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BIOENERGETICS OF PLATELET AND ALVEOLAR MACROPHAGE
FUNCTION

By

Welivitiya K. Ajith Karunarathne

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Chemistry

2009

ABSTRACT
BIOENERGETICS OF PLATELET AND ALVEOLAR MACROPHAGE FUNCTION
By
Welivitiya K. Ajith Karunarathne
Part 1: Platelet bioenergetics

In modern day life, risk factors associated with life style such as high calorie
diet, unhealthy eating habits, mental stress, and lack of exercise substantially increase
the risk of cardiovascular diseases and diabetes. An important feature of platelets in
both of these conditions is their enhanced sensitivity to the activation stimuli that can
results in life threatening conditions such as heart attacks or strokes. Although platelet
activating factors account for the larger fraction of this systemic thrombus formation,
they do not explain the entire spectrum of platelet hyperactivity. We observed a
correlation between; red blood cell (RBC) derived ATP release and platelet
hyperactivity in the circulation. Therefore, it was predicted that platelet hyperactivity
may be related to the physiological behavior of RBCs and their ATP release.
Furthermore, the potential of ATP and its primary receptor on platelets, P2X1 have
been underestimated as a result of the lack of available analytical tools and
methodologies.

The work shown here demonstrates the biphasic nature of ATP and P2Xl
receptor on platelet inhibition and aggregation in a concentration-dependent manner.
We found that incremental increases of ATP concentration initially reduce the platelet
aggregation, with a gradual increase in platelet Ca2+z’ and NO production. However,
with fiirther increase in ATP, platelets began to aggregate. Therefore our data explains

the behavior of extracellular ATP on platelets: an antagonist at low concentrations and

an agonist at high concentrations. We also found that the most commonly used ATP
analogue to study P2X1 function, a,B—methylene ATP, fails to induce the actual P2X1
receptor potential, dUe to its chemical structure and the behavior which abolishes the
P2X1 fimction. In summary, investigations in chapter 2 and 3 of this dissertation
demonstrate the biphasic nature of extracellular ATP as a platelet antagonist and an
agonist. Also we were the first to demonstrate the actual potential of the P2X1 receptor
and bioavailable ATP in platelet activation and aggregatiOn which may be useful in
future antiplatelet therapy.

Part 2: Bioenergetics of alveolar macrophages in cystic fibrosis pathogenesis

Cystic fibrosis (CF) is a genetic disorder associated with mutations in the cystic
fibrosis transmembrane conductance regulator (CF TR) gene and its protein product,
CFTR. Although, the fatal CF lung disease is characterized by chronic pulmonary
infections and inflammations, the available molecular level understanding cannot
explain the failures in the lung defense against the pathogen invasion, which results in
lung infections. Despite the fact that alveolar macrophages are the first defense in the
lungs against particulate matter and pathogen invasion, there have never been
correlations described between defective CFTR on macrophage function and defective

macrophage function on CF lung defense failure.

Our investigations reveal the correlation betWeen CFTR dysfunction and
macrophage glucose uptake which eventually regulates the alveolar macrophages
phagocytosis. We also found that Zn2+ activated C-peptide, a known glucose uptake
sensitizer for RBCs, completely reversed the reduced glucose uptake and phagocytosis

due to the CFTR inhibition.

ACKNOWLEDGEMENTS

Completion of a doctoral degree would be rewarding for anybody, but
for me it is more than that. I have earned every single step of this long journey from a
rural farming village in Eastern Sri Lanka to prestigious Michigan State University. 1
would not be able to make it up to chemistry graduate school without my sister and
mother who spent most of their lives for my education. I think the hardships I have
faced during my childhood made me strong to stand in front of every single challenge

faced later.

At this point, it is my pleasure to acknowledge most of my teachers back in the
school and college. I especially admire Mr. Thilak Tennekoon, my mid school teacher
for identifying my talents and weaknesses and helping me. I really appreciate the
guidance and encouragement gained from my mentor, Professor Dana Spence. I believe
that his enormous tolerance to discuss every single possibility in my scientific research
immensely assisted me to be a better scientist. He is the one who encouraged me to set

my goals to be an academic researcher with my own research group someday.

I was fortunate to be a member of a wonderful research group that felt much like
a family. I have never seen such caring people as in the Spence’s group. Chia. Nicole,
Jen and Andrea: thank you for being such cooperative colleagues. I will never forget the
assistance I had from Steve, Adam, Paul, Kari and Suzanne during my dissertation
writing. I really appreciate Steve’s immense support during these years and I would not
be able to drive my car after skidding on the icy road without him. Although we were

quarrelling all the time, I never forget my graduate school life with Wathsala.

iv

I have met very special characters like Lasantha and Nalaka who would do
anything to make other people’s lives better. Finally my incredible love goes to my wife

Sumithra. and sweetheart Diniti. You are the reasons for my life and work.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. xi
LIST OF FIGURES ................................................................................ iv
CHAPTER 1 .
1 .1 INTRODUCTION ......................................................................... 1
1.2 PLATELET STRUCTURE FUNCTION AND REGULATION .................... 4
1.2.1 Platelet structure ................................................................... 4
1.2.2 Hemostasis: platelet in bleeding prevention ................................... 7
1.2.3 Mechanisms of platelet function regulation .................................... 9
1.2.4 Platelet associated disorders ................................................... 14
1.3 PLATELETS IN CARDIOVASCULAR DISEASES AND
ANTIPLATELET THERAPY ........................................................... 16
1.3.1 Antiplatelet therapy associated with purinergic signaling ................... 17
1.3.2 F ailures/ insufficiency in current antiplatelet therapy- necessity of
new drug targets .................................................................. 18
1.4 A BRIEF INTRODUCTION TO THE EFFECT OF DEFECTIVE CFTR
ON MACROPHAGE FUNCTION IN CYSTIC FIBROSIS ........................ 20
1.4.1 CF pathogenesis and CFTR ...................................................... 20
1.4.2 Defective CFTR and alveolar macrophages .................................. 22
1.5 ANALYTICAL TOOLS TO EVALUATE PLATELET AND
MACROPHAGE FUNCTION .......................................................... 23
1.5.1 Chemiluminescence assays for cellular ATP release measurements. . ...24
1.5.2 Fluorescence determination of intracellular NO and Ca2+i ................. 24
1.5.3 Light transmittance aggregometry for platelet aggregation ................ 28
1.5.4 Liquid scintillation counting for cellular glucose uptake
measurements .................................................................... 28
1.6 THESIS OBJECTIVE .................................................................... 32
1.6.1 Role of ATP and its receptor, P2X1, in platelet function ................... 32
1.6.2 Inhibition of CFTR on glucose uptake and phagocytosis by alveolar
macrophages ...................................................................... 34
REFERENCES ...................................................................................... 37
CHAPTER 2
2.1 EXTRACELLULAR BIOAVAILABLE ATP AND PLATELET
FUNCTIO .................................................................................. 49
2.2 A RELATIONSHIP BETWEEN P2X1 P2Y1 PLATELET
RECEPTORS ............................................................................. 50
2.2.1 P2X, P2Y receptor synergy............................... ...................... 50
2.2.2 Common components in platelet purinergic signaling ...................... 51
2.3 INTERCELLULAR COMMUNICATIONS OF PLATELETS IN THE
CIRCULATION ........................................................................... 52
2.4 F ATES OF Ca2+i MEDIATED BY ATP .............................................. 55
2.4.1 ATP mediated Ca2+i as a platelet antagonist ................................. 57
2.4.2 ATP mediated Ca2+i as a platelet agonist ..................................... 57

vi

2.5 EXPERIMENTAL ........................................................................ 58
2.5.1 Platelet and RBC preparation ................................................... 59
2.5.2 ADP induced platelets activation in terms of platelet granular
ATP release ...................................................................... 59
2.5.3 Extracellular NO mediated platelet inhibition-as a measure of ADP
induced ATP release ............................................................. 61
2.5.4 Effect of RBC derived ATP on platelet NO production in a micro
flow system ........................................................................ 62
2.5.5 Fluorescence determination of platelet NO ................................... 65
2.5.6 Fluorescence determination of platelet intracellular Ca2+ .................. 66
2.5.7 Atomic absorption spectroscopy for platelet total Ca2+. . . . . . . . . . ...........67
2.5.8 Platelet aggregation measurements ............................................ 68
2.6 RESULTS AND DISCUSSION ........................................................ 71
2.6.1 ADP induced platelet activation and granular ATP release ................ 71
2.6.2 Inhibition of ADP induced platelet activation by extracellular NO ...... 71
2.6.3 ATP, a communicator between RBC and platelet ......................... 73
2.6.4 ATP and P2X1 in the regulation of platelet NO and cytosolic Ca2+. .....73
2.6.5 ATP and P2X1 in the regulation of platelet aggregation ................... 79
2.7 CONCLUSIONS AND OTHER CONSIDERATIONS ............................. 85
LIST OF REFERENCES ........................................................................ 92
CHAPTER 3
3.1 PLATELET PURINERGIC RECEPTORS ........................................... 100
3.2 POSSIBLE EXPLANATIONS FOR ATP-P2X1 FUNCTION
UNDERESTIMATION ................................................................. 104
3.3 EXPERIMENTAL ...................................................................... 106
3.3.1 Measurement of P2X1 and P2Y1 induced platelet Ca2+i increase
and their differential regulation with P2X1 and P2Y1
antagonists ............................................................................................. 1 07
3.3.2 PRP aggregation measurements .............................................. 108
3.3.3 ATP degradation assay with 0.5 U/ml apyrase ............................. 109
3.4 RESULTS AND DISCUSSION ....................................................... 110
Phosphoenolp ruvate-private kinase system and P2 Y1 behavior ................. 11 1
Regulated Ca +studies-the P2X 1 response .................................... - ....... 116
Inhibition of P2Y1 with MRS 2179 ................................................... 116
Determination of P2Y1 independent ATP induced P2X1 response. . . . . . . . .......1 18
ATP (5 ,uM) decay assay with 0.5 U/ml apyrase ..................................... 120
Reasons behind the underestimation of the role of extracellular
ATP andP2X1 ........................................................................... 121
Utilization of optimized P2 Y1 inhibitory conditions to compare the eflect
of A T P and a,,B—methylene A TP on P2X 1 mediated platelet Caz ' influx. . . 1 28
CONCLUSIONS AND OTHER CONSIDERATIONS ..................................... 129
LIST OF
REFERENCES ....................................................................................... 131

vii

CHAPTER 4

4.1 THE EFFECT OF CFTR DYSFUNCTION IN ALVOLAR
MACROPHAGES ON CYSTIC F IBROSIS LUNG DISEASE .................. 135
4.1.1 Macrophages in brief ........................................................... 138
4.1.2 Pulmonary alveolar macrophages ............................................. 142
4.2 ALVEOLAR MACROPHAGE FUNCTION, ENERGY METABOLISM
AND CFTR .............................................................................. 142
4.2.1 Dependency of Macrophage energy metabolism on the
CFTR function .................................................................. 144
4.3 EXPERIMENTAL ...................................................................... 1 50
4.3.1 Isolation and purification of rabbit alveolar macrophages ................ 150
4.3.2 Liquid scintillation determination of macrophage glucose uptake....... 151
4.3.3 Fluorescence spectroscopy determination of opsonized bacteria
particle ingestion ............................................................... 152
4.3.4 Single cell fluorescence imaging for RAM phagocytosis ................. 153
4.3.5 Macro fluorescence microscopy evaluation of C-peptide mediated
recovery of the inhibitory effect of glybenclamide on RAM
phagocytosis .................................................................... 1 53
4.4 RESULTS AND DISCUSSION ...................................................... 156
4.5 CONCLUSIONS AND OTHER CONSIDERATIONS ........................... 163
LIST OF REFERENCES ........................................................................ 168
CHAPTER 5
5.1 OVERALL CONCLUSIONS FROM PLATELET STUDIES .................... 175
5.2 FUTURE DIRECTIONS FOR PLATELET STUDIES“ ...180
5.3 OVERALL CONCLUSIONS FROM MACROPHAGE STUDIES ............. 182
5.4 FUTURE DIRECTIONS FOR MACROPHAGE STUDIES ..................... 184
REFERENCES .................................................................................... 186

viii

LIST OF TABLES
Table 1.1 Classification of platelet disorders63 .............................................. 15

Table 4.1 Data summarizes the CFTR regulated the key transport
processesls’lg’36 ..................................................................... 141

ix

LIST OF FIGURES

Figure1.1 Detailed schematic of a platelet cross section .................................... 6
Figure 1.2 Platelet purinergic signaling and associated pathways .......................... 8
Figure 1.3 Platelet function regulation in the vascular system .............................. 10
Figure 1.4 Electron transfer pathway and NO production in eNOS ....................... 12
Figure 1.5 Schematic representation of luciferin luciferase bioluminescence

reactionl ............................................................................. 26
Figure 1.6 Fluorescence probes used in the determination of a) NO, and b)

Ca2+i. .. ........................................................................................................ 27
Figure 1.7 Working principle of an aggregometer .......................................... 29
Figure 1.8 Basics of liquid scintillation counting (LSC) .................................... 31
Figure 2.1 Effects of nitric oxide on platelet inhibition .................................... 54
Figure 2.2 Ca2+ signaling in platelet homeostasis ........................................... 56
Figure 2.3 Two tee setup for investigation of platelet ATP release ...................... 63
Figure 2.4 Three tee setup for nitric oxide mediated platelet inhibition ................ 64
Figure 2.5 ATP release determination of ADP activated Platelets ...................... 69
Figure 2.6 Inhibition of ATP release from ADP activated platelets by ................. 72
Figure 2.7 Effect of P2X1 inhibition on platelet derived NO ............................. 75
Figure 2.8 Influence of ATP on P2X1 mediated Ca2+ influx in platelets ................ 77
Figure 2.9 Influence of ATP on P2X1 mediated Ca2+ influx with AAS ................ 78
Figure 2.10 P2X1 inhibition on ATP and ADP induced platelet aggregation ............ 81
Figure 2.11 P2X1 inhibition on ATP and ADP induced platelet aggregation ............ 82
Figure 2.12 ATP as a platelet antagonist and agonist ......................................... 83
Figure 2.13 Dual nature of ATP as a platelet antagonist and agonist ...................... 84

Figure 3.1 P2X architecture based on trimeric AP2X4-B receptor ...................... 102

Figure 3.2 Heterotrimeric model of P2X1 receptor ........................................ 103
Figure 3.3 Structures of ATP and oc,[3—methy1ene ATP .................................. 105
Figure 3.4 Agonist induced PRP aggregation .............................................. 112
Figure 3.5 PRP aggregation with ATP, ADP and a,B—methy1ene ATP ................ 113
Figure 3.6 Pyruvate kinase reaction ......................................................... 115
Figure 3.7 The effect of PEP-PK reaction on PRP aggregation ........................ 1 15
Figure 3.8 Optimization of P2Y1 inhibition on ADP and ATP induced platelet
cytosolic Ca2+ increase .......................................................... 1 19
Figure 3.9 Platelet cytosolic Ca2+ increase upon addition of ADP and ATP .......... 121

Figure 3.10 ATP breakdown profile of 5 11M ATP treated with 0.5 U/ml apyrase ...... 122
Figure 3.11 ATP breakdown profile constructed using the fitted decay curve .......... 124

Figure 3.12 Differentiation of the magnitude of ATP and (LB-methylene ATP
induced, P2X1 mediated cytosolic Ca2+ influx ............................... 125

Figure 3.13 Averaged normalized Ca2+i increase in ADP, ATP and ot,[3-MeATP
treated platelets in the presence of 0.5 U/ml
apyrase ........................ 1270

Figure'4.1 Proposed structure ofCFTR protein which contains 1480 amino acids... 137

Figure 4.2 Differentiation of myeloid progenitor cells into different mononuclear
cell types ........................................................................... 139

Figure 4.3 Airway epithelium in dysfunction in cystic fibrosis .......................... 143
Figure 4.42 Carruthers model of ATP controlled GLUTI modulation in RBCs.. . . . 1 46

Figure 4.5 Proposed signal transduction pathways associated with stimulated
glucose transport and CF TR activation ....................................... 149

Figure 4.6 Effect of CF TR inhibition on RAM glucose uptake .......................... 155

Figure 4.7 Influence of CF TR inhibition on opsonized bacteria particle
phagocytosis .......................................................................................... 1 57

xi

Figure 4.8 Effect of CFTR inhibition on RAM glucose uptake and the

enhancement of glucose transport by Zn2+ activated c-peptide ......

