U. K.)
2001, 126, 1257-1260.

Moehlenbrock, M. J .; Price, A. K.; Martin, R. S. Analyst (Cambridge, U. K.) 2006,
131, 930-937.

Sprague, R. S.; Stephenson, A. H.; Dimmitt, R. A.; Weintraub, N. A.; Branch, C.
A.; McMurdo, L.; Lonigro, A. J. Am. J. Physiol. 1995, 269, H1941-Hl948.

Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J. Am. J. Physio].
1996, 271, H2717-H2722.

Bogle, R. G.; Coade, S. B.; Moncada, 8.; Pearson, J. D.; Mann, G. E. Biochem.
Biophys. Res. Commun. 1991, 180, 926-932.

Rubanyi, G. M.; Romero, J. C.; Vanhoutte, P. M. Am. J. Physiol. 1986, 250,
H1145-H1149.

Buga, G. M.; Gold, M. E.; Fukuto, J. M.; Ignarro, L. J. Hypertension (Dallas)
1991, 17, 187-193.

Kennedy, C.; Delbro, D.; Burnstock, G. Eur.'J. Pharmacol. 1985, 107, 161-168.

Liu, S. F .; McConnack, D. G.; Evans, T. W.; Barnes, P. J. J. Pharmacol. Exp.
Ther. 1989, 251, 1204-1210.

129

(25)

(26)

(27)

(28)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

Allsup, D. J .; Boarder, M. R. Mo]. Pharmacol. 1990, 38, 84-91.

Dalziel, H. H.; Westfall, D. P. Pharmacol. Rev. 1994, 46, 449-466.

Communi, D.; Raspe, E.; Pirotton, S.; Boeynaems, J. M. Circ. Res. 1995, 76,
191-198.

McCullough, W. T.; Collins, D. M.; Ellsworth, M. L. Am. J. Physiol. 1997, 272,
H1886-H1891.

Sprague, R. S.; Olearczyk, J. J .; Spence, D. M.; Stephenson, A. H.; Sprung, R. W.;
Lonigro, A. J. Am. J. Physio]. 2003, 285, H693-H700.

Ellsworth, M. L.; Forrester, T.; Ellis, C. G.; Dietrich, H. H. Am. J. Physiol. 1995,
269, H2155-H2161.

Dietrich, H. H.; Kajita, Y.; Dacey, R. G., Jr. Am. J. Physiol. 1996, 271,
H1109—H1116.

Collins, D. M.; McCullough, W. T.; Ellsworth, M. L. Microvasc Res 1998, 56,
43-53.

Sase, K.; Michel, T. Life Sci. 1995, 5 7, 2049-2055.

Wallerath, T.; gath, I.; Aulitzky, W. E.; Pollock, J. S.; Kleinert, H.; Foerstermann,
U. Thromb. Haemostasis 1997, 77, 163-167.

Randriamboavonjy, V.; Fleming, 1. Pharmacol Rep 2005, 5 7 Suppl, 59-65.

Beigi, R.; Kobatake, E.; Aizawa, M.; Dubyak, G. R. Am. J. Physiol. 1999, 276,
C267-C278.

130

(37)

(3 8)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(43)

Olearczyk, J. J .; Stephenson, A. H.; Lonigro, A. J .; Sprague, R. S. Am. J. Physiol.
2004, 287, H748-H754.

Carroll, J. S.; Ku, C.-J.; Karunarathne, W.; Spence, D. M. Anal. Chem.
(Washington, DC, U. S.) 2007, 79, 5133-5138.

Meyer, J. A.; Froelich, J. M.; Reid, G. E.; Karunarathne, W. K. A.; Spence, D. M.
Diabetologia 2008, 51, 175-182.

Kovacs, I. B.; O'Grady, J. Agents Actions 1984, 14, 306-310.

Korbut, R. A.; Adamek-Guzik, T.; Madej, J .; Korbut, R. J. Physio]. Pharmacol.
2002, 53, 655-665.

Gunduz, Z.; Canoz, 0.; Per, H.; Dusunsel, R.; Poyrazoglu, M. H.; Tez, C.;
Saraymen, R. Renal Failure 2004, 26, 597-605.

Davila-Esqueda, M. E.; Martinez-Morales, F. Exp. Diabesity Res. 2004, 5,
245-251.

Kitabchi, A. E. Metab., Clin. Exp. 1977, 26, 547-587.

Steiner, D. F.; Rubenstein, A. H. Science (Washington, D. C.) 1997, 277,
531-532.

Meyer, J. A.; Spence, D. M. Metallomics 2009, 1, 32-41.

Sobol, A. B.; Watala, C. Diabetes Res. Clin. Pract. 2000, 50, 1-16.

Vinik, A. I.; Erbas, T.; Park, T. S.; Nolan, R.; Pittenger, G. L. Diabetes Care 2001,
24, 1476-1485.

131

(49)

(50)

(51)

(52)

(53)

(54)

(55)

(56)

(57)

(53)

Sprague, R. S.; Stephenson, A. H.; Bowles, E. A.; Stumpf, M. S.; Lonigro, A. J.
Diabetes 2006, 55, 3588-3593.

Sprung, R.; Sprague, R.; Spence, D. Anal. Chem. 2002, 74, 2274-2278.

Ku, C.-J.; Karunarathne, W.; Kenyon, 8.; Root, P.; Spence, D. Anal. Chem. 2007,
79, 2421-2426.

Freedman, J. E.; Loscalzo, J.; Barnard, M. R.; Alpert, C.; Keaney, J. F., Jr.;
Michelson, A. D. J. Clin. Invest. 1997, 100, 350-356.

Sprague, R. S.; Stephenson, A. H.; Ellsworth, M. L.; Keller, C.; Lonigro, A. J.
Exp. Biol. Med. (Maywood, NJ, U. S.) 2001, 226, 434-439.

Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Kleinhenz, M. E.; Lonigro,
A. J. Am. J. Physio]. 1998, 275, H1726-H1732.

Freitas, J. P.; F ilipe, P. M. Bio]. Trace Elem. Res. 1995, 47, 307-311.

Bhat, V. B.; Madyastha, K. M. Biochem. Biophys. Res. Commun. 2001, 288,
1212-1217.

Kosower, N. S.; Kosower, E. M.; Wertheim, B.; Correa, W. S. Biochem. Biophys.
Res. Commun. 1969, 37, 593-596.

Hu, H.; Li, N.; Ekberg, K.; Johansson, B.-L.; Hjemdahl, P. Thromb. Res. 2002,
106, 91-95.

132

CHAPTER 4 A CIRCULATORY MIMIC IN A MICROFLUIDIC
DEVICE INCLUDING MUTLIPLE CELL TYPES

4.1 INTRODUCTION TO MICROFLUIDICS

The first microfluidic construct, a miniaturized gas chromatograph, was
developed by Terry et a] in 1979.1 A few years later, small scale zone electrophoresis
was performed in glass capillaries2 followed by flow injection in microconduits.3 Other
early miniaturized devices included a coulometric acid-base titration system4 and
silicon-based micropump system.5 Early on, there were concerns with these devices
because peripheral pieces of equipment such as pumps, detectors, and chart recorders
were still required to complete the analysis. It was not until the early 1990’s that
lithographic technology was combined with capillary electrophoresis (CE) to create a
single device capable of sample injection, pretreatment, separation, and detection.6

As the popularity of genomics grew in the early 1990’s, so did interest in
developing technology for the analysis of complex molecules such as DNA and proteins.
Microfluidic-based devices offered improvements in analytical performance with the ease
of utilizing already developed technology. The leaders in microfluidics systems that

12-17 18-23
y, and

. . 7-11 . .
analyzed multifaceted aqueous solutions were Manz, Harrison, Ramse

24-27

Mathies. Although early work in microfluidics ranged from biological sample

. . . . , 22 . . 28 .
pur1ficat1on16 and amplificatlonlo to high-throughput screening and genomic

133

9

2 27 . . . . . . .
assays, other areas benefited from the potential of microfluidic applications. Pomt of

9,130

care diagnostics based on immunological assays and cell counting“ for clinical

analysis was expanded, while devices for in vivo drug delivery and monitoring for disease
.. 32 . . . . .
conditions were developed. The detection of Single molecules on a microfluidic

33-35

construct also became popular. Although at the beginning the analytical performance

of these devices was more desirable than their reduced size, miniaturization was the route
to shorter analysis time and versatility. However, the central technology for a number of
miniaturized systems was the microfluidic technology, where samples were manipulated

in channels with dimensions on the order of 10 — 100 um.

4.1.1 MATERIALS FOR MICROFLUIDIC DEVICES
Microsystems can be built on various substrates with a range of materials, from

37’ 38 metals,39 and organic polymers.40 Each of these materials

silicon,5’ 36 glass,2 quartz,
possesses certain advantages and disadvantages depending on the type of application.
Silicon processing was carried out by such conventional, planar fabrication
techniques as photolithography and etching adapted from the microelectronics industry.
Advantageous to silicon is its surface characteristics; it possesses a negative charge

thereby supporting electroosmotic flow (EOF) for electrophoresis application and,

because fabrication occurs by etching, the surface is cleaned as the channel is produced.

134

Moreover, silicon processing techniques are well developed, thereby enabling two and
three dimensional shapes that can be reproduced with high precision.“ 42 However,
silicon is not suitable for optical detection because it is opaque in the visible/UV region
of the spectrum and it conducts current, thereby limiting its usefulness for processes
involving electric field compared to glass. Another shortcoming to silicon is the sealing
process, which requires not only a clean room environment, but also high voltages or
temperatures.

Since the advent of microfluidics, a significant amount of research has focused on
introducing new types of materials into the field. Polymers43 make a suitable alternative
to silicon and glass because they are inexpensive, channels are easily formed by molding
or embossing as opposed to extensive etching processes, and the devices have the ability
to be sealed”.46 The surface chemistry of the polymer substrate is more easily modified
than that of silicon and glass, thereby making polymers more applicable to the biomedical
field.47

Two of the most actively developed polymers for microfluidics and other
applications are poly(dimethylsiloxane) (PDMS) and poly(methylmethacrylate)
(PMMA).48'50 The polymer mainly discussed in this thesis is PDMS due to its

transparency for optical observation and compatibility to biological samples. PDMS

became a popular polymer substrate for soft lithography in the late 1990’s when it was

135

applied in the fabrication of systems for biological and aqueous-based applications.47
PDMS consists of a repeating (—Si—O—) backbone and each Si atom has two methyl
(—CH3) groups covalently attached to it.

There are several properties of PDMS that make it suitable for chemical and
biological miniaturized systems. First, the elastomeric nature of PDMS allows it to
conform to nonplanar surfaces and release easily from delicate or fragile structures
without damaging the mold or itself. Second, PDMS is optically transparent down to
about 300 run51 so it is amenable to optical detection schemes. Third, PDMS is
chemically inert, non-toxic, and gas permeable providing a means to culture mammalian
cells directly to the surface. Fourth, PDMS is very hydrophobic as the result of the
methyl substituent groups attached to each silicon atom creating a polymer that is water
impermeable. However, the surface properties of PDMS can be modified with plasma
treatment. Finally, PDMS has the ability to seal reversibly to itself and other materials via
van der Waals interactions or it can be irreversibly sealed after exposure to plasmas}54

In contrast to the advantages of working with PDMS, its elastomeric properties
are problematic when creating patterned stamps. Elastomers are subject to deformations
such as pairing, sagging, and shrinking. Pairing occurs when either gravity or capillary
forces stress the elastomeric features and result in collapse. Sagging is the result of the

aspect ratio (length/height) of the relief structures becoming too small, compression will

136

occur between the stamp and the substrate to which it is adhered. Finally, the elastomer is
subject to distortions that occur during the fabrication process as the result of the
flexibility of the polymer.55’ 56 There are several other disadvantages that affect the
performance of PDMS for certain applications. It is known that PDMS is incompatible
with solvents such as toluene and hexane57’ 58 resulting in swelling of the elastomer. It
has also been reported that PDMS shrinks approximately 1% upon curing.S6 The
hydrophobicity of the polymer also creates some disadvantages, especially when dealing
with biological applications. There exist several proteins and cell types that experience
non-specific binding to PDMS that prevent the sample from flowing from one part of the
device to another. Another disadvantage is that EOF is difficult to generate in PDMS due
a lack of silanol groups (Si—OH) that are deprotonated at neutral and basic pH.

To date, a variety of polymeric materials have been reported for the fabrication of
microdevices. Besides PDMS, PMMA and polycarbonate (PC) are the most popular
polymer materials for microfabrication, either by embossing or injection molding,
however other standard polymers include polyethylene (PE), polypropylene (PP),

polystyrene (PS), and poly(etheretherketone) (PEEK).

137

4.1.2 PHOTOLITHOGRAPHY

Essential to the expansion of microelectronics, microfabrication - the generation of
small structures, has become the standard for the creation of microprocessors and other
microelectrical devices for the information and technology industry since its development
in the late 1950’s. The majority of the semiconductor circuitry was and continues to be
made by this technology, although the escalating need for smaller features has strained
the industry as smaller sizes of circuitry are increasingly difficult and expensive to
produce. Microfabrication also gained popularity in areas outside of microelectronics,
providing a means to miniaturize and construct devices that are more portable, produced

at a lower cost, require less reagents and sample volumes, and have improved detection

limits. The most popular method in microfabrication is photolithography,59’ 60

specifically, a transfer technique based on a projection-printing system in which an image
is projected onto a thin film of photosensitive material (also known as photoresist),
spin-coated on a wafer, and selectively expose to a radiation source. This exposed region
will contain the pattern of the transferred image and unexposed material can be washed
away with a developer solution. The portion of the negative photoresist unexposed to the
radiation is soluble thus create positive relief on the wafer while the positive photoresist

generates negative relief structures as illustrated in Figure 4.1.

138

Photoresist

- Silicon wafer

1 Exposure

llllllll

— — — Mask

 

 

Development
Negative Positive
photoresist photoresist
Positive relief Negative relief

Figure 4.1 Two type of photoresist used result in different relief structure on wafer. The
portion of the negative photosist exposed to the radiation is insoluble thereby create
raised features corresponding to the mask, while positive photoresist generate negative
relef structures after development

139

Though useful, photolithography has some disadvantages that limit its use in the
preparation of devices. These constraints include the lack of technology to create features
below 100 nm, expense, the complexity of patterning on non-planar surfaces, limited
control over the chemistry of the patterned surface, and the lack of variation in materials
used as photoresists. These limitations demonstrated a need for the development of

non-photolithographic techniques to fabricate micro- and nano-structures.

4.1.3 SOFT LITHOGRAPHY

Soft lithography, named for its use of flexible organic materials as opposed to rigid,
inorganic materials common with lithography fabrication, gained momentum in science
and industry with increasing needs to generate structures smaller than 100 nm. Soft
lithography is considered to be a non-photolithographic technique, and other
non-photolithographic methods to create nanoscale structures include injection molding,
embossing, and laser ablation. The strength of soft lithography is in replicating the master
as opposed to fabricating a new device for each experiment performed. A few examples
of soft lithography include microcontact printing (pCP), microtransfer molding (uTM),
and replica molding (REM). These techniques have been reported to generate structures
from 1 pM in p.TM61 to 30 nm in REM46 laterally. Soft lithography requires a much

smaller investment in materials as opposed to other lithographic methods and with the aid

140

of rapid prototyping and REM, it can take less than 24 hours to complete from design to
stamp.56 Moreover, preparation via soft lithography requires only an ambient laboratory
setting as opposed to a required clean room environment for photolithography. Most
importantly, soft lithography is not subject to limitations of optical diffraction and optical
transparency as other types of lithography often are. This method also enables pattern

transfer to curved materials.48

4.1.4 RAPID PROTOTYPING AND REPLICA MOLDING
Developed by the Whitesides group, rapid prototyping is a fabrication technique in
which a design is patterned onto a silicon wafer. These designs often have features
greater than 20 pm and their production is cost effective. The first step is to generate
patterns in a design related software such as Freehand or AutoCAD and print them to
polymer sheets. Photomasks contain the design pattern and are inexpensive to produce,
approximately $1 per square inch as compared to chrome masks that can range from $200
to $500 per square inch. Although these masks are not durable for manufacturing
microelectronic devices, they suffice for the rapid production of simple designs for
microanalytical devices.
In general, soft lithography starts with a photomask that is placed on top of a

silicon wafer coated with a thin film of photoresist; photolithography and developing

141

methods are used to transfer the design pattern to the wafer, thereby creating a master as
illustrated in Figure 4.2 (more detail will be described in the experimental section). Once
a master is complete, patterns can be formed in polymer substrates by a process known as
replica molding. Specifically, REM duplicates the shape and structure present in a design
by casting a polymer against a master. PDMS prepolymer, consisting of a particular ratio
of base to curing agent, is mixed, degassed, and poured onto the silicon master. The
polymer is cured in an oven in a period of time and the PDMS chip can be peeled away

from the. master.

4.2 CIRCULATORY MIMIC WITHIN A MICRODEVICE
RBCs are responsible for carrying oxygen from the lungs and delivering it to the

body’s tissues. Recent work has suggested that there are additional roles for the RBC in

9

addition to supplying oxygen.6 63 It is known that while traversing the circulation,
RBCs release nanomolar to micromolar amounts of ATP due to deformation and other
stimuli such as hypoxia, pharmacological agents, or more recently, metal-activated
C-peptide.63'69 The importance of this in vivo release of ATP from the RBC is that ATP

is a known stimulus of NOS, which catalyzes the production of NO in various cell types.