Figure 4.9 Confocal fluorescence microscopy images captured with 100X oil
immersion objectives. A dramatic reduction in phagocytosis was

observed after CFTR inhibition .............................................

Figure 4.10 Confocal macro fluorescence microscopy determination of

RAM-opsonized bacteria particle intake ...................................

Figure 4.11 Fluorescence imaging for the evaluations of RAM phagocytosis,

(Look at the Figure 12 for detailed figure caption) .......................

Figure 4.12 Fluorescence imaging for the evaluations of RAM phagocytosis. . . . . .

xii

....159

....160

....162

...164

165

CHAPTER 1

1.1 INTRODUCTION

The average adult has about five liters of blood circulating in the vascular
system. While traversing through vessels, blood is acting as a multifimctional tissue
which delivers essential elements such as nutrients and oxygen and removes harmful
wastes such as C02 and urea. Blood is the grth medium for the body’s cells and by
transporting nutrients from the digestive system, delivering regulatory hormones and
distributing disease fighting substances, blood circulation maintains the regular
dynamics of the body. Blood is considered as the largest tissue of the body containing

multiple cell types and substances.

Blood plasma is a yellow liquid in which the blood cells are suspended. Plasma
contributes 55% of the total blood volume. Although 90% of plasma is water by
volume, it contains proteins (8%), glucose, electrolytes, clotting factors, hormones and

carbon dioxide.

Cells are 45% of the total volume of the blood. The cellular component of the
blood is mainly red blood cells (RBCs), although other cell types are important. For an
example, white blood cells protect the body from infections and destroy unwanted cells
and particulate matter. There are two types of white blood cells: granulocytes
(neutrophils, basophils and eosinophils) and agranulocytes (monocytes, lymphocytes
and macrophages). Granulocytes differentiate in to mature cells that are responsible for

1

specific immune responses (T lymphocytes) and phagocytosis (macrophages).
Monocytes leave the circulation and become tissue macrophages. Macrophages are the
front line defense in the lungs against pathogen and other harmful materials. Platelets
are smaller anucleated cells that mediate the blood clotting mechanism, thereby
preventing blood loss during vascular injury. Although white blood cells and platelets
are smaller in percentage, they are equally as important as RBCs for a proper integrated

body function.

Either one or several components of blood interact with any given cell in the
body at all the times. Therefore any defect in function/structure of blood components
can influence one or many tissues. On the other hand, an abnormal behavior or function
of tissue or organ mostly manifests itself in blood. This is why blood is such an

important component of medical diagnostics.

Many diverse disease conditions, where entirely dissimilar abnormalities occur,
different cell types, tissues or an organ, give rise to common pathological conditions.
For example; in some disease states, platelets are hyperactive and unusually susceptible
for aggregation. Hyperactive platelets are prominent in diseases such as diabetes,
multiple sclerosis and cystic fibrosis and cause an imminent threat of thrombus
formation by blocking blood vessels. When analyzing the behavior of other cell types
interacting with hyperactive platelets, our group and others have observed that
bioavailable ATP released from RBCs are abnormal in these diseases due to either

decreased deforrnability (in diabetes) or increased cell lysis (multiple sclerosis).M

Investigations in recent years by the Spence group and others suggest the
potential involvement of RBCs in the regulation of energy in terms of glucose
consumption and ATP production/release. Being the most exposed cell type to the
abundant glucose absorbed from the digestive system, RBC glucose uptake may play a
major role in glucose homeostasis. Also, the significance of bioavailable ATP (mostly
derived from RBCs) in the circulatory system serves as a key regulator for
vasodilatation and platelet inhibition through stimulating production nitric oxide (NO)

in the cell types.

Although ATP was considered primarily as a simple energy molecule two
decades ago, its importance as a secondary messenger and a ligand in purine nucleotide
binding receptor signaling (purinergic signaling) has recently made it an important
molecule in biological research. Despite the fact that ADP has long been recognized as
one of the main platelet function regulators and ADP associated signal transduction
pathway currently being the main target for treating hyperactive platelets (antiplatelet
therapy), investigations described in this dissertation will demonstrate the significance
of bioavailable ATP as a potent platelet function regulator. This would also provide
another key signal transduction pathway for novel antiplatelet therapy that may
eventually help in preventing and managing cardiovascular diseases. Here, we also
investigated the ATP mediated regulation of macrophage glucose uptake and their
phagocytic ability which is very important in preventing lung infections in especially in
diseases like cystic fibrosis (CF). We believe that, molecular level picture of
macrophages function described in this dissertation will be able to enhance the

strategies in fighting against cystic fibrosis.

1.2 PLATELET STRUCTURE, FUNCTION AND REGULATION

1.2.1 Platelet structure

Platelets, or thrombocytes, are anucleated irregular shaped cellular bodies that
are 2-4 pm in diameter. Platelets are derived from megakaryocytes during hematopoisis.
Although the basic function of platelets is hemostasis, the prevention of bleeding during
vascular injuries, they are also critical in numerous and diverse pathological processes
including thrombus formation in cardiovascular disease, hemorrhage, inflammation-
immune disorders and tumor metastasis.5 Platelets are of great interest in the bio-
medical research community as they play a significant role in coronary artery disease
and other common diseases including stroke, peripheral vascular disease and diabetes.5
Although platelets have a short lifespan of 10 days, they are the source of many cellular
growth factors and cellular messengers. There are about 150,000 to 400,000 platelets
per micro liter of blood in a healthy person.5’6 Platelets in large excess (thrombocytosis)
or platelet hyperactivity could possibly lead to unnecessary thrombus formation leading
to fatal conditions. Similarly, platelets in low numbers (thrombocytopenia) or decreased

platelet activity can cause severe and fatal hemorrhagic situations.7

Platelet structure can be divided in to three key components: membrane
structure, cytoskeleton, and the secretory organelles system (Figure 1.1). The outermost
part of the platelet membrane is mainly comprised of glycoprotein and is collectively

called glycocalyx. Transmembrane and sub membrane structures mediate responses to

platelet stimulation, immune responses and express specific antigenic characteristics.
The platelet cytoskeleton is a detergent-insoluble, intricate three-dimensional network
of filamentous proteins. The filaments are composed of three types of polymeric
proteins called microfilaments, intermediate filaments and microtubules.8'9 Changes in
actin and myosin filaments associated with increased platelet cytosolic calcium (Ca2+i)
concentrations lead to platelet shape change and aggregationlo’”

Platelet alpha granules, dense granules and lysosomes are the main secretory
organelles.8 Alpha granules consist of a large spectrum of components that regulate a
diverse range of functions.12 These components include coagulation proteins
(fibrinogen, factor V-proaccelerin), soluble adhesion molecules (von Willebrand factor-
vWF, vitronectin), growth factors (PDGF -plate1et derived growth factor, epidermal
growth factor), protease inhibitors (plasminogen activator inhibitor-1, az-antiplasmin),
and membrane adhesion molecules (P-selectin, GPIIb/IIIa). Small molecules
(ADP/ATP-adenosine diphosphate/adenosine triphosphate) and important ions (calcium
and magnesium) that mediate platelets activation are stored in platelet dense granules.”

16 Secretory organelles are responsible for control of platelets adhesion, cell growth,

and cellular signaling cascades.'7

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1.2.2 Hemostasis: platelet in bleeding prevention

Hemostasis is the mechanism that prevents bleeding during vessel wall injury.
Upon vessel wall injury, an immediate reflex of vessel constriction and platelet
adhesion to the exposed collagen occurs to reduce blood loss. Subsequent activation and
platelet granular release of ADP, TXAz and serotonin further activate platelets and
recruit more platelets to the site of injury. Serotonin constricts the vessel, thereby
facilitating the formation of platelet plug.

Coagulation is the final step of the hemostasis and has two mechanisms,
extrinsic and intrinsic. In brief, the extrinsic mechanism starts with the release of factor
III18 from damaged tissues that activate factor VII by converting it to factor VIIa with
the help of tissue factor, a protein present in sub endothelial matrix.19 The intrinsic
mechanism forms the active factor XI with a cascade of reactions.20 Eventually, both
intrinsic and extrinsic mechanisms work together to activate factor X.21’22 Both active
factor VII and active factor XI will promote cascade reactions, finally activating factor
X. Active factor X, along with factor III, factor V, Ca2+, and platelet thermoplastic
factor (PF3), will activate the prothrombin activator that converts prothrombin to
thrombin.23’24 Finally, thrombin converts fibrinogen to fibrin and forms a loose
mesh.25’26 This loose mesh of fibrin network is covalently modified by factor XIII and

converts to a dense mesh of fibers, trapping activated platelets and red cells.27’28

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1.2.3 Mechanisms of platelet function regulation

Platelet activation, aggregation and inhibition are highly regulated mechanisms.”32
Extensive activation or delayed response to activation stimuli would be fatal. Platelet
activation is a multi step process involved in multiple signaling cascades triggered by
binding soluble factors (mobilized by the vascular injury) or adhesive molecules to the
platelet surface. Most platelet activators (agonists) bind to the platelet membrane
receptors coupled to G proteins, which initiate the mobilizations of secondary
messengers leading to platelet activation. Purine nucleotides such as ADP and
adenosine have been recognized as critical platelet agonists. ADP released from initial
collagen-induced platelet activation propagates further platelet activation and
aggregation, until platelet inhibitors such as NO dominate and inhibit the process.

In brief, platelet dense granular ADP release dramatically intensifies platelet
activation and aggregation inducing the activation of P2Yl and P2Y12, two G protein
coupled receptors (GPCR). Activation of Gq, a smaller G protein, coupled to P2Y1
promotes the hydrolysis of Phospholipase C (PLCB) and generates inositol (1,4,5)-
triphosphate (1P3) and diacylglycerol (DAG).9 1P3 mobilizes the intercellular calcium
stores to initiate activation of the Cay-dependent integrin; GPIIb/IIIa. DAG activates
protein kinase C (PKC), phosphorylates myosin light chain kinase (MLCK) and
eventually activates GPIIb/IIIa.33 Activation of P2Y12 has an inhibitory action on
adenylyl cyclase and hence reduces the formation of cAMP (Figure 1.2).‘7 Binding ATP

to its receptor, P2X, can also increase the cytosolic calcium, Ca2+i, by allowing

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extracellular Ca2+ to flow in to the cytosol (Ca2+ influx) through the opened receptor
channel. Due to the issues related to the fast desensitizing nature of this receptor and
ATP’s ability to easily break down to ADP, the role of ATP in platelet aggregation has
not been completely explained. However, hyperactive platelets in conditions where
abnormal levels of ATP exist suggest a possible involvement of ATP in the platelet
aggregation process.

Platelets maintain continuous interaction with the endothelium and RBCs that
help to regulate the vascular properties. We have previously shown that ATP released
from RBCs while traversing through a resistance vessel mimic can elicit NO production
in circulating platelets and endothelial cells."34 It has also been reported that people
with diseases where RBCs release less ATP upon deformation or pharmacological
stimuli, have hyperactive platlets.2’4’35'37 A possible implication of this decreased RBC-
derived ATP release arises when considering that ATP is a recognized stimulus of NO
production in platelets and endothelium. Importantly, NO is a well-established inhibitor

of platelet aggregation.3840

Therefore the interesting features of the purinergic platelet activators are their
ability to stimulate NO production and induce platelet activation at the same time.
Natural platelet antagonists such as NO and prostacyclin (PGIZ) up regulate the
production of cyclic adenosine monophosphate (CAMP) and cyclic guanosine
monophosphate (cGMP). PG12 activates Gs coupled IP receptor, stimulates adenylyl
cyclase and increases the concentration of CAMP.41 NO increases cGMP and thereby
increases the cAMP concentration as illustrated in Figure 1.2.42 NO in the circulation is

mainly generated through endothelial nitric oxide synthase (eNOS) (NOS III, a

11

NO + Citruline

  

Red uctase

Figure 1.4 Electron transfer pathway and NO production in eNOS. Electrons (e-) are
donated by NADPH to the reductase domain of the enzyme (eNOS) and
proceed via FAD and FMN redox carriers to the oxygenase domain. There
they interact with the heme iron and BH4 at the active site to catalyze the
reaction of oxygen with L-arginine, generating citrulline and NO as
products. Electron flow through the reductase domain requires the
presence of bound calcium-calmodulin (Ca2+/CaM).43

constitutively expressed NOS) and the main source is the endothelium. Other cell types
with eNOS in the cardio-vascular system are red blood cells (RBCs), platelets, cardiac
myocytes and megakaryocytesfu’44 Endothelium derived NO prevents platelet adhesion
to the endothelium and helps in vascular tone regulation. However there is also a
considerable amount of evidence suggesting that platelet derived NO plays numerous
roles in platelet function and vascular tone regulation.1"‘5'49 NO life span in the
circulation is limited to 3-10 seconds.50 NO bio-reactivity should rapidly dissipate and
only therefore affects cells within close diffusible range of the site of production.
Therefore the diffusion-mediated delivery of NO from the endothelium to circulating
platelets may be limited implying that platelet derived NO should be significant in
platelet behavior. Therefore, purine nucleotides like ATP and ADP, which are stimuli
for NO production and platelet activation, may be able to inhibit platelet activation or

activate platelets depending on the local concentration of each.

eNOS regulation and NO production in platelets: Platelet eNOS is thought to be
regulated in a manner similar to the eNOS in endothelial cells, which is primarily
activated an increase in Ca2+i and the formation of the calcium calmodulin complex (Ca-
CAM). Ca-CAM binding facilitates electron transfer from the reductase domain to the
oxygenase domain of eNOS.51 The reductase domain consists of FAD and FMN
cofactors and transfers electrons from NADPH to the oxygenase domain. The
oxygenase domain has binding sites for heme and arginine and facilitates the conversion

of L-arginine to L-citrulline producing NO as a byproduct (Figure 1.4).43’52’53

13

It has been shown that increased NO production by the endothelium exposed to
shear stress is Ca2+ independent.54’55 Bradykinin induced endothelium NO production
can be blocked by calmodulin inhibitors and seems to be calcium dependent.S6 Also
ATP and ADP induced Ca2+i increase in platelets stimulate platelet NO production.
Therefore, eNOS is possibly regulated by both the Ca-CAM dependent mediated
electron flux as well as the shear stress induced activation of eNOS.54’57 eNOS is a
membrane associated protein and its localization depends on the co-translational
modifications and post translational modifications.55 This facilitates eNOS attachment
to golgi and caveolae regions. Golgi and caveolae are frequently subjected to
movements and therefore these localizations of eNOS provide more evidence for shear

induced eNOS activation.58

1.2.4 Platelet associated disorders

Platelet associated disorders can be can be attributed to unusual platelet
availability, unusual platelet function or both. Some of these conditions are acquired
while others are congenital. Broadly, defined conditions where unusually low platelet

”’60 while high count conditions are

counts prevail are called thrombocytopenia
classified as thrombocytosis. (”’62 Platelet function disorders are very diverse and
heterogeneous. The dysfunction can be a result of the receptors, signal transduction
pathways, granule contents, cytoskeletal proteins or platelet procoagulant activity.“66 A

detailed classification of platelet function disorders id shown in table 1.1.