NO is not only responsible for blood vessel dilation/0'73 but is also an inhibitor of platelet

. . . 74-77
activation and subsequent aggregation.

142

8:53.: coma: @860:
5:5 83.2: 05 £830 98 56890:: uomtofibomg 2.: 96:8: guano—gum A8 .550: @3098 05 6.57386 63898

>3va .Eogmza x38 5:833“; A8 .EE 2: 38:88 :5 E028 88233 8 .815 3V 3.35 :02? a: 05 :o 36882.3
#25 E: o3 bofiEMxoaam 5 33mm: .38-:Em A3 domfiomfifi Emma: 05 :8 Emawofizouona mo $38: 2:. Nd gamma

8%?
Emefionm

 

.1 . .J. ..,.... . J.
. “1*... w x.

32: and—cram A3 oimomwm AB _ . 3.0.0. :26 A3

143

Although platelets flow through the circulation and do not generally adhere to the
endothelium, upon vascular injury, subendothelial collagen is exposed and stimulates
platelet activation. Upon activation, the platelet shape changes allowing for adhesion to
the vascular walls and even subsequent recruitment of additional platelets. However, left
uncontrolled, the over-aggregation of platelets results in a thrombus formation, which if
broken off, can become lodged in a small arteriole or capillary resulting in an embolism.

According to pieces of information described above and evidence presented in
previous chapters, it is hypothesized that RBC-derived ATP plays an important role not
only in controlling vascular caliber, but also in platelet function, especially for platelet
adhesion to endothelial cells. Interestingly, it is known that hyperactive platelets are
observerd in conditionsm'80 where circulatory RBC-derived ATP levels are either low
(e.g., diabetes,“ 82 cystic fibrosis,83 pulmonary hypertension”) or high (e.g., sickle cell
disease, multiple sclerosis), thus, people with these abnormal circulatory ATP levels may
have higher rates of stroke and associated complications. However, to date,
ATP-mediated platelet function has not been completed investigated.

There have been previous reports of microfluidic systems that enable certain
features of the circulation to be investigated. Models of the blood brain barrier have been

reported and successfully implemented for studies involving transport across an

endothelium layer.84 Other reports of a cell culture analogue, a device containing

144

 

different tissue types connected by a series of fluid channels, have also been reported.85’

86 . . . .
Many groups, 1nclud1ng our own, have demonstrated the ablllty to culture or

immobilize cells in the channels of various microfluidic devices. Previously, the ability to
image NO production by endothelial cells immobilized in the channels of a microfluidic
device,87 as well as determine the concentrations of ATP released from RBCs flowing
through microfluidic channels,88 was demonstrated by our group.

In an extension of previous reports involving immobilized endothelial cells in
microfluidic channels, the results in this chapter demonstrate the ability of platelets to
adhere directly to endothelial cells in a microfluidic device. In this study, rather than
immobilizing the cells directly onto the microfluidic channel, the microfluidic device was
reversibly sealed to a petri dish. In this construct, the microfluidic device channels are
simply used to direct reagent and fluid flow while the endothelial cells are immobilized to
the surface of a petri dish. Upon immobilization, these cells then have platelets and RBCs
directed over them through the channels of the microfluidic device under various
conditions. Importantly, the channels are in parallel creating a high-throughput device
capable of optimizing cell immobilization conditions. Therefore, the work presented here
demonstrated the ability of multiple cell types (platelets, RBCs, and endothelial cells) to
interact directly in the channels of a circulatory mimic device (Figure 4.3). Such a device

should prove useful for investigating those pharmaceutical substances whose mechanism

145

nose—mcowg Ea “negate H2033 we 5:535 8 £62 32:? aouosuoa
OZ Sszofiouno EB “2033 5 £32 momm Sea 3322 A.;. .883 coo—n 08. 5 no wEow momwoooa .Ho :23ng me oEmE

.. 28:8 58:5

 

 

     

.. .. . «mofiowfinsm
...:oxéosouam

 

 

   

oz .I.. ...._...,....§o§m

8§za< 95.1 g =8 82an

. I ’ 365m

 

146

of action is to prevent platelet activation in vivo or certain physiological interactions in

the circulation.

4.3 EXPERIMANTAL METHODS

Preparation of a microfluidic device. Microfluidic devices were fabricated using
standard soft lithographic technology. PDMS channel structures were produced following
previously published methods.48’ 89 Briefly, masters for the production of PDMS
microchannels were made by coating a 4 inches silicon wafer (Silicon, Inc., Boise, ID)
with SU-8 10 negative photoresist (MicroChem Corp., Newton, MA) using a spin coater
(Brewer Science, Rolla, MO) operating with a spin program of 2000 rpm for 20 seconds.
The photoresist was prebaked at 95°C for 5 minutes prior to UV exposure with a
near-UV flood source (Autoflood 1000, Optical Associates, San Jose, CA) through a
negative film (2400 dpi, PageWorks, Cambridge, MA), which contained the desired
channel structures drawn in Freehand (PC version 10.0, Macromedia, Inc. San Francisco,
CA). Following this exposure, the wafer was postbaked at 95°C for 5 minutes and
developed in Nano SU-8 developer (Microchem Corp.) (PGMEA, propylene glycol
monomethyl ether acetate). The thickness of the photoresist was measured with a
profilometer (Alpha Step-200, Tencor Instruments, Mountain View, CA), which

corresponded to the channel depth of the PDMS structures. A 10:1 mixture of Sylgard

147

184 elastomer and curing agent (Ellsworth Adhesives, Germantown, WI) was used to
increase the adhesiveness of the PDMS to aid in the reversible bonding procedure. This
degassed mixture was poured onto the master and cured at 75°C for approximately 10 —
15 minutes. After this time, the PDMS layer was removed from the master and inlet holes
were punctured using a 20 gauge luer stub adapter through the chip as well as 1/8” exit
holes. A chip containing channels of 100 um depth x 200 um width x 2 cm length was
used for all studies reported here. The channel depth corresponds to the height of the
master, which was measured with the aforementioned profilometer.

Cell culture. Unless otherwise stated, all chemicals and reagents for cell isolation
and culture were purchased from Lonza (Walkersville, MD), fluorescent dyes were from
Invitrogen (Carlsbad, CA), and all other materials were purchased from Fisher Scientific
(Pittsburgh, PA). Bovine pulmonary artery endothelial cells (bPAECs) were thawed and
expanded in Endothelial Cell Basal Medium (EBM) supplemented with 0.1% gentamicin
sulfate/amphotericin (GA- 1000), 0.1% human epidermal grth factor (rhEGF), 0.1%
hydrocortisone, 0.4% bovine brain extract (BBB), and 5% fetal bovine serum (FBS). All
reported experiments used bPAECs between passages 2 and 10. Once a confluent layer of
cells has been obtained in a culture flask, the cells are ready to be subcultured or obtained.
Cells were rinsed with 5 mL of HEPES for about 1 minute. The HEPES solution was

then aspirated off, replaced with 5 mL of Trypsin/EDTA, and placed into a NUAIRE IR

148

Autoflow C02 water-jacked incubator (model NU-8500 at 5% C02, 37°C and humidified)
for 1 minute. Trypsin is a protease that cleaves proteins that are used by the cells for
adhesion to a substrate, however trypsin is not specific and will also cleave any other
proteins it can find. As a result, the trypsin was only in contact with the cells for a
minimal amount of time, approximately 1 - 2 minutes. As trypsin cleaves cellular
proteins, the cell morphology is altered and the shape changes from an elongated
cobblestone to a rounded sphere. Once approximately 90% of the cells have become
dislodged (the solution will become cloudy as this process occurs), 10 mL of trypsin
neutralizing solution (TNS) was added to stop the trypsin activity. This concentrated
suspension was centrifuged at 1500 rpm. (revolutions per minute) for 5 minutes at 25°C
in order to separate the cells from the solution. After centrifugation, the supernatant was
aspirated out of the tube with care as not to disturb the cell pellet. Finally, 1 mL of
equilibrated medium is added to the tube to resuspend the cells. This concentrated cell
suspension is then equally pipetted into the previously prepared culture flasks
(approximately 3 — 4 flasks). All handling and use of reagents were carried out under a
NUAIRE Class 11, type A-B3 biological safety cabinet with filtered laminar airflow to
ensure safety and sterility of chemicals. All objects were sprayed with a 70% ethanol
solution before entry into the hood to achieve and maintain a sterile environment.