14

Disorders with known or presumed defects intrinsic to platelets

 

Primary defects of
adhesion

(a) Bernard—Soulier Syndrome
(b) platelet-type von Willebrand disease

 

Primary defects of
aggregation

Glanzrnann thrombasthenia
Defects of secretion and/or signal transduction
(excluding granule deficiencies) affecting:
(a) platelet-agonist receptors for thromboxane A2, ADP or collagen
(b) thromboxane generation (impaired liberation of arachidonic acid, or
deficiencies
of cyclooxygenase-l or thromboxane synthase)
(c) G-protein activation
(d) phosphatidyl inositol metabolism defects, such as phospholipase C deficiency
(e) calcium mobilization
(1) protein phosphorylation

 

Disorders
affecting platelet
granules or their
contents

(a) d-granule deficiency, including forms with pigment abnormalities (e.g.
Hermansky—Pudlak Syndrome)

(b) gray platelet syndrome (a-granule deficiency)

(0) combined ad-granule deficiency (ad-storage pool deficiency)

(d) Quebec platelet disorder (increased platelet u-PA and a-granule protein
degradation)

 

 

 

 

Defects of platelet (a) Scott Syndrome
procoagulant (b) factor V New York
function
Defects in (a) MYH9-related disorders (formerly known as May Hegglin anomaly, Sebastian
structure or in syndrome, Alport’s or Fechner syndrome)
cytoskeletal (b) Wiskott—Aldrich Syndrome protein defects (includes some forms of X-linked
proteins thrombocytopenia)
(c) platelet spherocytosis
(d) macrothrombocytopenia with cytoskeletal abnormalities and absent shape
change
(6) microvesicle generation defects
Miscellaneous (a) Montreal Platelet Syndrome
disorders (b) X-linked macrothrombocytopenia (not linked to the WASP gene)

 

(c) Paris-Trousseau Syndrome

(d) thrombocytopenia with absent radius syndrome

(e) familial thrombocytopenia with predisposition to leukemia
(t) congenital amegakaryocytic thrombocytopenia

 

Disorders with defects extrinsic to platelets

 

 

 

von Willebrand factor deficiency
afibrinogenemia

 

Table 1.1 Classification of platelet disorders63

15

 

 

1.3 PLATELETS IN CARDIOVASCULAR DISEASES AND

ANTIPLATELET THERAPY

Platelets play a critical role in the development and pathology of cardiovascular
disease including atherosclerosis, coronary artery disease (CAD), hypertension, heart
attack and stroke. Conditions such as diabetes mellitus, of which a symptom is
hyperactive platelets,67 has been shown to have a strong link with cardiovascular
disease.68

Hyperactive platelets in arterial thrombus formation are one of the major
concerns in cardiovascular therapy.”71 Antiplatelet drugs are frequently used to reduce
the platelet hyperactivity and the cost of antiplatelet therapeutics in the year 2005 was
US$ 6.2 billion.72 Existing pre and post thrombosis treatments commonly, known as
antithrombotic drugs, can be classified in to three basic classes: anticoagulants,
thrombolytics and antiplatelet.73 Anticoagulants are used to prevent blood coagulation
mechanisms often used in blood transfusion and surgical procedures. Thrombolytic
drugs are used to dissolve thrombi after a thrombotic condition in the circulation such as
myocardial infarction or ischemic strokes. Out of the three, antiplatelet therapy is of

greater concern as it is used not only to prevent the initiation of thrombus formation but

also regulate platelet mediated inflammatory conditions.

16

1.3.1 Antiplatelet therapy associated with purinergic signaling

Despite the fact that current antiplatelet therapeutics serve remarkably in
vascular disease management, there are limitations due to drug efficacy in different
clinical circumstances. Lack of direct evidence for cardiovascular event prevention and
insufficient signal transduction information of currently marketed antiplatelet drugs are
the major concerns in novel antiplatelet target identification and drug development.
Current antiplatelet drugs can be broadly categorized to four different groups based on
their targeted signal transduction pathway: cyclooxygenasel (COXl) inhibitors, P2Y12
inhibitors, orIIBIII inhibitors and PDE inhibitors.71 Except COXl inhibitors, the others

directly regulate purinergic signal transduction pathways.

P2Y12 inhibitors: P2Y12 is GPCR associated with Gai which inhibits adenylyl cyclase

upon ADP binding, regulating cAMP generation. Inhibition of this receptor results in I
increased cytosolic cAMP, which eventually inhibits Ca2+i mediated platelet activation
and aggregation.”74 Even under P2Y12 inhibition, P2Y1 still mediates platelet dense
granular Ca2+ mobilization leading to Ca2+i increase. Increased adenylyl cyclase activity
and subsequent CAMP increase results in Ca2+i granulation preventing OLIIBIII
activation. An example of a P2Y12 inhibitor is ticlopidin, an older drug belonging to the

thienopyridine family, it was later replaced by Plavix (clopidogrel).75’76

PDE inhibitors (dipyridarnoles): Clinical usage of dipyridamole started in the early

19605 as a coronary vasodilator. Dipyridarrlole’s ability to inhibit platelet adhesiveness

17

to glass (ex-vivo) in patients with coronary artery disease led Boehringer Ingelheim to
introduce it as an antithrombotic agent.77 By inhibiting the enzyme cyclic guanine
monophosphate (cGMP) phosphodiesterase (PDE), bipyridamole inhibits the
breakdown of cGMP and cAMP, resulting in increased levels of CAMP and cGMP.7"78‘
80 Also combinatorial therapy of bipyridarnole in combination with acetyl salicylic acid

has been found much more effective as an antiplatelet agent than individual therapies.“

aIIflIII inhibitors: Glycoprotein IIb/IIIa inhibitors prevent platelet aggregation and
adhesion by preventing fibrinogen, vWF and fibronectin binding to OLIIBIII. GP IIb/IIIa
heterodimeric protein belongs to the family of integrins consist of a 02- and [33-
subunit.82 Binding three or four cations (Ca2+) to the (12 sub unit initiates
conformational changes required to stabilize the GPIIb/IIIa heterodimer. Ability of
GPIIb/IIIa antagonists to bind to both resting and activated platelets is a very important
property to retard ongoing platelet activation and prevent activation initiation.83
GPIIb/IIIa inhibitors can broadly be divided two categories, inhibitors with affinity to

the receptors and competitive inhibitors that compete with natural ligands such as

fibrinogen for binding.84

1.3.2 Failures/insufficiency in current antiplatelet therapy- necessity of new drug

targets

Existence of hyperactive platelets in entirely different clinical situations such as

type 1 and type 2 diabetes, cystic fibrosis, and primary pulmonary hypertensiongs'gg,

18

may explain the lack of universal antiplatelet therapeutics and failures of current
therapeutics in some clinical situations. For an example, aspirin and clopidogrel
resistance in diabetes is a growing concern in the clinical community.”92 Although
there are different explanations for this phenomena, it has never been viewed as
preventing ADP-induced essential NO production. In diabetes, RBCs are less
deformable and release less ATP in to the circulation.35 Lack of bioavailable ATP leads
decreased NO production in endothelial cells and in platelets, making platelets more
susceptible to activation.

When clopidogrel binds to the P2Y12 ADP receptor, Ca2+i concentrations may
be decreased even below the basal level. Although clopidogrel would work as an
effective antiplatelet agent in a clinical situation where bioavailable ATP is abnormally
high, in a diabetes-like situation, it would make the situation worse. Therefore,
antiplatelet therapy should be developed as customized therapy after evaluating certain
parameters like RBC deformability and ATP bioavailability. However, this only can be
achieved by identifying new targets of signal transduction pathways that are involved in
platelet function. ATP activated P2X signal transduction pathway has never been

investigated as a potential drug target in antiplatelet therapy.

19

1.4 A BRIEF INTRODUCTION TO THE EFFECT OF DEFECTIVE CFTR

ON MACROPHAGE FUNCTION IN CYSTIC FIBROSIS

Cystic fibrosis (CF) is a disease where bacterial infections in the lungs and
associated inflarnmations are the main causes of death (CF lung disease).93 Lung
macrophages (alveolar macrophages) are mainly responsible in protecting lungs from
pathogen invasion and harmful substances.94 CF is a result of a genetic mutation in the
fibrosis transmembrane conductor regulator (CFTR) gene and its product, CFTR
protein.95 To date, CFTR mutations in CF macrophages have not been correlated to

alveolar macrophage function linked to CF lung disease.

A portion of this dissertation describes the correlation between cellular glucose
uptake and subsequent ATP release that influences the phagocytosis process of alveolar
macrophages, providing the significance of defective macrophage-CFTR in this
process. CFTR is a protein directly facilitating or assisting cytosolic ATP release to the

extracellular matrix.

1.4.1 CF pathogenesis and CF TR

Cystic fibrosis (CF) is one of the most common lethal autosomal recessive
disorders especially, in the Caucasian population96. Statistical data shows that nearly 1

in every 2500 Caucasian children is diagnosed with CF in their early childhood. People

20

of European descent carry one gene for CF, making it the most common genetic disease

among this group.

CF is a genetic disorder caused by a mutation in a gene, Cystic Fibrosis
Transmembrane Conductance Regulator or commonly known as CFTR gene96. CFTR
protein is identified as a cAMP regulated chloride channel present in the cell
membrane97’98. Certain mutations of the CFTR protein make it inactive, which affects
its functions, including chloride and adenosine triphosphate (ATP) conductance. ENaC,
an epithelial sodium ion channel, regulated by CFTR is essential for the re-absorption of
electrolytes in the sweat duct and the respiratory epithelia”. Disturbances to this re-
absorption process in CF result in complications in the epithelial lining of organs such
as the lungs, pancreas, intestine, liver, male reproductive tract, and sweat glands. As
water follows the same direction as salts,rCF patients are found to have airways with

unusually dehydrated mucous secretions that obstruct the luminal space.

Lungs are the largest exposed organs in the body to the outside environment.
Innate immunity is the mechanism that keeps lungs free from infections, which is
primarily governed by macrophages. Defective CFTR in pulmonary epithelia disturbs
the salt balance, reduces airway surface liquid (ASL) hydration.100 Reduction of ASL
volume leads to accumulation of thick mucus layer on the top of the epithelia and
prevents airways surface clearance making it more vulnerable to infections. As a result
of decreased mucus clearance, CF airways become fertile ground for bacteria and
viruses. Bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa have a
fertile environment in these mucus layers and grow into infections and cause

inflammations in CF lungsml’102 These infections are the prime cause of death in people

21

with CF, although advancements in CF therapy has significantly increased the life span

of CF patients.103

1.4.2 Defective CFTR and alveolar macrophages

Macrophages are mononuclear phagocytes derived from blood monocytes.'04’106

Alveolar macrophages are the front line defense system in the lungs against pathogen
and particulate matter invasion. Other than its main function, phagocytosis, alveolar
macrophages are involved in numerous functions, such as bactericidal activity, antigen
presentation, turnor cytotoxicity, removal of aged or damaged cells, repair of injured
tissue, bone resorption, and special lipid metabolism.105 A large number of macrophage
destruction due its defense activities is predicted by considering 1.5 X 106 monocyte
production in mouse bone marrow a day.107

Ingestion of pathogens or phagocytosis by alveolar macrophages is the one of
the foundation mechanisms of pulmonary innate immunity against pathogen invasion.
There are conflicting evidences in the literature about the effect of ‘ CFTR on
macrophage bacterial killing ability. One such study demonstrated the requirement of
CFTR for phagosomal acidification and subsequent bacterial killing in 1996.108 Similar
kind of studies performed later denied the involvement of CFTR in macrophage
phagosomal associated bacterial killing.”109 In brief, the role of defective CFTR in
alveolar macrophage on its function as well as if such dysfunction exists, the effect of

that particular macrophage dysfunction on CF pathogenesis has not been explained.

22

It has been shown that the macrophage glucose transport mainly mediated
through facilitative glucose transporter GLUTI and P. aeruginosa ingestion is glucose
dependent.110 Reduced glucose uptake in any cell should results in decreased reducing
power which may results in reduced bactericidal ability in macrophages. Failure to
release ATP into the extracellular matrix due to the CFTR dysfunction can create a
negative feed back on glucose uptake and also it has been shown that the accumulation
of ATP in the cytosol leads to GLITl inhibition.lll Theoretically it is clear that
dysfunction if CFTR in CF may result in reduced ATP release which eventually inhibit
macrophage glucose uptake leading to the decreased phagocytic activity. In an attempt
to reconcile these diverse findings and to mimic the alveolar macrophage CFTR
dysfunction in CF lung disease, investigations in chapter 4 were performed. Here we
examined the effect of CFTR inhibition on rabbit alveolar macrophage (RAM)
phagocytosis and glucose transport. Previously developed glucose sensitizer for RBCs,
Zn activated C-peptide,112 has been used to investigate whether it can enhance the
glucose uptake and improve the opsonized bacteria particle ingestion by alveolar

macrophages.

1.5 ANALYTICAL TOOLS TO EVALUATE PLATELET AND

MACROPHAGE FUNCTION

Although the problems that are investigated in this dissertation are biological,
highly selective analytical tools described here enabled us to take measurements earlier

described as difficult measurements in the literature. The reason behind our success

23

may be due to the lack of customizing ability in most of the automated instruments
available in conventional biomedical laboratories. Here, we briefly describe the basic
principles of analytical techniques used to evaluate key components of the signal
transduction pathways in platelets and macrophages such as ATP, intercellular NO,

Ca2+i, platelet aggregation and cellular glucose uptake.

1.5.1 Chemiluminescence assays for cellular ATP release measurements.

Evaluation of standard ATP assay and the ATP released from platelets or RBCs
were conducted with the luciferin/luciferase chemiluminescence reaction related to the
reaction shown in Figure 1.5.1 '3 ATP concentration is proportional to the
chemiluminescence intensity. The sensitivity of the assay is enhanced by addition of 2
mg of D-luciferin (Sigma, St. Louis, M0) to the crude firefly extract (Sigma, St. Louis,
MO). The luciferin/luciferase solutions were prepared by diluting the luciferin in 5 m1
of distilled; deionized 18.2 M!) water (DDW) and adding it to a vial containing 100 mg

luciferase. The luciferin/luciferase mixture was prepared on the day of use.

1.5.2 Fluorescence determination of intracellular NO and Ca2+i

Fluorescence imaging and spectrosc0py is being used widely to study living
cells due to its sensitivity, ease of use, rapidity, reproducibility and adaptability.

Detection of picomolar concentrations of analytes in a single cell is currently achievable

24

with the appropriate fluorescence probe and the instrumentation.114 A cell permeable
florescence probe, DAF-FM diacetate (4-amino-5-methylamino- 2', 7'-
difluorofluorescein diacetate) has been used to detect intracellular NO production in
platelets. Once in the cell, cellular esterases cleave the ester moiety and forms DAF-
FM, which is the active probe. Although the quantum yield of DAF-FM is ~0.005, the
fluorescence intensity increases about 160-fold, to ~0.81, after reacting with nitric

oxide.1 15

The ability of use in several instruments such as flow cytometers,
microscopes, fluorescent plate readers and fluorometers with excitation/emission
maxima of 495/515 nm, make DAF-FM DA a versatile fluorescence probe.

An acetomethyl ester of fluo 4, fluo 4 AM, (4-(6-acetoxymethoxy-2,7-difluoro-
3-oxo—9-xanthenyl)-4'-methyl-2,2'-(ethylenedioxy)dianiline-n,n,n',n'-tetraacetic acid
tetrakis (acetoxymethyl)ester) has been used to determine Ca2+i in platelets. The
quantum yield of DAF-FM is ~0.14, Kd for Ca2+ of 390 nM, the fluorescence intensity
increases about 100-fold after reacting with Ca2+ and the Ca2+ free indicator in non

fluorescent (Figure 1 .6). 1 16’1”

25

HO
D-Luciferin

N\ s
I H ,COOH
, \l

H

 

 

 

ATP
M g2+
l
g <\: xCXDOAMP
+PPi
D-Luciferin sdenylate
O2

 

HO
Oxyluciferin

l
N s
H +AMP+C02+hv
S N
0

Figure 1.5 Schematic representation of luciferin luciferase bioluminescence
reaction113

26

esterases

   

DAF-FM
DAF'FM DA C611 membrane Weakly fluorescent
Non fluorescent cell permeate

 

 

DAF-FM T
Fluorescent

 

Figure 1.6 Fluorescence probes used in the determination of a) NO, and b) Ca2+i, their

cell permeation mechanism and active probe formed after cellular esterase
mediated cleavage of diacetate moiety in DAF FM-DA and
acetomethylester moiety in fluo 4 AM1 15" 16'1”

27

1.5.3 Light transmittance aggregometry for platelet aggregation

Platelet aggregometry is the most frequently used technique to screen patients
for inherited or acquired defects of platelet function ever since its introduction in 1962
by Born “8’119.Optical aggregometry measures the increase in light transmission
through platelet-rich plasma or washed platelet suspended in an appropriate buffer, that

occurs when platelets are aggregated upon agonist addition (Figure 1.7).120

(aggregation at start-aggregation maximum)
Maximum aggregation [%] = 100 % X

 

(aggregation at start-platelet poor plasma)

1.5.4 Liquid scintillation counting for cellular glucose uptake measurements

Liquid scintillation counting (LSC) was introduced in 1950 with the idea of
improving counting efficiency of low energy [3 emitters. Currently, LSC offers the
counting efficiencies of the order of 55 % for 3H and 95% for 140121 In LSC, fraction of
the energy of ionizing radiation emitted by the isotope is converted in to light by the
scintillation cocktail (Figure 1.8). Scintillation cocktail basically consists of scintillator
molecules dissolved in an organic solvent. Liquid scintillation cocktail composition
should enable efficient transfer of energy between the solvent and scintillation
molecules as well as coexistence of the aqueous radioactive solution with the organic
solvent. Earlier benzene and toluene were used as the solvent but less toxic solvents like

XYIene or pseudocumene are being used. In the last twenty years, a new

28

2)

  
 

Diode light Semiconductor

detefors

\l (MIN

 

b)
Addition of antagonists and/
or PEP-PK

-
Aggregation -
measurement start -

Test >

channel 1 Base line correction *7

 

Agonist 1 addition

 

 

  

Cloudy platelet
- o - a
suspensron or PRP a 0L 0 A» transmittance ......... - ,
37 °C am out}... ...".ng .
- ‘ «WWit't‘i’ithi‘tiat‘Wl‘v‘ithii" :
Reference 9 or 100 % transmittance
channel. 4 gtafi'riitfiry‘in‘ir ”1.1%, £§%;#cq';l_ s’i”“flligtll‘~,

Buffer or PPP at 37 °c ‘

Aggregation
measurement start

-
Base line correction I

Agonist 2 addition m

channel 2

 

Figure 1.7 a) Schematic diagram of a Chronolog 490-2D aggregometer b) Working
principle of an aggregometer, in brief, tests in each channel are considered

as 100 % translucent suspensions compared to the reference sample which
is considered as 100 % transparent.

29

-generation of aromatic solvents like di-isopropylnaphthalene (DIN), phenylxylylethane
(PXE) or dodecylbenzene (LAB) has been developed. When radionuclide decay
radiation passes through the solvent, incident electrons, or secondary electrons created
by the interaction of radiation with the solvent. Approximately 10% of the energy is
transferred to excited singlet and triplet states. Relaxation of excited singlet state to very
quickly to their ground state S]. Triplet states lose their energy by internal conversion
and cannot directly emit light. The energy migrates from one solve'nt molecule to
another on a sub-nanosecond time scale until the energy is trapped by a solute molecule
or dissipated as heat. Energy trapped in primary scintillation molecules than transfer to
the secondary scintillation molecules which shift the wavelength and emit the light in

the detection range of the PMT.(adapted from reference 122)122

30

Scintillation cocktail

 

 

 

Solvent Scintillatiors
[3 -interaction

3 OO

toluene

I 2- [4- -biphenylyl]- -5- [4- tert-butylphenylj- -1 ,3 4- oxadiazole

Energy transfer
phenyl xylylethane

/2, 5- diphenyloxazole

_ Light emission
/ naphthalene

 

Photomultiplier tube

 

Figure 1. 8 Basics of liquid scintillation counting (LSC), the solvent portion of an LSC
cocktail comprises from 60 - 99% of the total solution When a
radioisotope dissolved in the cocktail undergoes an emission event, solvent
molecules act as efficient collectors of energy. The energy from these
molecules passes back and forth among the solvent ring systems, allowing
efficient capture by dissolved phosphors (primary and secondary
scintillation molecules). Primary Scintillatiors provide the conversion of
captured energy to the emission of light which is detected at the
photomultiplier tube.

1.6 THESIS OBJECTIVE

1.6.1 Role of ATP and its receptor, P2X1, in platelet function

Purinergic signaling plays a very important role in platelet homeostasis.
Particularly the complex multiple signal transduction pathways associated with ATP
gated P2X1 ionotropic (conduct ions) channel family receptors, and ADP gated
heptahellical metabotropic( do not conduct ions) receptors are key regulators of platelet
functionm However, the involvement of the ATP-gated P2X1 receptor in platelet
homeostasis is controversial. ATP induced platelet shape change and ATP upregulated-
collagen induced platelet activation reported in the literature are critical evidence for
ATP-P2X1 aggregatory effect on platelets.3’124‘126 In vivo studies conducted on the
P2X1 knockout mice (PZXl'l') has shown reduced mortality due to systemic
thromboembolism (a Lodgement of a blood clot causing blockage of a blood vessel),
decreased size of mural thrombi (a blood clot in a large blood vessel) upon laser-
induced vessel wall injury and the rapid thrombus clearance indicating the importance

of P2X1 in cardiovascular diseases.127

There are specific difficulties associated with P2X1 receptor function assessment that
prevents a complete understanding of its behavior. The major constraint is the ligand
occupied rapid desensitization of the receptor. In the presence of ATP, the reported
desensitization time of the P2X1 receptor is 47-107 msm’129 It has been shown that
stirring washed human platelets at 37 °C for 10 minutes causes a release of 10 attomoles

of ATP per platelet without adding any agonist.130 Therefore it is fair to estimate that

32

the ATP release from resting washed platelet over a course of time can easily
desensitize its own P2X1 receptors. One of our major concerns on previous P2X1-ATP
functionality analysis was the utilization of a,B—methylene ATP, a stable analogue of
ATP. Although the characterization data for cab—methylene ATP on human urinary
bladder cell showed high specific binding to P2X1 compared to ATP( binding affinity
order, a,B-methylene ATP > [3,y-methylene ATP > suramin > 2-methylthio ATP > ATP

> ADP >> adenosine),13 I

the subsequent receptor recycling and the overall receptor
kinetics have not been explored well. Furthermore, the inability of suramin, a potent
P2X antagonist to overcome the receptor mediated cab—methylene ATP induced
depolarization of P2X1 in rat isolated vagus nurves explains that, this unusual binding
of a,B—Methylene ATP which may retard the receptor function and the recycling.132
The dilemma of the use of apyrase to keep the P2X1 receptor in the functional state and
the use of apyrase-sensitive ATP to stimulate P2X1 encouraged scientists to use
a,B-—methylene ATP, instead of a form of ATP quite similar to the bioavailable ATP. It
has been shown that a,B—methylene ATP ATP is able to induce a small calcium influx
just enough to elicit a platelet shape change but not the aggregation.83 '133’134

Based on these findings, and others reporting a dual nature of ATP and its
effects on platelet function, we anticipated that ATP has an anti-platelet effect at low
levels of ATP due to its ability to increase platelet NO production and an aggregatory
effect at higher concentrations of ATP due to increased Ca2+i.13 5 ’136 In addition we also

provide data suggesting that the ATP gated P2X1 receptor activity is a major contributor

to platelet aggregation.

33

In the present study also we investigated the behavior or P2X1 in the presence of
exogenous ATP very closely. We have optimized conditions to complete platelet P2Y1
receptor inhibition, employed an enzymatic system to eliminate the accumulation of
ADP due to ATP break down, and sensitize the platelets P2X1 receptor to evaluate its
natural performance in the circulation. We also extended our study to evaluate the
clinical significance of bio available ATP on platelet function and as well as the cross

talk between P2X1 and P2Y types receptors.

Findings of the investigations described in this chapter 2 and 3 of thesis would
bring more attention to the underestimated clinical significance of bioavailable ATP and
the P2X1 receptor. We also believe that our finding will be very useful in future patient
specific and customized antiplatelet therapy development. Due to overwhelming
dependency of platelet filnction with RBCs and endothelial cells described here may be

useful for platelet function regulation by RBC targeted therapeutics.

1.6.2 Inhibition of CFTR on glucose uptake and phagocytosis by alveolar

macrophages

In recent years, CFTR has been widely investigated as a dynamic regulator in the
cardiovascular system due to its involvement in RBCs derived ATP releasez’137
HoWever, in most of the CF investigations, CF pathology has been linked to the
defective salt balance due to CFTR dysfunction in the airway epithelia.102 But, it is

impossible to explain the pathophysiology of CF lung disease associated with extensive

bacterial infections and inflammations only by considering defective CFTR in lung

34

epithelial cells. It is dubious that CF lung disease is almost independent of the alveolar
macrophage function, which is generally considered as the front line defense in the
lungs against pathogen and particulate material intake. Therefore the investigations
described in the chapter 4 of this dissertation were primarily designed to examine the
effect of the CFTR dysfunction in alveolar macrophage fimction.

A substantial decreased phagocytic activity has been observed when RAM
CFTR is inhibited. This was just a result of the initial effort of correlate CF
pathogenesis with alveolar macrophage activity. It was speculated that the lack of
CFTR activity retards the cytosolic ATP clearance and led to ATP accumulation in the
macrophage cytosol. Accumulated ATP may result in GLUTl inhibition and lead to
decreased macrophage glucose uptake. Therefore, CFTR inhibition on macrophage
glucose uptake was investigated. Here, CFTR inhibition has retarded the macrophage
glucose uptake to a large extent which is similar to the reduction observed in
macrophage bacteria particle ingestion after CFTR inhibition. At this point, it is clear
that CFTR function is essential for cellular energy dynamics and CFTR failure leads to
reduced glucose uptake eventually retarding CFTR phagocytosis against pathogen
invasion.

Application of previously reported glucose sensitizer RBCs, Zn activated C-
peptide112 to the CFTR inhibited macrophages revealed the ability of Zn activated C-
peptide to completely reverse the CFTR blockage elicited glucose uptake inhibition as
well as opsonized bacteria particle ingestion. Therefore, investigations described in the
chapter 4 of this dissertation have been able to correlate the effect of the mutated CFTR

in CF macrophages with their dysfunction and with the extensive bacterial infections

35

and colonization in CF lungs. Although the mechanism is so far unclear, the ability of
Zn activated C-peptide to enhance the macrophage glucose uptake and phagocytosis
may lead to a novel aerosol type therapeutic agent that can be used as a macrophages

sensitizer in CF lung disease.

36

10.

11.

12.

13.

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cardiac diseases. Acta Pharmacol Sin. 2005;26:265-278.

48

CHAPTER 2

2.1 EXTRACELLULAR BIOAVAILABLE ATP AND PLATELET

FUNCTION

Hyperactive platelets are associated with many different disease states. For
example, people with type 1 and type 2 diabetes, cystic fibrosis, and primary pulmonary
hypertension are reported to have hyperactive platelets."5 It has also been reported that
people with these diseases have red blood cells (RBCs) that release less adenosine
triphosphate (ATP) upon being subjected to deformation or pharmacological stimuli.6'9
A possible implication of this decreased RBC-derived ATP release arises when
considering that ATP is a recognized stimulus of nitric oxide (NO) production in the
platelet. Importantly, NO is a well-established inhibitor of platelet aggregationm’12

Adenine receptors found on platelets are major determinants in platelet
function.”’14 P2Y receptor family is for adenosine diphosphate (ADP), while the P2X
receptor family is for ATP.”'” These receptors can be subdivided into 7 distinct P2X
receptors (P2X1—7) and eight different P2Y (PZYl, P2Y2, P2Y4, P2Y6, P2Y11,
P2Y12, P2Y13 and P2Y14) receptors.18 Two ADP receptors, the Gq-protein-coupled
P2Y1 and Gi-protein-coupled P2Y12, and one ATP receptor, the P2X1 ion channel,
have been identified on platelets.19 Both the P2X1 and P2Y-type receptors are thought
to contribute to platelet aggregation, although the exact role for the P2X1 receptor is not

as well-defmed as those for the P2Y —type receptors. In fact, some reports suggest that

the P2X1 receptor is not required for platelet aggregation.20

49

2.2 A RELATIONSHIP BETWEEN P2X1 P2Yl PLATELET RECEPTORS

2.2.1 P2X, P2Y receptor synergy

Although cross-talk between P2X ionotropic receptors and P2Y type
metabotropic receptors has been shown for other cell types such as cells in the central

”3’21 such a relationship has never been established for platelets.

nervous system (CNS),
When P2X1 is co-expressed with P2Y] in Xenopus oocytes, it elicited more
pronounced P2X1-mediated currents, suggesting that P2Y may be assisting optimum
performance of P2X1. This work further suggested that P2X1 activation may not result
from direct phosphorylation, but more likely a staurosporine-sensitive (staurosporine is
a natural alkaloid which inhibits protein kinases through the prevention of ATP binding
to the kinase) phosphorylation of an accessory protein in the P2X1 receptor
complex.22'23

Other than the cross-talk with P2Y, there is emerging evidence for P2X receptor
cross-talk with other types of receptors. Reports suggesting the possibility of ATP or
ATP derivatives as antagonists for P2Y type receptors appeared a decade ago at a time

when understanding of these receptors was limited.24'26

Application of these non-
hydrolysable ATP analogues as P2Y antagonists suggest two basic possibilities. The
first one would be P2X1 may act as an ATPase, which would generate ADP in close
proximity to the P2Y receptors, which induce P2Y activation. The other possibility is

that there may be not direct P2Y1 inhibition; instead the lack of ADP generation with

these ATP analogues leads to non-responsive P2Y receptors. However there is no direct

50

evidence to deny the possibility of the blockage of both P2X and P2Y type receptors by
these ATP analogues. Therefore it is possible that both P2X and P2Y receptors work
together to increase the Ca2+i, and also to regulate the Ca2+i by CAMP-mediated Ca2+

down regulation and PKC mediated PLCB inhibition”:28

2.2.2 Common components in platelet purinergic signaling

The first common component in P2Y1 and P2X1 signaling is Ca2+i. ATP ligand
binding associated conformational changes lead to P2X1 cation channel opening
resulting in Ca2+ influx. ADP binding to the P2Y] receptor activates a small G-protein,
Gq. Activation of Gq leads to the activation of phospholipase-0132 (PLCB2), which
eventually results in inositol triphosphate (1P3) and diacyl glycerol (DAG) production.
1P3 and DAG catalyze the mobilization of the stored Ca2+ in the dense tubular system to
the cytosolw’30 Although DAG activates PKC mean while inducing Ca2+ mobilization,
PKC-associated direct inactivation of PLCB through a phosphorylation reaction has
been observed in several cell types, which helps to regulate the entire mechanism.3 1’32
Therefore, elevated concentrations of Ca2+i can inhibit further ADP-mediated Ca2+i
increase, but there is no evidence for increase in Ca2+i or on associated signaling
pathway that can inhibit ATP mediated Ca2+ influx. It seems the down regulation of
Ca2+i is mainly controlled by CAMP-associated Ca2+i granulation or PKC-associated
direct PLCB inhibition.