Cell immobilization. The microfluidic device was placed with the array of

149

 

channels down into a 100 x 20 mm petri dish as shown in Figure 4.4. The petri dish is
of the same nature to flasks used to culture bPAECs. Prior to bPAEC loading, the
microchannels were coated with a cell adhesion protein, bovine plasma fibronectin
(FN, Invitrogen). FN was prepared in 1 mL of DDW to a concentration of 1000 pg
mL.1 and further diluted to a concetration of 100ug mL'1 in phosphate buffer solution
(PBS) as working solution. To coat the microchannels, 100 uL of FN were added to
the top of the inlet of the microchannel device and vacuum was applied at the exit
hole to draw solution into each microchannel. An additional 10 uL of FN solution was
added to the exit reservoir to keep the channel from drying out due to evaporation.
The device was incubated at 37°C for 45 - 55 minutes, allowing for sufficient
adsorption of the protein onto the surface. After removing excess FN solution from
the inlets and reservoirs by pipet, the channel was dried with a stream of clean dry air
(AirGas, East Lansing, MI), covered, and exposed to UV light for approximately 10
minutes. Cell pellet was obtained as described above and the pellet was suspended
into 200 - 500 uL of equilibrated EBM. The concentrated cell solution was loaded
into the microchannels in the same manner as FN and the device was incubated at
37°C and 5% C02 for 60 minutes. The channels were rinsed with equilibrated EBM 1
hour after initial seeding to remove any non-adherent cells. Additional cells were

loaded into the channel and the above was repeated until the channels were >75%

150

20mm? cognac“
mo memomEmE com 83% 222.85g m E mom<n3 mo cob: 28338 < 3 .832? 06 3 So: @3863 8m 83: ES can 5::
55 .35 mEQm EV Show was 523 E... o3 .«o 333086 was Egan. :23 £95.. BER—@238 mo 2288 380 A3 we 2sz

2o; Em

56 can

 

 

g

65:23 22.6805 mzom

 

3 5V 3

151

confluent. On average, the time elapsed was approximately 4 - 8 hours from intital
seeding of the channel with cells.

Isolation and purification of platelets and RBCs. All procedures followed
previously described methods in Chapter 2.

Reagent preparation. Reagents were purchased from Sigma Chemical Co. (St.
Louis, MO) and used as received without further purification unless otherwise noted.
Reagents (ATP, ADP, L-NAME, C-peptide, and glybenclamide) were prepared following
previously reported methods in Chapter 2 and 3.

Clopidogrel, (also as known as Plavix) was prepared as a 1.0 mM stock solution
by dissolving a 35 mg tablet in 100 mL of diluted hydrochloric acid solution (pH ~4),
while the working solution reported here was prepared by several dilutions of the stock
into HBSS and the final concentration of clopidogrel with platelets was 40 uM.

Fluorescence labeling of platelets. Regarding the platelet adhesion studies,
platelets were incubated with Cell Tracker Green CMFDA (1 ,uM as final concentration
in HBSS) (Invitrogen) approximately 1 hour and extra fluorescence probe was removed
by centrifugation. CMFDA can freely pass through cell membrane, but once inside the
cell, is transformed into the cell-impermeant product due to reaction with thiols (e.g.,
glutathione) (Figure 4.5). Once the channels were confluent, platelets were

hydrodynamically pumped over endothelium layer for 10 minutes at 37°C, 0.2 uL/min

152

 

8:005 00080000: 0 mama—00.00
800.8000 to 0.820 80800800 028030 Ea: #:080000m00: 0:0 8010—00 mm <Dm§0 0009000 «REED mo mcouomom m6 2&5

200320 - $00

   

Tl

 

153

and channels were rinsed with equilibrated EBM. Images were acquired using an
Olympus 1X71 microscope (Olympus America, Melville, NY) with an electrotherrnally
cooled CCD (Orca, Hamamatsu) and Microsuite software (Olympus America). The
microscope is fitted with a FITC filter cube (Chroma Technology Corp.) containing the

excitation (460-500 nm) and emission (505-560 nm) filters.

4.4 RESULTS AND DISCUSSION

The effect of NO on platelet adhesion. As described in previous chapters, our
quantitative research involving platelet activation was confined to cuvettes90 or even
in a flow system, was in the absence of endothelial cells.” These techniques focus on
the platelet only and, while important in understanding platelet activity in the form of
aggregation and molecules secreted by activated platelets, do not enable the
monitoring of actual adhesion to an endothelium under the conditions of flow.

To demonstrate the ability of platelets to adhere to an immobilized
endothelium upon activation, platelets were incubated in solutions containing various
concentrations of ADP and hydrodynamically pumped through an endothelium-lined
microchannel for 10 minutes as described above. Following this flow period, the
channel was washed with equilibrated EBM and the resultant number of adhered

platelets was determined by counting under fluorescent image conditions. A

154

 

representation of platelets adhering to the immobilized endothelial cells in the
presence and absence of ADP is shown in Figure 4.6. In Figure 4.6, the images on the
left are bright field images of the immobilized endothelium while the images on the
right are the corresponding fluorescent images of the platelets adhering to the
endothelium. In addition, the images were acquired for platelets alone and platelets in
the presence of ADP. However, to demonstrate the importance of
endothelium-derived NO on platelet adhesion, images were also obtained after the
endothelial cells in the microfluidic channels had been incubated with L-NAME, a
recognized inhibitor of eNOS and subsequent NO production. An examination of the
fluorescent platelets in each image clearly shows the increased platelet adhesion due
to ADP activation. Moreover, the data also show that a decrease in NO production by
the endothelium increases the number of platelets adhering to the endothelial cells.
The data in Figure 4.7 was the summary of the results represented in Figure 4.6 and
additional concentrations of ADP that were investigated. The number of platelets
adhering to the L-NAME treated immobilized endothelium in the absence of ADP was
35.2 i 0.9, however, the number of adhered platelets was approximately the same to
35.4 i 5.2 at ADP concentration of 1.0 pM but with untreated endothelium suggesting
the importantance of endothelium-derived NO on inhibition of platelet adhesion to the

endothelium

155

 

Bright field Fluorescent image

 

(a) Platelet

   

(b)+ 5.0 uM ADP

  
  

  
  

(c) Platelet in L-NAME treated bPAEC

4 -...

(d) Platelet + 5 .0 W ADP in L-NAME tdreate bPAEC

Figure 4.6 The effect of ADP activation and L-NAME on platelets pumped over a
confluent channel of bPAECs. A stream of equilibrated medium was pumped over the
endothelium to ensure any nonadherent platelets were removed before averaging the
platelet count. Images in the lefi column represent bright field images, while those on the
right represent the corresponding fluorescence images. (a) Untreated platelets, (b)
platelets incubated in 5 ,uM ADP, (c) untreated platelet adhering to a 10 mM L-NAME
treated endothelium, and (d) platelets activated with 5 ,uM ADP adhered to a 10 mM
L-NAME treated endothelium

 

300 _
. — Untreated bPAECs

250 L-NAME treated bPAECs

   

N

o

o
I

...

o

o
1

Average number platelets
C:
O

   
 

  

 

 

   

50 {
Plt 50 nM 100 nM 500 nM1.0 uM 5.0 [J.M 10.0 uM
[ADP]

Figure 4.7 Varying concentrations of ADP from 50 nM to 10 E] M effects the number of
platelets adhered to an untreated and L-NAME treated bPAEC monolayer. Non-activated
platelets were also examined on similar untreated and treated surfaces. Error bars
represent SEM. p < 0.05 (n = 5)

157

In contrast to endothelium NO, it has been hypothesized that platlet NO can
inhibit platelet recruitment when adhere to the endothelium,75 but this has never been
proven. As shown in Figure 4.8, the number of platelets adhering to endothelium was
17.8 i 0.5, however, the number increased approximately doule to 30.5 d: 0.6 when
platelet NO was inhibited by L-NAME further proved the hypothesis by Freedman et
al back to 1997. Collectively, no matter where NO is released from, it plays an
important role in platelet adhesion. As shown dark gray bars in Figure 4.8, when
platelets were pumped through the endothelium layer along with 40 nM exogenous
NO, the number of platelets adhering to the endothelium decreased to 9.5 d: 0.6 and
23.5 d: 1.4 in the absence and presence of 5.0 uM ADP, respectively.

Applications for drug discovery. To demonstrate the utility of the microfluidic
device for future studies involving drug discovery or drug efficacy, platelets were
activated with increasing concentrations of ADP in the presence and absence of
clopidogrel, a pharmaceutical often prescribed to prevent platelet aggregation.
Clopidogrel is an inhibitor of P2y12 acceptor which is a ADP receptor on platelet
membrane and is though to irreversibly block the ADP uptaking further inhibit platelet
aggregation. As shown in Figure 4.9, platelet adhesion studies that were performed over
the course of 3 days (using platelets from 3 different rabbits) resulted in an adhesion

number of 15.3 :1: 1.4. However, when this study was repeated using an aliquot of the

158

 

180

 

— Normal plt
160 '2 L-NAME treated plt

I _
140 _. NO treated plt

p—A

FA
A O\ 00 O N
O O O O O
.l.t..lt...l....l....l..