The second common component for P2X1 and P2Y1 associated signal

transduction is NO. Cay-calmodulin (Ca-CaM) activates eNOS in platelets to produce

51

NO, which prevents platelet activation. Under normal conditions with basal Ca2+i, all
the Ca2+i available for CAM should be consumed by eNOS”. The resultant NO
facilitates high CAMP concentrations in the cytosol that is associated with Ca2+i re-
granulation. Therefore, the basal levels of Ca2+i seem capable of mediating enough NO
production to prevent any GPIIb/IIIa activation by the CAM.”36 In the case of
substantial Ca2+i build-up, even in the presence of maximum inhibitory conditions
elicited by NO, the Ca2+i surplus can induce platelet shape change through a myosin
light chain kinase (MLCK) mediated mechanism and binds to the GPIIb/IIIa, both

through a CAM mediated mechanism(Figure 2.2).”:38

2.3 INTERCELLULAR COMMUNICATIONS OF PLATELETS IN THE

CIRCULATION

When evaluating platelet function it is very important to consider its
surrounding environment in the circulation and its communication with other cell types.
RBCs and endothelial cells are the two most abundant, and interacting cell types with
platelets. Therefore, platelet function may highly depend on the metabolic and physical
characteristics of these two cell types. Under normal circulatory conditions, vascular
endothelium is antithrombotic (favors prevention of thrombus formation) but under
certain conditions, it is prothrombotic (favors thrombus formation). Coagulation factor
XIII, a transglutaminase has been shown to modulate platelet GPIIb/Illa and endothelial
integrin, aVBIII, leading to increased platelet adhesion to the endothelium.”40 Both

plasma and platelet XIIIs are activated by thrombin.“ NO plays major role in

52

cardiovascular tone regulation by inducing vasorelaxation, inhibiting platelet activation
and subsequent aggregation. Although NO synthesis occurs in a range of cell types and
tissues, including the vascular endothelium, macrophages, and platelets, endothelium
derived NO is the main platelet inhibitor.“43 Binding of ATP to P2Y type receptors in
the endothelium increases Ca2+i and activates eNOS, which produces No.44
Endothelium derived NO may pay an important role in preventing the adhesion of
platelets traversing much closer to the endothelium. However, there should be another
source of NO for platelets traversing away from endothelium to prevent their activation
and aggregation. It has been shown that platelets produce their own NO and even
increase the NO production upon activation.44 The ability of NO released from platelets
to inhibit platelet activation and further platelet recruitment for the aggregation process
has been shown.45

One source for the endothelium to increase Ca2+i and eventually produce NO is
ATP. The main extracellular ATP source in the circulation is RBCs. RBCs contain
membrane-bound glycolytic enzymes and hold millimolar amounts of ATP, and release
ATP upon hypoxia and deformation.“47 When traversing through resistance vessels,
RBCs are subjected to mechanical deformation, which results in ATP release.”49 ATP
released from RBCs stimulates endothelial NO production, which eventually leads to
smooth muscle relaxation essential to regulate blood pressure. This ATP also interacts
with platelets, stimulate their NO production and prevent platelet aggregation.
Therefore, it is very important to have proper chemical communication between RBCs,
endothelium, and platelets, as RBCs possess a remarkable physiological sensitivity to

the changes in the cardiovascular system.50

53

Agonist

Receptor

E-arginine\ 0.3% /L-citrulinem """Hm'flm.

ll"
recepxtAzflor “mum” \JK'II 1‘ bun,"
WW 1.,GPllb/llla
i

Nob

Dense granular/
tubular

 

 

Figure 2.1 Effects of nitric oxide on platelet inhibition. Nitric oxide (NO) plays a
central role in the inhibition of platelet function. NO, formed as a by-product
of the conversion of L-arginine to L-citrulline, is involved in many
mechanistic pathways contributing to platelet inhibition. In reaction (1), NO
binds the heme moiety of soluble guanylyl cyclase, leading to the catalysis
of guanosine triphosphate (GTP) to 3,5-cyclic guanosine monophosphate
(cGMP): cGMP acts as NO’s principal second messenger, leading to platelet
inhibition. Nitric oxide has been associated with decreased intracellular Ca2+
levels through the inhibition of Ca2+ release from the dense tubular system
(2). Decreased Ca2+ levels inhibit cytoskeletal rearrangement, a- and dense-
granule release, and overall platelet activation. The thromboxane A2
(TXA2) receptor represents a complementary pathway involved in platelet
activation. NO acts to catalyze the phosphorylation of the TXA2 receptor,
preventing TXA2-mediated activation (3). The expression of P-selectin is
required to facilitate platelet adhesion. NO leads to the down regulation of
this protein, leading to inhibition of platelet adhesion (4). Fibrinogen, a
bivalent molecule, promotes cross-linking between platelets via the
GPIIb/IIIa receptors present on the platelet surface. NO attenuates platelet
aggregation by decreasing the total number of GPIIb/IIIa receptors available
to bind to fibrinogen (5), in addition to increasing the dissociation constant
of these receptors for fibrinogen. Along with these various mechanisms
through which NO exerts its inhibitory effects on platelets, it also 5diffuses
through the platelet membrane (6) and inhibits neighboring platelets.“

54

Investigating one particular cell type would help to understand only a partial
behavior of that particular cell type. It is very important to consider all possible
combinations of effects that can be elicited by other cell types in the circulation
simultaneously for a better understanding the behavior or one particular cell type. In
order to predict the behavior of a platelet in traversing through an arteriole, it is
necessary to consider the pressure exerted on the RBCs, RBC deformability, the extent
of ATP release, and endothelial and platelet NO production, which eventually leads to

vasodilation and platelet inhibition.
2.4 FATES OF Ca2+i MEDIATED BY ATP

Although its ATP-binding abilities may result in the subsequent production of
NO, the P2X1 receptor has also been shown to participate in platelet activation and
subsequent aggregation; through a mechanism which is not completely understood.52 It
has been reported that “priming” of the P2X1 receptor with a P2X1 agonist often
sensitizes the platelet to expedited activation and aggregation through the P2Y—type
receptors.53 For example, it has been reported that addition of 01-13 Me-ATP, a stable
ATP analogue, to the platelets will lead to an increased level of aggregation upon

54 While many theories exist for this

stimulation of the P2Y—type receptors.
phenomenon, definitive proof is elusive due to the difficulty in studying the P2X1
receptor and the numerous sub types from P2X1 to P2X7 in addition to the variety of
methods by which platelets are prepared from whole blood.”57 Therefore, identifying

all of the connections between the P2X1 and P2Y—type receptors has been difficult.

55

Ca2+

“if:

lllllllllllllllllj: firnmmmmmmmm g- f;; 353:: g , ‘

ATP
ADP l Thrombin

GPCRs

Plasma
membrane

132x1

   

 

    

 

 

 

Dense granular and
tubular Ca2+ release

 

 

 

2
F;
.C'.
U
H
..C
C?
2
"<7:
0
3,,
E

Platelet
aggregation

 

Figure 2.2 Both Ca2+ influx and stored Ca2+ mobilization increase Ca2+i. Ca2+ carrier
protein, calmodulin (CAM) then binds to the Ca2+i and transport and
facilitate calcium binding to the myosin light chain kinase (MLCK) and
glycoprotein IIb/IIIa (GPIIb/IIIa). Activated MLCK phosphorylates myosin
light chains (MLC) eventually contacting them. MLC contraction results
platelet shape change. Binding calcium activates GPIIb/IIIa. Activated
GPIIb/IIIa provides binding sites for fibrinogen and vWF and initiates
platelet aggregation.”38

56

2.4.1. ATP mediated Ca2+i as a platelet antagonist

Recently, in a study designed to quantitatively determine platelet nitric oxide
(N 0) production using fluorescence spectrophotometry, an interesting feature of the NO
production by platelets was observed.58 Specifically, when the platelet NO production
was stimulated using ATP, a steady increase in the fluorescence emission intensity
(indicative of the NO production) was measured as a function of the concentration of
ATP added to the platelets.

However, beyond a certain level of added ATP, the platelet NO production did
not increase, rather, it reached a steady value, even in the presence of additional ATP. It
has been shown previously that activated platelet derived NO can prevent neighboring
platelet activation.45 Substantial and sudden increases in Ca2+i is one of the features of
platelet activation and aggregation. Considering the fact that platelets produce NO
through Ca2+ dependent eNOS activation”, it is easy to correlate the activation induced
by Ca2+i increase and the subsequent NO production.60 Therefore, under normal
circulatory conditions, at a certain level of basal NO production, platelets may be able

to inhibit their own aggregation.
2.4.2 ATP mediated Ca2+i as a platelet agonist

ATP induced and P2X1 mediated transient Ca2+i increase and the reversible
decrease in light transmission corresponding to platelet aggregation61 suggested the

possibility of P2X1 mediated platelet aggregation. However, the extent of the Ca2+i

57

increase and the reversible nature of the aggregation excludes the possibility of P2X1 to
be a receptor responsible for platelet aggregation. Furthermore ATP induced P2X1
mediated Ca2+i increase and platelet shape change, but not the aggregation, have also
been observed.61 Interestingly in all these investigations, instead of ATP, a stable
analogue of ATP, cab-methylene ATP has been employed. Reasons to avoid ATP have
been described in chapter 1. Here it is suspected that although call-methylene ATP has
a higher binding affinity towards P2X1 than ATP, it may not serve as an effective P2X1
agonist. Therefore, if ATP is a more effective agonist than call-methylene ATP and
capable of inducing higher Ca2+i increase, it is quite possible for ATP to be a more
potent platelet agonist, rendering P2X1 to be an important target in antiplatelet therapy.
Based on these facts and other reports that suggests a dual nature of ATP toward
platelet function,62 we suspected that ATP has an anti-platelet effect at low levels of
ATP (due to its ability to increase platelet NO production) and an aggregatory effect at
higher concentrations of ATP.63 Here, we provide evidence suggesting that the ATP-

gated P2X1 receptor activity contributes to platelet inhibition and aggregation.

2.5 EXPERIMENTAL

Platelet purinergic signal transduction pathways seem to be interconnected with
numerous secondary messengers like Ca2+, Ca-CaM and NO. Evaluation of changes in
these secondary messengers upon binding agonists such as ATP and ADP to their
corresponding receptors on platelets would be important in understanding platelet

homeostasis properly. Furthermore, the quantification of agonist induced platelet

58

granular secretions and the effect of platelets inhibitors like NO on platelet activation in
terms of granular secretion would also assist the above purpose. Application of P2X1
specific inhibitors and evaluation of the concentration dependent effects of ATP on
platelet Ca2+i, NO and aggregation would provide more complete picture of the roles

ATP and P2X1 in platelet function.
2.5.1 Platelet and RBC preparation

Rabbits were anesthetized with ketamine (8 ml/kg, i.m.) and xylazine (1 mg/kg,
i.m.) followed by pentobarbital sodium (15 mg/kg iv.) 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 and for phlebotomy. After heparin (500
units, i.v.), animals were exsanguinated and the whole blood collected in 50 ml tubes.
Generally, 70 ml of blood was collected from the animal. Blood was centrifuged at 500
x g at 37 °C for 10 min. Packed RBCs were resuspended and washed three times in the
physiological salt solution (PS8; in mM; 4.7 KCl, 2.0 CaClz, 1.2 MgSO4, 140.5 NaCl,
21.0 tris(hydroxymethyl)aminomethane, and 11.1 dextrose with 5% bovine serum
albumin, pH adjusted to 7.4). The platelet rich plasma (PRP) was decanted for the
subsequent isolation of platelets. Platelets were isolated from the PRP by adding 1 ml of
acid citrate dextrose (ACD) to 9 ml of the PRP and centrifuging at 37 °C for 10 min at

1500x g.

59

2.5.2 ADP induced platelets activation in terms of platelet granular ATP release

Binding ADP to receptors P2Y1 and P2Y12 results in an increase in platelet
Ca2+i that subsequently initiates platelet shape change and aggregation through the
mechanisms shown in Figure 2.2. As a result of ADP induced platelet activation, the
contents of platelet storage granules such as ATP stored in platelet dense granules are
released in to the extracellular matrix. Therefore, the concentration of ATP released
from platelets is a measure of platelet activation.

The amount of ATP released from platelets activated with ADP was determined
by using fused-silica tubing that was intended to resemble vessels in vivo. The
. instrumentation employed consisted of a syringe pump (Harvard Apparatus, Boston
MA) equipped with two 500 all syringes (Hamilton, Fisher Scientific), two pieces of
~40 cm long microbore tubing with an internal diameter of 75 um and an outer diameter
of 362 um (Polymicro technologies, Phoenix, AZ). The platelet suspensions or ATP
standards loaded in one of the syringes was pumped through the tubing at a rate of 3.35
til min]. At the first tee, a stream of HBSS (for ATP standards) or ADP standard
solutions (10-40uM) in HBSS (for platelet suspensions) was combined. This stream
was then mixed at the second mixing tee with luciferin/luciferase (6.7 uL/min), which
resulted in a chemiluminescence reaction that was detected with a photomultiplier tube
(PMT) housed in a light excluding box. Two sets of experiments were conducted on
each day using two different lengths of mixing tubing (100 um ID), 12.5 and 18.5 cm,
from first tee to second tee. The length of the tubing (100 um ID) from second tee to the
PMT window was kept at 5 cm. All other parameters in Figure 2.3 were kept constant.

A working curve was constructed using five ATP standards between 0 and 1.5 uM. The

60

ATP standards were prepared from a stock 100 11M ATP in HBSS. All solutions were

prepared on the day of the experiment.

2.5.3 Extracellular NO mediated platelet inhibition-as a measure of ADP induced

ATP release

Although the NO mediated inhibition of platelet activation is well-known,
quantitative estimation of such inhibition in terms of the reduction of ATP release from
ADP activated platelets in a microflow system has never been reported. Nitric oxide
was prepared as a 38 mM stock solution from spermine NONOate (Cayman Chemical,
Ann Arbor, MI) by dissolving 10 mg of solid spermine NONOate in 1 ml of 0.01 M
sodium hydroxide (NaOH) solution. Working solutions were prepared by dilution of the
alkaline NONOate solution in 0.1 M phosphate buffer saline (PBS, pH 7.4). Sperrnine
NONOate is reported to have a half-life of 39 min at 37°C in 0.1 M phosphate buffer
and follows first order kinetics for its rate of decay.36 The 0.38 uM working NONOate
solutions were incubated in a water bath at 37°C for 15 min to ensure NO release from
the donor

The experimental setup was designed to let the platelets to incubate in a solution
that contains NO and then activate them with ADP while traversing through the micro
bore tubing. A platelet suspension was mixed with an NO donor (spermine NONOate)
at the first tee and then pturlped through the 75 nm diameter, 0.5 m long fused silica coil

(Figure 2.4). The tubing segment connecting second and the third tee was to activate the

61

platelets with ADP. This activated platelet suspension was then combined with

luciferin/luciferase at the third tee prior to moving through the detector assembly.

2.5.4 Effect of RBC derived ATP on platelet NO production in a micro flow

system

Although evaluation of individual cellular responses upon the addition of an
agonist is useful, multi cellular experimental platforms are more dynamic and provide
real time information about cell behavior. As discussed in section 2.3, RBCs interact
with the platelet in the circulation during normal hemostasis. In this experiment, the
objective was to investigate the effect of deformation-induced ATP release from RBCs
on platelet NO production. Demonstration of ATP mediated intracellular chemical
communication between RBC derived ATP and platelet, is a critical part of mimicking
the blood circulation in vitro. This experiment not only evaluates the effect of ATP on
platelet-derived NO, which can easily be performed with ATP standards, but also many
other known or unknown interactions between RBCs and platelets that might have an
influence on platelet NO production. Therefore, we anticipated the changes of NO
production in platelets interacting with RBCs to be proportional to the extent of the
ATP release from interacting RBCs.