Average number platelets

205

 

 

 

Platelet + ADP (5.0 uM)

Figure 4.8 In contrast to inhibition of endothelium NO, platelets were incubated with 10
mM L-NAME prior to flowing through the bPAEC layer channel. The effect of
exogenous NO was also examined shown as dark gray bars. Error bars represent SEM.
p < 0.05 (n = 4)

159

 

   

 
 
 

         

 

 

   

70_
60:
a :
g 50_
E :
a, .
40
l: ; l
530:
§ .
E202
< :
10:
3 0° Y9 t9 v9 v9
N
9 @ Vi” V1” 9 V“
\V XV ‘3' x‘).
6’ Q @ Q
0° 0°
@ 6‘

Figure 4.9 The effect of an anti-platelet drug, clopidogrel (Clop), on platelet adhesion to a
bPAEC monolayer. Platelets were incubated with clop for 30 minutes before being
pumped over the endothelium — (b), (d), and (f). In (d) and (t), after clop incubation and
prior to being pumped over the endothelium, platelets were treated with 1.0 and 5.0 uM
ADP. Error bars represent SEM. p < 0.05 (n = 3)

160

platelets that had been incubated in 40 uM clopidogrel for 30 minutes, the number of
platelets adhering to the endothelium decreased to 9.7 i 1.4. This decrease in platelet
adhesion was even more pronounced in the presence of higher concentrations of
platelet-activating ADP. For example, in the presence of 1.0 uM ADP, the number of
platelets adhering to the endothelium was 31.3 :1: 2.0. In the presence of clopidogreal and
1.0 uM ADP, the number of platelets adhering to the endothelium was decreased to a
value (16.7 d: 1.3) that statistically overlaps with the number of platelets adhering to the
endothelium in the absence of any ADP. Similar effects from the clopidogrel are also
seen at higher concentrationso of ADP.

The effect of A T P on platelet adhesion in a multiple cell types system. People

,

have abnormal circulatory ATP levels such as diabetes,8 82 cystic fibrosis, pulmonary

hypertension83 (decreased ATP amount in the circulation) or multiple sclerosis, sickle
cell disease (increased ATP level), they also have been found having more readily active
platelets”.80 Our group have hypothesized that ATP might have the dual effect on
platelet adhesion and aggregation. Therefore, subsequent studies were performed to
determine the role of ATP in platelet adhesion to an immobilized endothelium in the
presence of flow. A microfluidic device was employed to investigate the effects of
exogenous ATP, and ATP released from RBCs, on platelet adhesion to endothelial cells.

As reported in Chapter 2 (Figure 2.6, also shown in Figure 4.10a), platelet NO production

161

 

3.5 1
3.4 1

3.2 1
3.1

IAILALLJIA

3.0
2.9 -j

Fluorescence intensity (a.u.)

2.8 -j
2.7 ~j

 

 

2.6 ' . , . . . . , . . . . . . . . . . . . . - j
0.00 0.25 0.50 0.75 1.00
[ATP1(l1M)

 

(b) 80
70 «j
60 .5
50 :
40 3

30-j

Average number platelets

20:

    

 

 

Plt 20 nM 50 nM 100 nM 10 uM

Figure 4.10 (a) Platelet NO production as a function of concentration of ATP. As ATP
concentration was about 50 nM, platelet NO stopped increasing. Error bars represent
SEM. (n = 4) (b) Platelets were treated with various concentrations of ATP (and were
centrifuged to remove excess ATP) prior to flowing through a channel coated with a
confluent layer of bPAECs. Error bars represent SEM. p < 0.05 (n = 5)

162

stopped increasing when ATP concentration was about 50 nM which perfectly fit in the
platelet adhesion studies. Figure 4.1% summarizes the number of platelets adhering to
the endothelium in the presence of various concentrations of exogenous ATP added to the
platelets prior to pushing the platelets over the immobilized endothelium. The ATP that
was added to the platelets was washed prior to adding to the microfluidic device, thus
ensuring that the effect seen was from the ATP acting on the platelet as opposed to the
ATP stimulating endothelium-derived NO that could also inhibit the platelet production.
It was clearly evident that the adhesion trend as a function of ATP concentrations
explained the curve in Figure 4.10a, when platelet NO stopped increasing, platelet
adhesion started increasing. The data in Figure 4.11 also demonstrated the importance of
considering sources of ATP in the bloodstream and possible effects on platelet behavior.
Specifically, the platelet adhesion was monitored without the addition of any exogenous
sources of ATP to the flowing stream. Rather, platelets were pumped over the
endothelium along with RBCs in the solution. It is well-established that flowing RBCs
subjected to shear-induced stress will release ATP;68’ 92 moreover, this ATP release can
be inhibited or stimulated with glibenclamide and zinc-activated C-peptide, respectively.
As shown in Figure 4.11, the platelet adhesion to the endothelial cells (15.7 :t 1.2) was
decreased in the presence of RBCs (8.2 :t 1.4), but increases to a value of 18.6 3: 1.2when

the RBCs were first incubated with an ATP release inhibitor prior to flowing with the

163

 

 

25.

20{

Average number platelets

 

 

 

 

 

Q0 x~ \V x‘

Figure 4.11 Platelets were incubated with RBCs in the absence and presence of
glybenclamide (an ATP release inhibitor) or Zinc-activated C-peptide (an ATP release
stimulus) prior to flowing over the confluent bPAECs layer. Error bars represent SEM.
p < 0.05 (n = 6)

164

platelets through the endothelium layer. More specifically, platelets treated with RBCs
incubated with zinc-activated C-peptide adhered less to an immobilized endothelium with
only 4.8 :t 0.4, suggesting the importance of RBC-derived ATP for maintenance of

normal platelet adhesion function.

4.5 CONCLUSIONS

Here, we report the creation of an in vivo process on an in vitro mimic and
successfully used this device to monitor the physiological interaction (adhesion) of
platelets to endothelial cells. The construction of the device is rather simplistic.
Specifically, the use of a petri dish as the substrate to which endothelial cells are
immobilized requires no pretreatment of the substrate (such as plasma oxidation).
Moreover, the PDMS-chip was sealed to the substrate in a reversible manner, which also
simplified construction of the microfluidic system. The device incorporates an important
constituent of the bloodstream (platelets, RBCs, and endothelial cells) and enables the
direct monitoring of their adhesion when activated. The importance of such a device is
exemplified when considering work by Freedman et al75 resulting in the conclusion that
NO production from the endothelium prevents platelet adhesion to the endothelium. In
turn, NO production by platelets may prevent platelets aggregating to each other, a

process known as platelet recruitment. Moreover, the work presented here involving the

165

dual role of ATP as a platelet inhibitor and platelet activator is in concert with established
findings involving hyperactive platelets, clinical outcomes, and certain types of disease as
mentioned above. The work employed three different cell types in a single microfluidic
channel, suggesting a possible method to investigate the communication between
different cell types which such measurements would not be possible with current platelet
aggregation or adhesion study technologies.

Therefore, such a device will enable future studies involving pharmaceutical
candidates for reducing platelet activation to be investigated in a high-throughput manner
using conditions that more closely approximate conditions in the bloodstream in vivo.
Moremore, the changes in platelet behavior as a function of ATP concentrations
monitored within the device, may serve as a starting point for explaining some of the

clinical observations involving hyperactive platelets.

166

(1)

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

LIST OF REFERENCES

Terry, S. C.; Jerman, J. H.; Angell, J. B. IEEE Trans. Electron Devices 1979,
ED-26, 1880-1886.

Jorgenson, J. W.; Lukacs, K. D. Clin. Chem. (Winston-Salem, N. C.) 1981, 27,
1551-1553. ’

Ruzicka, J .; Hansen, E. H. Anal. Chim. Acta 1984, 161, 1-25.
Van der Schoot, B.; Bergveld, P. Sens. Actuators 1985, 8, 11-22.
van Lintel, H. Sens. Actuators, A 1988, 15, 153-167.

Hanison, D. J.; Manz, A.; Fan, Z.; Luedi, H.; Widmer, H. M. Anal. Chem. 1992,
64, 1926-1932.

Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators, B 1990, B], 244-248.

Manz, A.; Verpoorte, E.; Effenhauser, C. S.; Burggraf, N.; Raymond, D. E.;
Harrison, D. J .; Widmer, H. M. J. High Resolut. Chromatogr. 1993, 16, 433-436.

Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481-1488.

Kopp, M. U.; De Mello, A. J .; Manz, A. Science (Washington, D. C.) 1998, 280,
1046-1048.

Manz, A. J. Pharm. Pharmacol. 1998, 50, 42.

Harrison, D. J .; F luri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science
(Washington, D. C., 1883-) 1993, 261, 895-897.

167

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

(24)

Fluri, K.; Fitzpatrick, G.; Chiem, N.; Harrison, D. J. Anal. Chem. 1996, 68,
4285-4290.

Liang, Z.; Chiem, N.; Ocvirk, G.; Tang, T.; F luri, K.; Harrison, D. J. Anal. Chem.
1996, 68, 1040-1046.

Chiem, N.; Harrison, D. J. Anal. Chem. 1997, 69, 373-378.

Li, P. C. H.; Harrison, D. J. Anal. Chem. 1997, 69, 1564-1568.

Salimi-Moosavi, H.; Tang, T.; Harrison, D. J. J. Am. Chem. Soc. 1997, 119,
8716-8717.

Hadd, A. G.; Raymond, D. E.; Halliwell, J. W.; Jacobson, S. C.; Ramsey, J. M.
Anal. Chem. 1997, 69, 3407-3412.

Ermakov, S. V.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1998, 70,
4494-4504.

Jacobson, S. C.; Culbertson, C. T.; Daler, J. E.; Ramsey, J. M. Anal. Chem. 1998,
70, 3476-3480.

Kutter, J. P.; Jacobson, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70,
3291-3297.

Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.;
Ramsey, J. M. Anal. Chem. 1998, 70, 5172-5176.

Khandurina, J.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal.
Chem. 1999, 71, 1815-1819.

Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680.

168

 

(25)

(26)

(27)

(23)

(29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

Simpson, P. C.; Woolley, A. T.; Mathies, R. A. Biomea’. Microdevices 1998, 1,
7-26.

Woolley, A. T.; Lao, K.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1998, 70,
684-688.

Liu, S.; Shi, Y.; Ja, W. W.; Mathies, R. A. Anal. Chem. 1999, 71 , 566-573.

Brenan, C. J. H.; Morrison, T.; Stone, K.; Heitner, T.; Katz, A.; Kanigan, T. S.;
Hess, R.; Kwon, S.-J.; Jung, H.-C.; Pan, J .-G. Proc. SPIE-Int. Soc. Opt. Eng. 2002,
4626, 560-569.

Kikuchi, K.; Nagano, T.; Hirobe, M. Biol. Pharm. Bull. 1996, 19, 649-651.

Colyer, C. L.; Tang, T.; Chiem, N.; Harrison, D. J. Electrophoresis 1997, 18,
1733-1741.

Mulchopadhyay, R. Anal Chem 2007, 79, 2628.

Santini, J. T., Jr.; Cima, M. J .; Langer, R. Nature (London) 1999, 397, 335-338.

Dorre, K.; Brakmann, S.; Brinkmeier, M.; Han, K.-T.; Riebeseel, K.; Schwille, P.;
Stephan, J .; Wetzel, T.; Lapczyna, M.; Stuke, M.; Bader, R.;. Hinz, M.; Seliger, H.;
Holm, J .; Eigen, M.; Rigler, R. Bioimaging 1997, 5, 139-152.

Effenhauser, C. S.; Bruin, G. J. M.; Paulus, A.; Ehrat, M. Anal. Chem. 1997, 69,
3451-3457.

Chou, H.-P.; Spence, C.; Scherer, A.; Quake, S. Proc. Natl. Acad. Sci. U. S. A.
1999, 96, 11-13.

Jansen, H.; Gardeniers, H.; de Boer, M.; Elwenspoek, M.; Fluitman, J. J.
Micromech. Microeng. 1996, 6, 14-28.

169

 

(37)

(38)

(39)

(40)

(41)

(42)

(43)

(44)

(45)

(46)

(47)

(43)

(49)

(50)

Leistiko, O.; Jensen, P. F. J. Micromech. Microeng. 1998, 8, 148-150.

Buettgenbach, S.; Michalzik, M.; Wilke, R. Chem. Ing. Tech. 2005, 77, 833-839.

Piotter, V.; Benzler, T.; Hanemann, T.; Wollmer, H.; Ruprecht, R.; Hausselt, J.
Proc. SPIE-Int. Soc. Opt. Eng. 1999, 3680, 456-463.

Becker, H.; Gartner, C. Electrophoresis 2000, 21, 12-26.

Varadan, V. K.; Varadan, V. V. Proc. SPIE-Int. Soc. Opt. Eng. 2001, 4592, 9-20.

Waits, C. M.; Ghodssi, R.; Ervin, M. H.; Dubey, M. Symp. Proc. - Int. Semicond.
Device Res. Symp. 2001, 182-185.

Frazier, A. B.; Ahn, C. H.; Allen, M. G. Sens. Actuators, A 1994, A45, 47-55.

Emmelius, M.; Pawlowski, G.; Vollmann, H. W. Angew. Chem. 1989, 101,
1475-1502.

Chou, S. Y.; Krauss, P. R.; Renstrom, P. J. Science (Washington, D. C.) 1996, 272,
85-87.

Xia, Y.; Kim, H.; Zhao, X.-M.; Rogers, J. A.; Prentiss, M.; Whitesides, G. M.
Science (Washington, D. C.) 1996, 273, 347-349.

Quake, S. R.; Scherer, A. Science (Washington, D. C.) 2000, 290, 1536-1540.

McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O.
J. A.; Whitesides, G. M. Electrophoresis 2000, 21 , 27-40.

McDonald, J. C.; Whitesides, G. M. Acc. Chem. Res. 2002, 35 , 491-499.

Becker, H.; Locascio, L. E. T alanta 2002, 5 6, 267-287.

170

(51)

(52)

(53)

(54)

(55)

(56)

(57)

(58)

(59)

(60)

(61)

(62)

(63)

Clarson, S. J .; Semlyen, J. A. Siloxane Polymers, 1993.

Morra, M.; Occhiello, E.; Marola, R.; Garbassi, F.; Humphrey, P.; Johnson, D. J.
Colloid Interface Sci. 1990, 13 7, 11-24.

Chaudhury, M. K.; Whitesides, G. M. Langmuir 1991, 7, 1013-1025.

Chaudhury, M. K.; Whitesides, G. M. Science (Washington, D. C., 1883-) 1992,
255, 1230—1232.

Rogers, J. A.; Qin, D.; Schueller, O. J. A.; Whitesides, G. M. Rev. Sci. Instrum.
1996, 67, 3310-3319.

Xia, Y.; Whitesides, G. M. Angew. Chem, Int. Ed. 1998, 37, 550-575.

DeBolt, L. C.; Mark, J. E. Macromolecules 1987, 20, 2369-2374.

Kunzler, J. F. Trends Polym. Sci. (Cambridge, U. K.) 1996, 4, 52-59.

Moreau, W. M. Opt. Eng. 1983, 22, 181-184.

Brambley, D.; Martin, B.; Prewett, P. D. Adv. Mater. Opt. Electron. 1994, 4,
55-74.

Zhao, X. M.; Xia, Y.; Whitesides, G. M. Adv. Mater. (Weinheim, Ger.) 1996, 8,
837-840.

Ellsworth, M. L.; Forrester, T.; Ellis, C. G.; Dietrich, H. H. Am. J. Physiol. 1995,
269, H2155-H2161.

Sprague, R. S.; Olearczyk, J. J .; Spence, D. M.; Stephenson, A. H.; Sprung, R. W.;
Lonigro, A. J. Am. J. Physiol. 2003, 285, H693-H700.

171

 

(64)

(65)

(66)

(67)

(68)

(69)

(70)

(71)

(72)

(73)

(74)

(75)

Bergfeld, G. R.; Forrester, T. Cardiovasc. Res. 1992, 26, 40-47.

Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J. Am. J. Physiol.
1996, 271 , H27l7-H2722.

Edwards, J.; Sprung, R.; Spence, D.; Sprague, R. Analyst (Cambridge, U. K.)
2001, 126, 1257-1260.

Sprung, R.; Sprague, R.; Spence, D. Anal. Chem. 2002, 74, 2274-2278.

Price, A. K.; Fischer, D. J.; Martin, R. S.; Spence, D. M. Anal. Chem. 2004, 76,
4849-4855.

Meyer, J. A.; Froelich, J. M.; Reid, G. E.; Karunarathne, W. K. A.; Spence, D. M.
Diabetologia 2008, 51 , 175-182.

Furchgott, R. F.; Zawadzki, J. V. Nature (London) 1980, 288, 373-3 76.

Pahner, R. M. J.; Ferrige, A. G.; Moncada, S. Nature (London) 1987, 327,
524-526.