Measurements were performed using the diaminofluorofluorescein diacetate
(DAF-FM DA) fluorescent probe for NO. A stock solution of DAF-FM DA (Molecular

Probes/Invitrogen, Eugene, OR) was prepared by dissolving the DAF-FM DA in 20 uL

of DMSO. Next, a working solution of DAF-FM DA was prepared by diluting 2 11L of

62

 

Platelet suspensron In HBSS 150 “m, X cm 150mm 5.5 cm
3.35 til/min

 

 

 

 

 

 

 

(D

Q.

E

g ADP

g 3.35 til/min 75 “m 75 pm
'5

U)

Luciferin / Luciferase

 

 

 

6.7ul/min

 

 

 

Figure 2.3 Two tee setup for investigation of platelet ATP release upon ADP addition.
Platelets were allowed to mix with the agonist ADP at the first tee.
Depending on the experiment, the length of the output tubing of the first tee
was changed and platelets were expected to release ATP depending on the
mixing time/the length of the mixing tubing. The released ATP was then
reacted with luciferin/luciferase to produce chemiluminescence and the
length of this tubing was kept 5.5cm, the least possible length which could
go through the PMT window. All solutions were prepared on the day of the
experiment.

63

Platelet suspensnon In HBSS 75pm coil 150nm,18.5cm 150nm,5.50m

 

 

 

 

 

 

 

 

 

 

 

3.35pl/min
NONOate in Phosphate buffer

a:

Q- .

E 2.0pl/mln

:

o.

a) ADP

a:

.g 3.35ullmin .
>
(D

 

 

 

Luciferin / Luciferase

 

 

 

6.7ullmin

 

 

 

Figure 2.4 Three tee setup for the investigation of the nitric oxide inhibition of ADP-
induced platelet activation. This setup facilitates platelets to initially
interact with nitric oxide and then react with the ADP. ADP concentrations

are 5-40 uM. In control experiment 0.1M phosphate buffer is pumped in
place of NONOate.

64

the stock DAF-FM DA solution to 1 ml in HBSS. Next 100 uL of 2:108 platelet
suspensions were individually mixed with 50 uL of 10 uM DAF-F M DA in 2m] vials,
diluted up to 500 uL and incubated for 10mins. Platelet mixtures without DAF-FM DA

were prepared by mixing 100 pL of z108 platelets in suspension with 400 uL of buffer.
RBCs were separately incubated in the absence or presence of 80 nM iloprost.

Platelets were passed through a microbore tubing system consisting of three
major components, namely, a dual syringe pump, the sections of microbore tubing, and
a flow-through fluorescence detector (Jasco, Easton, MD) housed with a capillary flow
cell. DAF-FM DA-loaded platelets and RBCs were 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 is a conventional syringe pump where the syringe is easily accessible.
Previous experience with this type of syringe has shown 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 Lab View

(National Instruments).

2.5.5 Fiuorescence determination of platelet NO

As experiments in section 2.5.4 aimed to demonstrate the effect of RBC derived

ATP on platelet NO production and the platelet sensitivity to the changes in

65

extracellular ATP, investigations described in this designed to correlate the interaction
of ATP with its receptor, P2X1, and stimulation of platelet NO production.
Measurements were performed using the diaminofluorofluorescein diacetate
(DAF-FM DA) fluorescent probe for NO. A stock solution of DAF-FM DA (Molecular
Probes/Invitrogen, Eugene, OR) was prepared by dissolving the DAF-FM DA in 20 uL
of DMSO. A working solution of DAF-FM DA was prepared by diluting 2 pL of the
stock DAF-FM DA solution to 1 ml in HBSS. Aliquots of 100 uL of this working
solution of DAF-FM DA were added to 400 uL of the platelet solution (3 x 108 platelets
mL'l) and allowed to incubate for 15 min. These platelets were washed twice with
HBSS by centrifuging at 1500 g for 10 min. Finally, 100 uL of the washed, DAF-FM
DA-loaded platelets were then resuspended in 400 uL of HBSS that contained 10 1.1M
ATP. After 20 min, the fluorescence emission, resulting from NO production and the
subsequent reaction with the fluorescence probe in the platelet, was determined.
Fluorescence measurements were performed on a Fluoromax-4 spectrofluorometer
(HORJBA Jobin Yvon, Edison, NJ) at room temperature. Emission spectra were
obtained in a quartz cuvette with excitation at 495 nm and emission at 515 nm. Both
excitation and emission slit widths of 2 nm were used in all experiments with sampling

interval of 0.2 nm.

2.5.6 Fluorescence determination of platelet intracellular Ca2+

Although sections 2.5.4 and 2.5.5 correlate the ATP binding to P2X1 and the

subsequent platelet NO production, it does not provide the information about ATP

66

mediated P2X1 activation and related extracellular Ca2+ flux in to the cytosol. In this
experiment, intracellular Ca2+ concentrations were determined using fluorescence
spectroscopy. The expected result of this experiment was an increase in Ca2+i upon ATP
binding and decrease in Ca2+i when platelet P2X1 is inhibited.

The fluorescence determinations were performed using F luo 4 AM, an
intracellular fluorescence probe for Ca2+. The F luo 4 AM stock solution was prepared
by dissolving in 20 uL of DMSO. The working solution was prepared by diluting 5 uL
of the stock solution in 1 ml of Ca2+- free HBSS. To prepare the platelets for the
determination of Ca2+, the harvested platelets were sensitized with 0.28 U/ml apyrase in
Ca2+ free HBSS. The sensitized platelets were then washed 2 times in a Ca2+- free
HBSS solution that did not contain apyrase in order to remove any residual apyrase
from the initial wash. A 1 ml portion of these platelets (in the Ca2+- free HBSS) was
incubated with 500 uL of the working Fluo4-AM solution for 2 hours, centrifuged and
again washed twice with Ca2+- free HBSS. Next, 750 uL of this platelet sample were
combined with 300 uL of Ca2+- free HBSS that contained 0.67 mM NF 449. The
remaining 750 uL of the platelet sample were treated with 300 uL of Ca2+- free HBSS
alone. Finally, 100 uL of NF 449-treated or non-treated platelets were added to 250 uL
of HBSS that contained 40 mM Ca2+ and 10 “M ATP and allowed to incubate for 30

min before measuring the fluorescence emisSion from the sample at 516 nm (with 496

excitation) using the fluorescence parameters described in 2.5.6

67

2.5.7 Atomic absorption spectroscopy for platelet total Ca2+

Although with fluorescence spectroscopy, we were able to see the reduction in
Ca2+i upon P2X1 inhibition, we were not able to observe a significant increase in Ca2+i
upon ATP binding. It was speculative that this may be a result of the fast kinetics of
Ca2+i regulation in the platelet cytosol. Therefore, to further verify the effect of ATP on
platelet Ca2+i, atomic absorption spectroscopy has been used to detect changes in total
platelet Ca2+ concentration.

For Ca2+ influx measurements using atomic absorption, platelet suspensions
were prepared in Ca2+- free HBSS as described above for those measurements involving
fluorescence determination of Ca2+ influx. The platelet samples were fixed with
buffered Para formaldehyde (1.8%) and washed three times with Ca2+- free HBSS by
centrifugation at 2200 g. Before the final spin, the platelets were counted to ensure the
platelet number for subsequent data analysis. After the final spin, platelet pellets were
dissolved in 2.5 ml of distilled de-ionized water (DDW) and subjected to freezing with
liquid nitrogen and thawing at 37°C three times to ensure complete lysis of the platelets.
The lysed platelet samples were centrifuged at 3000 g for 15 min and the supernatant

was analyzed for Ca2+ using a Hitachi Z-9000 atomic absorption spectrophotometer.

2.5.8 Platelet aggregation measurements

Experiments 2.5.4-2.5.7 were designed to asses the purinergic signal

transduction pathway associated with ATP binding to P2X1. Although increases in

68

5
O
m

L l
A
8

Chemiluminescence lntensuty
O
O)
1 1 A I 1 l A
E E

4
A
D)
V

0.4 -

0.2

1L4 L41

 

 

0.0I""I

 

 

 

0 50 100 150 200
Time / Seconds
11)
- 12.5 cm mixing tubing 1
18.5 cm mixing tubing
1.2 4 l
E
‘7’ 1.0 - a;
$ . _
m 0.8 d '
£9
9
n. 0.6 a
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0.2 .. i
0.0 a: l ; T

 

 

 

 

 

 

 

 

 

Figure 2.5 a) Real time curves corresponding to the platelet ATP release upon ADP
addition. (a)-HBSS only (b) to (e) are platelets incubated with 10-40nM
ADP respectively. b) Averaged standardized ATP release from platelet
activated with ADP, a- + buffer, b- + 10uM, c: + 20 nM, d: + 30uM, e-+ 40
uM ADP. Here the results are presented, using ADP as an efficient agonist
for platelet activation demonstrating the ability to use platelet derived ATP
as a measure of platelet activation n=5, error bars: standard error of the
mean

69

platelet Ca2+i and subsequent NO production correlates the extracellular ATP
concentration with platelet inhibition, the outcome of the same Ca2+i on MLCK induced
platelet shape change and GPIIb/IIIa mediated aggregation were not conclusive. As
platelet aggregometry is the most frequently used technique to screen patients for
inherited or acquired defects of platelet function in terms of MLCK induced platelet
shape change and GPIIb/IIIa mediated aggregation, platelet aggregation. is used to
interconnect extracellular ATP concentration with platelet activation. Due to the ability
of both ATP and ADP to induce an increase in platelet Ca2+i, increased aggregation was
anticipated for both agonists. Inhibition of the P2X1 receptor was expected result in
reduced ATP induced aggregation but not with ADP. The ability of increased Ca2+i to
induce NO production, as well as integrin activation, made predictions very difficult for
concentration dependency of ATP on platelet aggregation.

A Chronolog 490-2D aggregometer was employed for all platelet aggregation
measurements. For each aggregation study, a 100 uL aliquot of platelets (3 x 108
platelets mL'l) Were added to 400 uL of buffered reagents (described for each study
below). Reference and test cuvettes were analyzed during each aggregation trial; the
contents were constantly stirred using P/N 311 stir bars and all studies were performed
at 37 °C. Data was obtained at 10 data points per second for 30 min. For studies
involving P2X1 inhibition, HBSS with 10 uM ATP was prepared. When investigating
the effect of P2X1 inhibition, the HBSS also contained 20 uM NF 449, a competitive
inhibitor of the P2X1 receptor. In separate studies involving the concentration
dependency of ATP on platelet aggregation, the HBSS contained ATP concentrations

ranging from 0 to 4 nM in ATP. For those studies involving the effect of ATP on

70

platelet aggregation in the absence of Ca2+, Ca2+- free HBSS was used to prepare the 10

uM ATP-containing solutions.
2.6 RESULTS AND DISCUSSION
2.6.1 ADP induced platelet activation and granular ATP release

ADP is one of the most potent agonists for platelet activation and aggregation.64
Platelet activation has long been measured in terms of platelet ATP release.“68
Although there are different methods employed for platelet ATP detection,65 the
luciferin/luciferase ATP specific enzymatic assay is widely accepted as the most
reliable method.”68 Figure 2.5 shows the platelet ATP release data obtained by using
the two tee microbore tubing assembly described in Figure 2.3. When traversing

through a 75 uM microbore fused silica tubing for 32.9 seconds, while reacting with 10

uM ADP, platelets release 42.7% higher ATP than platelets interacting with buffer
alone. Increasing ADP exposure time from 32.9 to 42.4 seconds by lengthening the

tubing resulted in a 2.4 fold increase in ATP release.
2.6.2 Inhibition of ADP induced platelet activation by extracellular NO

The effect of NO on platelet inhibition has been discussed under sections 2.3
and 2.4. Although the involvement of extracellular NO on platelet endothelial adhesion

and changes in the components of signal transduction pathways have been reported

71

a)
4.0 1 + Platelets

-o— Platelets in NONOate

3.5 —

3.0 -

2.5 -

2.0 -

ATP release - (pM)

 

 

 

b)

0.5 -

Fluorescence intensity

 

 

Figure 2.6 a). Inhibition of ATP release from ADP activated platelets in the presence of
~200 nM NO. Experiment was conducted with the use of the “3” microbore
tubing assembly described in figure 2.4. a- platelets, b- platelets + 5 uM
ADP, c- platelets + 10 uM ADP, d- platelets + 20 p.M ADP, e- platelets +
30 uM ADP, f- platelets + 40, 200 nM NO was able to inhibit the ADP
induced ATP release by 43%. Platelets concentration ~108 /ml b). RBC
derived ATP on platelet NO production in a flow through system: a-
Platelets, b-RBCs + Plt, c-RBCs (incubated with trental) + Platelets, d-
RBCs (incubated with Diamide and then Trental) + Platelets, n=3, error
bars: standard error of the mean

72

“"70 the effect of extracellular NO on ATP release from ADP activated

previously,
platelets in a flow system has never been investigated. The three tee assembly in Figure

2.4 provided the essential elements to create a multi component flow base reactor.

2.6.3 ATP, a communicator between RBC and platelet

Previously, we described a fluorescence based method that enabled the
quantitative determination of basal levels of NO in platelets and the amount of NO
produced after stimulation with the platelet aggregation agonists ADP or ATP.58 The
collaborative study in this paper suggests a possible unique relationship between RBCs
and platelets in the circulation. Specifically, RBCs may be able to communicate with
the platelet through RBC-derived ATP, stimulating NO. In order to verify this
relationship, a fluorescence based microflow experiment described in 2.2.4 was
conducted.

Here, the ability to measure platelet-derived NO upon stimulation with ATP
released 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 or absence of iloprost.
Iloprost is a prostacyclin analog that has been shown to increase the ATP release from
RBCs and here iloprost resulted in a 9 fold increase in platelet NO production compared
to the untreated RBCs. When RBCs were treated first with diamide, a known oxidant
that reduces RBC membrane deformability, and then with iloprost, a 60 % reduction of

platelet NO compared to the untreated RBCs, has been observed.

73

2.6.4 ATP and P2X1 in the regulation of platelet NO and cytosolic Ca2+

The increase in platelet NO production upon addition of ATP was measured
using DAF-FM DA, a fluorescent probe for NO (Figure 2.7). The percent increase,
normalized to the fluorescence intensity for platelets with the probe, but no agonist, was
26.7 :t 7.7% for ATP, a value representing a significant increase in platelet NO
production (p < 0.001). Though not quantitatively determined here, this 26.7 :t 7.7%
increase corresponds to approximately 2.2 x 10'8 moles of NO per 3 x 108 platelets
based on a previous report.58 A decrease in fluorescence intensity was measured when
the platelets were incubated with L-NAME, a competitive inhibitor of nitric oxide
synthase (NOS), to verify that the increase in fluorescence intensity was indeed due to
the production of NO. The black bars in Figure 2.7 demonstrate the ability of the ATP

to increase NO production in the platelets.