Ignarro, L. J .; Buga, G. M.; Chaudhuri, G. Eur. J. Pharmacol. 1988, 149, 79-88.

Moncada, S.; Palmer, R. M. J.; Higgs, E. A. Hypertension (Dallas) 1988, 12,
365-372.

Radomski, M. W.; Palmer, R. M. J.; Moncada, S. Br. J. Pharmacol. 1987, 92,
181-187.

Freedman, J. E.; Loscalzo, J.; Barnard, M. R.; Alpert, C.; Keaney, J. F., Jr.;
Michelson, A. D. J. Clin. Invest. 1997, 100, 350-356.

172

(76)

(77)

(73)

(79)

(80)

(81)

(82)

(83)

(84)

(85)

(86)

(37)

Wang, G.-R.; Zhu, Y.; Halushka, P. V.; Lincoln, T. M.; Mendelsohn, M. B. Proc.
Natl. Acad. Sci. U. S. A. 1998, 95, 4888-4893.

Sogo, N.; Magid, K. S.; Shaw, C. A.; Webb, D. J.; Megson, I. L. Biochem.
Biophys. Res. Commun. 2000, 279, 412-419.

Vinik, A. I.; Erbas, T.; Park, T. S.; Nolan, R.; Pittenger, G. L. Diabetes Care 2001,
24, 1476-1485.

O'Sullivan, B. P.; Linden, M. D.; Frelinger, A. L., 111; Barnard, M. R.;
Spencer-Manzon, M.; Morris, J. E.; Salem, R. O.; Laposata, M.; Michelson, A. D.
Blood 2005, 105, 4635-4641.

Villagra, J .; Shiva, S.; Hunter, L. A.; Machado, R. F .; Gladwin, M. T.; Kato, G. J.
Blood 2007, 110, 2166-2172.

Carroll, J.; Raththagala, M.; Subasinghe, W.; Baguzis, S.; Oblak, T. D. a.; Root,
P.; Spence, D. Mo]. BioSyst. 2006, 2, 305-311.

Sprague, R. S.; Stephenson, A. H.; Bowles, E. A.; Stumpf, M. S.; Lonigro, A. J.
Diabetes 2006, 55, 3588-3593.

Sprague, R. S.; Stephenson, A. H.; Ellsworth, M. L.; Keller, C.; Lonigro, A. J.
Exp. Biol. Med (Maywood, NJ, U. S.) 2001, 226, 434-439.

Freed, A. L.; Audus, K. L.; Lunte, S. M. Peptides (N. Y., NY, U. S.) 2002, 23,
157-165.

Shuler, M. L.; Ghanem, A.; Quick, D.; Wong, M. C.; Miller, P. Biotechnol.
Bioeng. 1996, 52, 45-60.

Park, T. H.; Shuler, M. L. Biotechnol. Prog. 2003, 19, 243-253.

D'Amico Oblak, T.; Root, P.; Spence Dana, M. Anal Chem 2006, 78, 3193-3197.

173

(38)

(89)

(90)

(91)

(92)

Genes, L. 1.; Tolan, N. V.; Hulvey, M. K.; Martin, R. S.; Spence, D. M. Lab Chip
2007, 7, 1256-1259.

Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem.
1998, 70, 4974-4984.

Ku, C.-J.; Karunarathne, W.; Kenyon, S.; Root, P.; Spence, D. Anal. Chem. 2007,
79, 2421-2426.

Carroll, J. S.; Ku, C.-J.; Karunarathne, W.; Spence, D. M. Anal. Chem.
(Washington, DC, U. S.) 2007, 79, 5133-5138.

Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Kleinhenz, M. E.; Lonigro,
A. J. Am. J. Physio]. 1998, 275, H1726-H1732.

174

CHAPTER 5 CONCLUSIONS AND FUTURE DIRECTIONS

5.1 CONCLUSIONS
The discovery of NO as the endothelium-derived relaxing factor (EDRF) by
Furchgott and Zadawaski has had a major impact,1 not only on the cardiovascular system,

2’ 3 The fact that NO is Synthesized by

but also in other field of scientific investigation.
platelets, as well as endothelial cells, distinguished this vasoactive messenger from other
' factors such as prostacyclin or endothelin. However, platelet NO has been hypothesized
to inhibit platelet recruitment4 and sustain the suppression of aggregations in a paracrine
manner. On the other hand, it has been shown that RBCs are able to release high
nanomolar amount of ATP subjected to mechanical deforrnation.6'8 The importance of
this release of ATP in vivo is that ATP is a recognized stimulus of nitric oxide synthase
(NOS) in endothelial cells.9 Although a relationship between ATP released from RBCs
and endothelium-derived NO has been established, there have been no reports of the
synergy between ATP derived from RBCs and the ability of platelets to produce NO.

In this thesis, a spectrofluorometric method to monitor NO production in platelets
using a molecular probe was developed.10 NO is quantitatively determined in platelets
prior to, and after, stimulation with ATP or activation with ADP. Platelets obtained from

the whole blood of rabbits were loaded with the fluorescence probe DAF-FM DA, and

the subsequent NO production was measured as a fluorescent benzonitriazole.

175

Experiments were performed to determine the effect of probe concentration and probe
incubation time in the platelets prior to measurement of the fluorescence. This
information, combined with the method of multiple standard additions, was then
employed to determine the moles of intracellular NO in the platelets (2.7 i 0.3 x 10'16
moles of NO/platelet) and the basal level of extracellular NO in the platelet sample (9.9 :t
2.2 x 10.18 moles of NO/platelet). Moreover, this method was used to quantitatively
determine the amount of NO released from platelets whose NO production was
stimulated with ATP or ADP. The ability to monitor this NO production using a
fluorescence probe may be advantageous over other forms of measuring NO production
from platelets (such as amperometry) because, once activated, a platelet’s ability to
adhere to surfaces is dramatically increased.

Secondly, the communication between RBCs and platelets was successfully
established using a flow through flourometer.11 It has been shown that RBC-dervied ATP
results in endothelium NO production and subsequent vasodilation suggesting that RBC
may be a determinant of vascular caliber in the microcirculation. Moreover, studies have
shown that patients with certain diseases such as pulmonary hypertension, cystic
fibrosis12 and diabetes,8’ 13 or complications arising from these diseases, have RBCs that
release less ATP than RBCs obtained from healthy controls. To date, these reduced values
of ATP release from the RBC have been discussed in terms of the endothelium and its

inability to produce the proper levels of NO needed for vasodilatory purposes. In contrast

176

to endothelium NO, once produced by the platelet, NO acts as a platelet inhibitor,
reducing the activation and aggregation of platelets. Here, we provided evidence that
platelet NO is stimulated through RBC-derived ATP, suggesting an important role of ATP
in platelet function. Interestingly, patients with diseases mentioned above also have
hyperactive platelets,14’ ‘5 therefore, decreased levels of RBC-derived ATP and more
active platelet may result in increased possibility of thrombus formation, further, stroke.
Finally, the creation of an endothelium mimic within a microdevice was
successfully implemented to monitor multiple cell types in a single channel.16 Here, the
creation of a device to monitor an in vivo process on an in vitro mimic has been used to
monitor the physiological interaction (adhesion) of platelets to endothelial cells. The
device incorporated an important constituent of the bloodstream (platelets, RBCs, and
endothelial cells) and enabled the direct monitoring of their adhesion when activated.
This work also revealed the dual effect of ATP on platelet adhesion. In addition to the
aforementioned diseases, there are other patient groups with hyperactive platelets who
may have excessive extracellular ATP levels. For example, people with sickle cell disease
whose cells are prone to hemolysis, are known to suffer from complications (e.g., stroke)
associated with hyperactive platelets.l7 Moreover, our group has found that people with
multiple sclerosis release excessive amounts of ATP from their RBCs in comparison to
controls (unpublished results) and also have been reported to be more susceptible to deep

vein thrombosis. Therefore, the changes in platelet behavior as a function of ATP

177

concentrations monitored with microfluidic technologies, may serve as a starting point

for explaining some of the clinical observations involving hyperactive platelets.