74

 

   

150 ' - Platelets
c m P2X inhibited platelets
,g 140 d - Platelets with L-NAME
S
E 120 -
o.
O
z. 100 -
a)
o
a“:
o 80 ..
a)
9
8 60 r
c -. .. I'd-'14.;
8 net .
.5 40 -i I g.
E . ‘
g
O 20 ‘ 12’
Z |

O l I r

 

 

 

Figure 2.7 Effect of P2X1 inhibition on platelet derived NO. Platelets treated with DAF
‘ PM DA were investigated for intracellular NO with (gray bars) or without
(black bars) P2X1 inhibition. As expected, the basal NO production
significantly increased in the presence of ATP ("LP < 0.001). Inhibition of
P2X] significantly reduced NO production in the platelets, even in the
presence of this agonist (*P < 0.001). L-NAME, a competitive inhibitor for
NOS has been used to demonstrate that the measured fluorescence signal is

due to NO. n=3, error bars: standard error of the mean

75

When the platelets were incubated with the NF 449, a selective P2X1 receptor
inhibitor, the NO production was significantly decreased (p <0.001) upon addition of
ATP. Based on the results shown in Figure 2.7 suggesting a Ca2+ influx in the presence
of ATP, experiments were performed to examine the Ca2+ influx into the platelets in the
presence of various agonists and antagonists of the P2X1 receptor. It is known that

“'73 and the data

stimulation of this receptor will result in Ca2+ influx into the platelets,
in Figure 2.8 demonstrates that this increase in Ca2+ flux into the platelet only occurs
when the P2X1 receptor is open. Specifically, the addition of Ca2+ to the platelet
suspension when the P2X1 receptor is inhibited with NF 449 resulted in a decreased
Ca2+ flux into the platelet (even in the presence of ATP).

While inhibition of P2X1 significantly decreased Ca2+ influx, the addition of
ATP to the platelets in the absence of the NF 449 did not significantly enhance the Ca2+
influx into the platelet. It was anticipated that this may be due to Ca2+ binding to
calmodulin (resulting in no increase in fluorescence intensity) as opposed to the
fluorescent probe. Therefore, Ca2+ influx studies were performed with atomic
absorption spectrophotometry in order to measure the total Ca2+ influx into the platelet.
This technique could measure influx even if the Ca2+ were to bind to calmodulin or any
other substance in the platelet. As shown in figure 2.9, measurement of Ca2+ by atomic
absorption did reveal a significant increase in Ca2+ flux into the platelet. Furthermore,

this Ca2+ influx measured by atomic absorption was inhibited in the presence of the NF

449 inhibition of P2X1.

76

- Normal platelets

120 _‘ P2X inhibited platelets

influx

100—:

2+

80-:
eol

405

Fluorescence intensity~Ca

20-:

  

P +ATP

 

 

Figure 2.8 Influence of ATP on P2X1 mediated Ca2+ influx in platelets. Platelets
treated with fluo-4 AM were investigated for Ca2+ influx into the platelets
with (gray bars) or without (black bars) P2X1 inhibition. In (A) the Ca2+
influx did not significantly increase in the presence of ATP. However,
inhibition of P2X1 significantly reduced Ca2+ influx (*P < 0.001). n=3,
error bars: stande error of the mean

77

 

Normalized Absoption

   

 

 

P P-ATP P-NF 449

Figure 2.9 Influence of ATP on P2X1 mediated Ca2+ influx in platelets. The total
intracellular concentrations of Ca2+ were determined with atomic
absorption spectroscopy. Here, a significant increase in Ca2+ influx into the
platelets was determined using either ATP as the agonist (*P = 0.004). As
expected, measurement of P2X1 inhibition of Ca2+ influx into the platelets
could also be determined with atomic absorption (last bar, P = 0.025). n=3,
error bars: standard error of the mean

78

2.6.5 ATP and P2X1 in the regulation of platelet aggregation

The results in Figures 2.9 and 2.10 suggest that ATP activation of the P2X1
receptor is required for both Ca2+ influx into the platelet and NO production in the
platelet, implying that significant platelet aggregation would only occur when the P2X1
channel is activated. Platelet aggregation was measured by adding ATP to the platelets
in the presence and absence of the NF 449, the P2X1 receptor antagonist.

The presence of 10 uM ATP in the HBSS solution to which the platelets were
added resulted in the expected increase in aggregation of the platelet solution, as shown
in Figure 2.10. In accordance with the results in Figures 2.8-2.10, when 10 pM ATP
was added to the platelets in the presence of NF449, the extent of aggregation was
decreased to level similar to the platelets incubated in buffer alone. Further evidence
for the importance of Ca2+ in platelet aggregation can also be seen in Figure 2.10.
Platelets in the bottom three traces were prepared in a Ca2+- free buffer and stimulated
with a solution of ATP, ADP or buffer that was also Ca2+- free. A comparison of the
aggregation profiles of these platelets in a Ca2+- free buffer to those platelets in Ca2+:
buffer (as shown in Figure 2.10 t0p 4 traces) reveals the importance of the Ca2+ for
platelet aggregation. The aggregation increases when Ca2+ is present in the buffer, even
in the absence of any type of P2X1 stimulus (ATP). The data in Figure 2.10 support the
importance of Ca2+ in platelet aggregation and the role of the P2X1 receptor in this
aggregation mechanism. In this experiment, ADP was used as a positive control for

platelet aggregation. Application of 10 uM NF 449 resulted in the inhibition of this

79

aggregation to a greater extent. Although NF 449 is a selective P2X1 inhibitor, the
concentration we used here is well above its IC50 value for P2Y1.

An appraisal of the results shown in Figures 2.8-2.11 does not explain the dual
nature of ATP in platelet aggregation. For example, through its ability to stimulate Ca2+
influx through the P2X1 receptor, maintain increased Ca-CAM concentration in the
cytosol and stimulation of eNOS activity, ATP can be regarded as a stimulus for NO
production, an inhibitor of platelet aggregation.”76 However, ATP also has the ability
to increase platelet aggregation through the same pathway, but with excessive Ca2+
influx.

Therefore, aggregation curves were measured in the presence of buffers
containing ATP from 0 to 4 uM (Figure 2.12). The aggregation curve for platelets in
buffer containing 0 nM ATP is labeled as (a) and was fairly constant for the entire 1500
seconds of measurement period. Interestingly, the addition of 10 and 20 nM ATP to the
platelets resulted in an apparent decrease in the aggregation. The addition of 40 nM
ATP increased aggregation in comparison to the 20 nM addition, although the value
was still lower than that of 0 nM ATP. Subsequent additions of 0.10, 1.0 and 4.0 uM
ATP resulted in significant aggregation of the platelets, which is summarized in Figure
2.13b as the final %T after 1200 seconds. The difference in aggregation profiles in
Figures 2.11 and 2.13 are due to the increased concentration of ATP used in Figure 2.10

(10 uM) resulting in a more rapid progression of aggregation.

80

 

 

P+ADP
30* P+ATP
(D
O
C:
(U
E
E 30-
U)
C
E
[...
e\°
10_ P+ NF+ADP
‘ ,, P+NF+ATP
,, , ' Platel-ts in Ca2+ free HBSS P
'-I‘"i‘.'“W“"F‘W‘S‘RM‘M t" “Hausa chw‘ awn; rag u been?» A P+ATP
0 -l " ‘ f U
P+ADP
200 400 600 * 800 1000
Time (s)

Figure 2.10 Representative curves for P2X1 inhibition. on ATP and ADP induced
platelet aggregation. Aggregation curves were measured for both normal
and P2X1 inhibited platelets in the presence of ATP and ADP (10 uM).

81

10-_

N
o
l

%Transmittance
OJ
0
l

405

    

 

P +atp +adp +nf+atp +nf+adp

Figure 2.11 P2X1 inhibition on ATP and ADP induced platelet aggregation.
Aggregation curves were measured for both normal and P2X1 inhibited
platelets in the presence of ATP and ADP (10 uM). Here is the summary of
the aggregation (reported as the % transmittance) from n = 3 rabbits. As
expected, there was a significant difference in the % transmittance
(aggregation) for platelets in the presence of ATP (*P < 0.001). P2X1
inhibition reversed the aggregation trend to values that were slightly lower

than normal platelets without any agonist (*P < 0.0024). n=3, error bars:
standard error of the mean

82

 

 

ix
8 101 \‘\
5 l \ _ (0.02)
e ‘ (0.01)
l- .
,\° 30; (0.00)
: (0.10)
40; (1.0)
‘ (4.0)
200 400 600 800 1 000 1200
Time(s)

Figure 2.12 Concentration-dependent dual nature of ATP as a platelet antagonist and
agonist. Representative aggregation curves for platelets in buffer
containing varying concentrations of ATP

83

w
40{
30{

20{

%Transmittance

 

b)

5I.‘. "i.

    
  

0 0010.02 0.04 0.1 1 4
WWW)

Average platelet counts
8 8 8 8 8 3

.a
O
1.. .|..

 

 

O
I

pit 20 nM 50 nM 100 nM 10 uM
[ATP]

Figure 2.13 a) Concentration-dependent dual nature of ATP as a platelet antagonist and

agonist. Averaged summary of these curves are shown in (B); note that
the aggregation begins to significantly decrease with incremental ATP,
prior to a significant rise as the ATP levels increase *P < 0.00. Error bars
are standard error of the mean for n = 3 rabbits. b) The effect of
exogenous ATP on platelet adhesion. Platelets were treated with various
concentrations of ATP (and were centrifuged to remove excess ATP) prior
to flow through a microfluidic channel coated with a confluent layer of
bovine pulmonery artery endothelial cells-bPAECs (A). A stream of
equilibrated medium was pumped over the endothelium to ensure any
non-adherent platelets were removed before averaging the platelet count.
n = 5 rabbits, error bars: standard error of the mean. (+p<0.05; *p<0.01)

84

The data in Figure 2.13a demonstrate that platelet exposure to varying
concentrations of ATP may play an important role in platelet aggregation. Therefore, _
subsequent studies were performed by in our laboratory to determine the role of ATP in
platelet adhesion to an immobilized endothelium in the presence of flow. A microfluidic
device, similar to that previously reported by our group,"7 was employed to investigate
the effects of exogenous ATP, and ATP released from RBCs, on platelet adhesion to
endothelial cells. Summarized averaged data representing 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 is shown in Figure 13b. It is clearly evident that the adhesion trend as a
function of ATP concentrations is strikingly similar to the aggregation curve summary
shown in Figure 2.13a. It is well-established that flowing RBCs subjected to shear-

induced stress will release ATP.78'79
2.7 CONCLUSIONS AND OTHER CONSIDERATIONS

The main objective of the experiments performed here were to provide further
evidence that the P2X1 receptor is a determinant in platelet aggregation and adhesion.
The P2Y] and P2Y12 receptors are generally thought to be important purinergic
receptors for platelet aggregation. However, there have been recent reports that the
P2X1 receptor may also be important. For example, Marhaut-Smith’s group has
reported that the P2X1 receptor does indeed participate in increase Ca2+ flux into the

platelet independent of P2Y-type receptor activity.80 Moreover, in vivo studies have

85

shown that P2X1 knockout mice have a decreased level of thrombus formation and
increased bleeding times,81 both indicators of lower platelet aggregation activity. Such
studies with P2X1 deficient mice also suggest that the P2X1 receptor is an important
component in platelet aggregation pathways.

However, there have been other reports that P2X1 activity is not required for
platelet aggregation.82 Further complicating the role of the P2X1 receptor in platelet
function, there are reports suggesting ATP as a platelet inhibitor,83‘84 due to its ability to
stimulate NO through eNOS while other suggested ATP as a platelet activator.

The work shown here demonstrates that both platelet NO production and
aggregation are affected by P2X1 activation with ATP, but in a concentration-
dependent manner (Figure 2.12 and 2.13). When ATP is added to the platelets, a
significant increase in NO production is measured as shown in Figure 2.1 and
elsewhere.58 However, results here show that blocking the P2X1 receptor with NF 449,
a potent P2X1 antagonist, results in NO production equivalent to that of platelets
without any added activators.

Another interesting feature of the work presented here is the form and manner
by which ATP was administered to the platelets. Most studies investigating the effect of
ATP on platelet physiology employ a-B-methyleneATP, a stable analogue of ATP that
does not break down even in the presence of enzymes known to break down ATP (e.g.,
apyrases). It is known that a-B-methylene ATP binds specifically to P2X1, although it
does not result in platelet aggregation, and its effect on Ca2+ influx, while significant, is
short—lived (5-10 3).”85 Moreover, a-B-methylene ATP is also known to “desensitize”

the platelet P2X1 receptor, thereby decreasing the overall Ca2+ flux into the platelet.86

86

In addition to the form of ATP added (e.g., the a-B-methylene ATP), many
researchers often use apyrase in their solutions when performing platelet studies. The
apyrase is often employed to sensitize the platelets, making them responsive to
purinergic agonists. In these studies, apyrase was also employed; however, after the
addition of apyrase, the platelets were washed twice in apyrase-free buffer. This
washing of the apyrase from the buffer was performed in order to guarantee that any
authentic ATP added to the platelets would have an opportunity to bind to the P2X1
receptor before being converted to ADP or other nucleotides form by the apyrase. Such
studies (in the absence of apyrase) need to be performed rather quickly (within ~ 2 hrs)
or the platelets do become desensitized; however, if the studies are performed soon after
washing from the plasma, the receptors respond quite strongly to purinergic agonists
such as ATP even without washing in an apyrase-containing buffer.

The results presented here suggest that the effect of ATP on platelet behavior is
concentration dependent, although the concentrations that have been reported are not
consistent (Figure 2.13).85 Here, our results suggest that zero to low levels of ATP result
in aggregation that is more pronounced (Figure 2.12). It does appear that increments of
ATP reduce platelet aggregation initially, only to begin increasing with continued
increments in ATP. We believe this may be due to the production of Ca2+-calmodulin
and saturation of eNOS within the platelet. Specifically, previous investigation on a
fluorescence method to quantitatively determine the amount of NO that was produced
and released from platelets as a result of ADP or ATP added to the platelet solution.58
The results from this particular study showed that NO production in the platelets

increased upon stimulation with ATP up to a value corresponding to 1.6 x 10'19 moles

87

of ATP added per platelet; beyond this amount of added ATP, the NO production
remained constant. Collectively, it may be possible that levels of RBC-derived ATP that
are abnormally low (resulting in low Ca2+ influx into the platelet) may subsequently
lead to insufficient eNOS activation and insufficient production of NO. Conversely, if
the Ca2+ influx into the platelet is abnormally high (due to exposure of the platelets to
large increments of ATP, such as hemolysis), our previous studies suggest that the
eNOS becomes saturated. If this saturation does occur, formed Ca2+-calmodulin may
then have an opportunity to diffuse to the platelet membrane and activate certain
integrins, resulting in an increase in aggregation. However, such a proposed mechanism
has yet to be validated.

This dual effect of ATP on platelet aggregation was verified by platelet adhesion
studies using microfluidic technologies. The data in Figure 2.13b clearly demonstrates
that there is an effect from ATP on the platelet’s ability to adhere to an 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 is from the ATP acting on the
platelet as opposed to the ATP stimulating endothelium-derived NO that could also
inhibit the platelet production. Importantly, the effect of ATP on both the aggregation
and adhesion are strikingly similar and both show a bi-phasic effect of ATP on platelet
function.

Another possible fate of the RBC-derived ATP is that it may be broken down by
ectopyrases located on the endothelium. A breakdown of ATP to other adenine
nucleotides (such as ADP) may lead to participation in platelet activation. However, if

this were occurring in the system described here, an increase in platelet adhesion would

88

be expected even at lower ATP concentrations, as opposed to the decreased levels of
platelet aggregation and adhesion shown in Figures 2.13a and 2.13b. Therefore, even if
some of the ATP released from the RBC is enzymatically degraded to ADP, it is not
enough to overcome the platelet inhibiting action of the NO production.

The work presented here involving the 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. For example, people with
diabetes, cystic fibrosis, and primary pulmonary hypertension all have RBCs that
release less ATP than healthy controls.7’8 Furthermore, all of these patient groups have
platelets that are more hyperactive than platelets obtained from controls. The data in
Figure 2.13b, which employs three different cell types in a single microfluidic channel,
suggests that a decrease in the RBC-derived ATP has a direct effect on platelet adhesion
to an endothelium. However, the authors also believe that deformation-induced ATP
release is not the sole source of ATP from the RBC. Other reagents (e.g., iloprost, C—
peptide) have been shown to induce ATP release in the absence of deformation.