5.2 FUTURE DIRECTIONS

The use of microfluidic devices will help improve our understanding of the
microcirculation. Moreover, microfluidic devices have shown the capacity to be used in
biomedical studies for diagnostic testing of certain disease states,18 as well as in the
pharmocokinetics field for drug efficacylg’ 20 NO generated by endothelial cells and
platelets has proven to be an important physiological and regulatory mediator modulating
vessel wall hemostasis and preventing thrombosis. The changes in its generation or
metabolism are likely to play role in the pathomechanism of vasospastic and thrombotic
disorders. Flow induced shear is largely responsible for the tendency of blood elements to
move towards the center of the flowing stream. If platelets were the only formed element
in the blood, they would occupy the axial stream and unlikely interact with the vessel
wall. However, in whole blood, numerous RBCs occupy the axial stream and force
platelets to assume the position close to endothelial cells. Under physiologic conditions,
platelets remain in a close contact with the endothelium and blood flow stimulates the
endothelium NO, which regulates platelet activity?“-23 The endothelium is an obvious
target organ of cardiovascular risk factors. Accordingly, functional alterations do occur

with aging, hypertension and hypercholesterolemia. All conditions are associated with a

178

decreased basal and stimulated release of endothelium NO. Clinical evidence
substantiates the antiplatelets role of NO. Thrombotic complications associated with
atherosclerosis, coronary artery disease, hypertension and diabetes mellituszii'26 have
been shown to involve impairment in NO synthesis and mechanism of action. Thrombi
are complex structures that are composed not only of fibrin meshwork, but also contain
blood-bome cellular elements such as platelets, leukocytes and RBCs and platelets play
an essential role in the initial response they adhere to vessel wall components.

Platelet activity is known to enhance the procession of coronary atherosclerosis
and to precipitate platelet-mediated coronary thrombosis and acute ischemic events (e.g.,
stroke).27 Over the past 40 years, considerable research has been conducted to examine
ways by which to inhibit abnormal platelet interaction with vessel wall in aforementioned
patients. Owing to the important role of platelet adhesion, aggregation, and growth factor
release on the damaged vessel surface (with the loss of functional endothelial cells and
NO release), platelet inhibitors such as aspirin, ticlopidine and clopidogrel have been
utilized to limit thrombosis. However, none of these inhibitors produced a completed
satisfaction. Moreover, with more potent drug dose, the risk of excessive bleeding
increased. The problem in finding a suitable platelet inhibitor is that a very high
concentration is needed systemically to have a major effect locally at the site of damage
or thrombus. Thus, there is a great interest in local delivery of substances with antiplatelet

and anticoagulant properties. One of the substances that would be very beneficial to apply

179

locally is the targeting clot-dissolving therapeutics.

Improvement, have been made toward developing a fibrin-specific nanoparticle as
a thrombolytic drug delivery system.28 Briefly, once the nanoparticle targeted a thrombus,
enzymes modified on particle surfaces can help dissolving the thrombus. However, more
effective therapeutic approaches might be possible if an effective model can be developed.
Thus, the device used in this work will be suitable for the study not only of the thrombus
formation but also of providing an in vitro platform for the drug delivery. Figure 5.1
illustrated the creation of a thrombus in the confluent bPAEC layer channel. More
specifically, the preparation of the microfluidic device was in the same manner as
previously established except a layer of collagen was coated in the channel before
fibronectin. Whole blood was pumped through while a 23 gauge needle poked through
PDMS chip to create an “injury” in the channel. Whole blood was used in this method
instead of RBCs suspension is because that species such as clot-factors facilitating
coagulation processes are presented in whole blood. However, the problem with this
method here is that it can’t provide both precise spatial and temporal controls.

Methods such as laser-induced injury29 or photothrombosis30 have been well
developed to generate a local disruption on the endothelium. The laser-induced method is
the direct contact of the laser beam with the target vessel (here, it will be the endothelium
layer), the prior fluorescently tagged platelets and proteins can then be pumped through

the channel to generate both bright field and fluorescence images for thrombus

180

 

 

 

 

Figure 5.1 A thrombus formation mimic in the microfluidic channel

181

 

development. On the other hand, the photothrombosis procedure utilizes a photosensitive
dye circulating in the channel. Once exposed to an external light beam, the dye turns into
highly reactive oxygen radicals to disrupt the endothelium layer, resulting in subsequent
endothelium injury. Thus, incorporation of any these methods with the device developed

in this work will provide a more beneficial and precise model for in vitro studies.

182

 

LIST OF REFERENCES

(1) Furchgott, R. F.; Zawadzki, J. V. Nature (London) 1980, 288, 373-3 76.

(2) Radomski, M. W.; Palmer, R. M. J.; Moncada, S. Br J. Pharmacol. 1987, 92,
181-187.

(3) Garg, U. C.; Hassid, A. Am. J. Physiol. 1989, 25 7, F60-F 66.

(4) Freedman, J. E.; Loscalzo, J.; Barnard, M. R.; Alpert, C.; Keaney, J. F., Jr.;
Michelson, A. D. J. Clin. Invest. 1997, 100, 350-356.

(5) Riddell, D. R.; Graham, A.; Owen, J. S. J. Biol. Chem. 1997, 272, 89-95.

(6) Sprague, R. S.; Ellsworth, M. L.; Stephenson, A. H.; Lonigro, A. J. Am. J. Physio].
1996, 271, H2717-H2722.

(7) Price, A. K.; Fischer, D. J .; Martin, R. S.; Spence, D. M. Anal. Chem. 2004, 76,
4849-4855.

(8) Carroll, J .; Raththagala, M.; Subasinghe, W.; Baguzis, S.; Oblak, T. D. a.; Root, P.;
Spence, D. Mo]. BioSyst. 2006, 2, 305-311.

(9) Bogle, R. G.; Coade, S. B.; Moncada, S.; Pearson, J. D.; Mann, G. E. Biochem.
Biophys. Res. Commun. 1991, 180, 926-932.

(10) Ku, C.-J.; Karunarathne, W.; Kenyon, S.; Root, P.; Spence, D. Anal. Chem. 2007,
79, 2421-2426.

(11) Carroll, J. S.; Ku, C.-J.; Karunarathne, W.; Spence, D. M. Anal. Chem.
(Washington, DC, U. S.) 2007, 79, 5133-5138.

183

 

(12)

(13)

(14)

(15)

(16)

(17)

(13)

(19)

(20)

(21)

(22)

(23)

(24)

Sprague, R. S.; Stephenson, A. H.; Ellsworth, M. L.; Keller, C.; Lonigro, A. J. Exp.
Biol. Med. (Maywood, NJ, U. S.) 2001, 226, 434-439.

Sprague, R. S.; Stephenson, A. H.; Bowles, E. A.; Stumpf, M. S.; Lonigro, A. J.
Diabetes 2006, 55, 3588-3593.

Vinik, A. 1.; Erbas, T.; Park, T. S.; Nolan, R.; Pittenger, G. L. Diabetes Care 2001,
24, 1476—1485.

O’Sullivan, B. P.; Linden, M. D.; Frelinger, A. L., III; Barnard, M. R.;
Spencer-Manzon, M.; Morris, J. E.; Salem, R. O.; Laposata, M.; Michelson, A. D.
Blood 2005, 105, 4635-4641.

Ku, C.-J.; D'Amico Oblak, T.; Spence, D. M. Anal. Chem. (Washington, DC, U. S.)

2008, 80, 7543-7548.

Villagra, J .; Shiva, S.; Hunter, L. A.; Machado, R. F.; Gladwin, M. T.; Kato, G. J.
Blood 2007, 110, 2166—2172.

Myers, F. B.; Lee, L. P. Lab Chip 2008, 8, 2015-2031.

Tolan, N. V.; Genes, L. 1.; Spence, D. M. Jala 2008, 13, 275-279.

Walsh, C. L.; Babin, B. M.; Kasinskas, R. W.; Foster, J. A.; McGarry, M. J.;
Forbes, N. S. Lab Chip 2009, 9, 545-554.

Pohl, U.; Busse, R. Circ. Res. 1989, 65, 1798-1803.

Cooke, J. P.; Santosa, A. C. Steroid Horm. Uterine Bleeding 1992, 225-240.

De Graaf, J. C.; Banga, J. D.; Moncada, S.; Palmer, R. M. J.; De Groot, P. G.;
Sixma, J. J. Circulation 1992, 85, 2284-2290.

Calver, A.; Collier, J .; Vallance, P. J Clin Invest 1992, 90, 2548-2554.

184

(25)

(26)

(27)

(23)

(29)

(30)

Cooke, J. P. Cardiovasc Clin 1992, 22, 3-17.

de Belder, A. J .; Radomski, M. W. J Hypertens 1994, 12, 617-624.

Vogel, J. H. Am J Cardiol 1987, 60, 68B-69B.

Marsh, J. N.; Senpan, A.; Hu, G.; Scott, M. J.; Gaffney, P. J.; Wickline, S. A.;
Lanza, G. M. Nanomedicine (London, U. K.) 2007, 2, 533-543.

Rosen, E. D.; Raymond, S.; Zollman, A.; Noria, F.; Sandoval-Cooper, M.;
Shulman,A.; Merz, J. L.; Castellino, F. J. Am. J. Pathol. 2001, 158, 1613-1622.

Watson, B. D.; Dietrich, W. D.; Busto, R.; Wachtel, M. S.; Ginsberg, M. D. Ann
Neurol 1985, I 7, 497-504.

185

 

° 3 I3063 1539