It should be noted that the cell sources used in these studies are mixed (rabbit
RBCs and platelets with bovine endothelial cells); however, human RBCs have been
shown to produce NO in rabbit pulmonary beds.87 Therefore, while not the ideal
scenario, the cells employed here are functional for the studies reported.

The data reported here suggests that there is indeed an “optimal” level of ATP
for reducing platelet aggregation. It may be somewhat difficult to determine that exact
level from the results presented in Figures 2.12 and 2.13a; specifically, in Figure 2.12,

the ideal concentration seems to be around 20 uM for the number of platelets studied.

89

However, this value appears to be higher when in the presence of RBCs. A possible
explanation for these results is that, in Figure 2.13a, the ATP was added to the platelets
as authentic ATP standards prior to pumping across the endothelium. In this construct,
there is really no “competition” for the ATP between the platelets and the endothelium.
In figure 2.5, the ATP source is the RBC and this ATP is not available until the
pumping mechanism is started. Therefore, in this scenario, ATP would be available for
platelets and the endothelium. Therefore, a 20 nM addition of authentic ATP to platelets
alone would quite possibly need to be increased in the presence of added cells. A
second important piece of information to consider is that the authors used a 3.5%
hematocrit, which is roughly a 50% decrease from studies to date employing RBCs
through such channels. Typically, the ATP release levels are in the low hundreds of
nanomolar range; here, that value may be less.

Importantly, the increase in platelet adhesion due to inhibition of RBC-derived
ATP does provide a model of “low” ATP release as would occur with the RBCs from
people with diabetes, cystic fibrosis, or primary pulmonary hypertension. Interestingly,
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. 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

90

concentrations monitored with microfluidic technologies, may serve as a starting point

for explaining some of the clinical observations involving hyperactive platelets.

91

10.

11.

12.

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99

CHAPTER 3

3.1 PLATELET PURINERGIC RECEPTORS

Purinergic signaling plays a very important role in platelet homeostasis by
acting as a platelet agonist or antagonist depending on concentration of the binding
molecule. Particularly, the complex multiple signal transduction pathways associated
with ATP gated P2X ionotropic channel family receptors, and ADP gated heptahellical
metabotropic (those that do not form an ion channel pore) receptors are the key
regulators of platelet function.1 However, the involvement of ATP in P2X activity in
platelet homeostasis is controversial. There are number of published investigations
suggesting the involvement of ATP and P2X1 receptor in platelet function.M Among
these are in vitro studies on ATP depletion attenuating platelet aggregation, ATP / von
Willebrand factor (vWF) mediated platelet aggregation due to shear forces, ATP
mediated enhancement of collagen induced platelet activation and, more prominently,
ATP induced platelet shape changes"8 All of these studies provide very strong evidence
for the involvement of ATP and P2X1 in platelet homeostasis. Interestingly, in support
of these in vitro studies, in vivo studies conducted on the P2X1 knockout mice (P2X1'l')
have shown reduced mortality due to systemic thromboembolism, decreased size of
mural thrombi upon laser induced vessel wall injury, and rapid thrombus clearance.9

Although the receptor structure and function are not well defined, there is
emerging evidence suggesting a trimeric architecture for the heteromeric P2X1

receptor.”12 Trimerization of the P2X1 monomers occur in endoplasmic reticulum

membranes and this polymerization seems to be occurring through disulfide bonding.

100

ATP binding induces conformational changes within and between subunits (Figure 3.1).
These changes propagate to the ion channel by conserved residues located at the
interface between the transmembrane domains leading to the opening of the P2X1 ion
channel pore (Figure 3.2).13

There are specific difficulties associated with P2X1 receptor function
assessment that prevent a complete understanding of its behavior. The major constraint
is rapid ligand occupied desensitization. In the presence of ATP, the reported
desensitization time is 47-107 ms.”15 Stirring at 37 °C for 10 minutes caused washed
human platelets to release 10 attomoles of ATP per platelet without adding any
agonist.l6 Therefore, prolonged storage of washed platelets in buffer results in platelet
P2X1 receptor desensitization. A major concern with previous P2X1-ATP functionality
analysis is the application of oc,B-methylene ATP, a stable analogue of ATP, to evaluate
the P2X1 activity. Although the characterization data for (LB—methylene ATP in human
urinary bladder cells showed high specific binding compared to ATP,17 the receptor
recycling after ligand binding and overall receptor function has not been reported. Also
the inability of suramin, a potent P2X competitive antagonist, to overcome the
0t,B—methylene ATP mediated depolarization of rat isolated vagus nerves (suggests an
unusually strong binding of 0t,B- methylene ATP to the P2X1 that may retard the
receptor function and the recycling.18 Difficulties in using ATP in the presence of
apyrase, an ATPase used to keep the purinergic receptors in the sensitized state, is the

reason behind oc,B—methylene ATP application in platelet investigation. Studies using

0t,B—methelene ATP suggest that this particular ATP analogue can induce a small

calcium influx to elicit a platelet shape change, but not full aggregation.8"9'20

101

 

Figure 3.1 a,“A plausible ATP-binding pocket located between two neighboring

subunits is highlighted in the black rectangle on an electrostatic potential
surface representation of the trimeric AP2X4-B receptor. The surface is
colored on the basis of the electrostatic potential contoured from -30 kT
(red) to +30 kT (blue). White denotes 0 kT. An ATP molecule, scaled
appropriately, is also shown. b, A surface representation viewed ~45° from
panel a. The head, dorsal fin and left flipper domains forming the jaw-
shaped ATP-binding pocket are colored. The putative ATP-binding residues
are in blue for subunit A (Asn 296, Arg 298 and Lys 316) and in red for
subunit B (Lys 70, Lys 72 and Thr 189). c, Close-up view of the highlighted
region in a illustrating subunits A (blue) and B (red). Conserved residues
implicated in ATP binding are labeled, and side chains are in stick
representation. Contours from a 2Fo - Fc electron density map drawn around
the side chains are in green. The electron density for the side chain of Lys 70
is weak and it has been built as an alanine. d, conserved residues, shown in
space-filling representation, are located between the ATP-binding site and
the transmembrane domain—extracellular domain interface. Only residues for
a single subunit are shown” 21 T Kawate et al. Nature 460, 592-598 (2009)
doi:10.1038/nature08198

102

  
     

Ion pore (closed)

Ion pore (open)

Figure 3.2 a) Heterotrimeric model of P2X1 receptor. b) and e), binding ATP to the
extracellular domain (ED) leads to conformational changes in all three
domains and opens up the channel.

103

3.2 POSSIBLE EXPLANATIONS FOR ATP-P2X1 FUNCTION

UNDERESTIMATION

Current understanding of P2X structure and function is not sufficient to explain
the behavior of the ATP binding cassette. It is not clear whether binding of ATP to
P2X1 phosphorylates the receptor that induces the conformational changes for channel
opening, or if ATP simply acts as a ligand for the P2X1 binding site that induces the
channel opening. Speculation suggests that binding of ATP to the putative binding
pocket, a jaw like conformation, closes the pocket inducing the conformation changes
necessary for the channel opening.21 It has not been explained how the channel is again
closed. It is possible that the ATP binding and the subsequent conformational changes
may lead to ATP hydrolysis, leaving ADP in the binding pocket. During this process the
channel may be sensitized, opened and closed and desensitized. This may be the
opportunity for apyrase to break down ADP and bring the receptor back to the
sensitized state.

Note the —CH2 group between first and second phosphate groups in
a,B—methylene ATP in Figure 3.3. Binding 0L,B-methylene ATP to the P2X receptor
would open the channel and induce Ca2+ influx, but in order to be re-sensitized, an???” M
may have to cleave it from the binding cassette. It is not clear how many phosphate
groups need to be cleaved from the ribose in the nucleotide in order be detached from

the P2X binding cassette. It is quite possible that once desensitized with 0L,B—methylene

ATP, P2X would not be sensitized with apyrase.

104

 

a)
N/ I N\>
|\\
o o o N N
ll ll ||
HO-P-O-P-o-P-o-HZC o
"O 'O HO X >
+ H
2N3 OHOH
NH2
b)

/ N
K
ii 8 ii N N
HO-lID-O-lf-HZC-FiLO O
HO HO HO V
.+ |-|
.xL1 OHOH
Figure 3.3 a) Adenosine 5’-triphosphate disodiurn salt, b) methyleneadenosine 5'-

triphosphate lithium salt (cull—methylene ATP), Note the -CH2- group
between first and second phosphate groups in a,B—methylene ATP.

105

In the present study, optimum conditions to completely inhibit the platelet P2Y]
receptor with the P2Y1 specific antagonist MRS 2179 (2'-Deoxy-N6-methyladenosine
3',5'-bisphosphate) have been investigated. Also, an enzymatic system that consumes
ADP and transforms it in to ATP has been employed to further eliminate the responses
from ADP (apyrase used in the platelet suspension to keep the platelet P2X1 receptor in
the sensitized state, also hydrolyzes ATP in to ADP initiating the ADP-P2Y signal
transduction pathway). This situation eliminates the effects of ATP-P2X1 association in

the ADP mediated Ca2+i increase in the platelet cytosol.

3.3 EXPERIMENTAL

Activation of both P2X and P2Y type purinoreceptors increase platelet Ca2+i.
Increased Ca2+i can induce platelet NO production by activating eNOS. Ca2+i can also
activate glycoprotein IIb/IIIa, which is able to induce platelet shape change and
aggregation. In this study, specific biological and analytical experimental platforms
have been used to investigate the differential regulation of platelet Ca2+z', NO and
platelet aggregation induced by P2X1 and P2Y] receptors. Analyses were performed
immediately after platelet rich plasma (PRP) separation or platelet isolation in order to
keep the maximum platelet responsiveness. Washed platelets were kept in 0.5 U/ml
apyrase, which prevents rapidly desensitizing P2X1-type receptors in the sensitized
state. Platelet responsiveness is measured in each experimental protocol by acquiring

the platelet response for ADP.

106

3.3.1 Measurement of P2X1 and P2Y] induced platelet cytosolic Ca2+ (Ca2+i)

increase and their differential regulation with P2X1 and P2Y] antagonists.

Binding of ATP and ADP to their corresponding platelet receptors, P2X1 and
P2Y1, respectively, increases platelet Ca2+z'. Although details of the associated signal
transduction mechanisms are described in chapter 2, in brief, binding ATP to P2X1
opens the Channel and allows the extracellular Ca2+ flux in to the cytosol. On the other
hand, binding ADP to P2Yl initiates signal transductions cascades that results in the
granulated Ca2+ in platelets to be released into the cytosol. When two different
mechanisms govern the same secondary messenger, Ca2+i concentration, it is
challenging to determine the differential regulation by individual agonists. The goal of
this particular series of Ca2+i determinations was to investigate the individual agonist
responses with the use of optimized concentrations of P2X1 and P2Y1 antagonists.

A 2.28 mM DMSO solution of fluo-4 AM (Molecular Probes, Eugene, OR,
F 14201) was prepared by adding 20 ul of anhydrous DMSO in to a vial containing 50
ug of fluo-4 AM (Molecular probes). Just before the preparation of buffered fluo-4,
equal volumes of fluo-4 were mixed with 200 mg/ml pluronic F-127 surfactant
(molecular probes) in anhydrous DMSO to enhance the probe penetration capacity.
Platelets were suspended in Ca2+ free modified tyrode buffer (CaFMTB) containing 0.5
U/ml apyrase, 10 % ACD (acid citrate dextrose buffer, a platelet antagonist) and 5 uM
fluo-4 AM earlier mixed with the pluronic F-127. After one hour incubation at room
temperature (all the incubations containing fluo-4 AM have been conducted in light

excluding containers), platelets were centrifuged and washed two times with CaFMTB

107

containing 0.5 U/ml apyrase and 10 % ACD. The final platelet suspension was prepared
in CaFMTB containing 0.5 U/ml apyrase to have a final platelet concentration of 1010
platelets/ml that was is kept on ice in a dark box until the analyses were performed.
Next, 20 ul of platelet suspension was added to a 21N-Q-10 far UV quartz
cuvette containing 1960 pl of modified tyrode buffer with 2 mM Ca2+ (CaMTB) kept at

37 °C. The cuvette was placed in its cuvette holder in a Fluoromax-4 spectrofluorometer
(HORIBA J obin Yvon, Edison, NJ) with a stirrer. Fluorescence baseline emission of the
sample was measured at 516 nm (with 496 excitation) with stirring for 10 seconds. A 20
pl portion of the agonist or the buffer was then added at 10 seconds and the
fluorescence scan was continued for up to 300 seconds. Agonists ADP and ATP,
inhibitors MRS 2179 (MRS) and NF 449, phosphoenolpyruvate and pyruvate kinase
enzyme mix (En-mix) were prepared in CaFMTB. Whenever applicable, inhibitors
and/or the En-mix were added to the CaMTB in the cuvette and the total volume kept

constant (2 ml) by adjusting with CaMTB.
3.3.2 PRP aggregation measurements

Agonist binding, platelet Ca2+i increase and the Ca2+-CAM mediated platelet shape
change and the aggregation are the steps of ATP and ADP induced platelet activation.
Although the separation of Ca2+i responses by individual agonists is critical, Ca2+i not
only induces platelet aggregation, but also induces NO production that prevents platelet
aggregation. Therefore a series of experiments were conducted to measure the

aggregation of PRP with ATP, a,B—methylene ATP and ADP. The earlier described

108

PEP-PK enzymatic reaction which converts ADP to ATP has been employed in order to
see the individual potential of ATP in PRP aggregation. Sustained aggregation was
expected for both ATP and ADP, as both can increase Ca2+i; no aggregation is expected
with or,B—methylene ATP due to its inability to assist continuous P2X1 operation.

A Chrono-Log 490-2D dual channel aggregometer (Chrono-Log Corporation,
Havertown, PA) was employed for all platelet aggregation measurements. For each
aggregation study, two aliquots of 460 uL PRP were incubated in P/N 312 cuvettes in
the aggregometer incubation wells. Platelet poor plasma (PPP) was used as the
reference. Contents of the test and control cuvettes were adjusted with either inhibitors
and or CaFMTB to 500 nM, constantly stirred using P/N 311 stir bars. All studies were
performed at 37 °C. Data was obtained at 10 data points per second for 6 min. For
studies involving P2Y] inhibition or P2X1 inhibition 10 ul of the appropriate
concentrated MRS 2179 or NF 449 were added to the PRP. In order to distinguish the
effect of ATP on P2X receptor, 10 ul of phosphoenolpyruvate-pyruvate kinase enzyme
system, PEP-PK (680 U/ml PK in 1.44 mM PEP) were added to PRP before the start of

the aggregation.

3.3.3 ATP degradation assay with 0.5 U/ml apyrase

Enzyme apyrase is an essential component of purinergic signaling assessments due to
its ability to keep purinergic receptors in their sensitized state. As described earlier,
apyrase prevented the use of ATP that resembles bioavailable ATP. The objective of
this experiment was to calculate the extent of the ATP hydrolyze by 0.5 U/ml apyrase (a

109

concentration equivalent to the concentration used to sensitize platelets in this chapter)
in to ADP. This piece of data is a very important component in resolving the individual
responses of ATP and ADP in platelet activation.

A calibration curve for different concentrations of ATP in modified tyrode
buffer (MTB) ranging from 1 uM to 10 11M was constructed. In brief 100 pl of ATP and
100 pl luciferin/luciferase (L/L) reagent mixture were combined in a cuvette and the
resultant chemiluminescence was measured exactly after 15 seconds of mixing. Each
measurement was performed in triplicate. For the ATP decay study, 5 uM ATP was
incubated with 0.25 U/ml apyrase, and the resultant chemiluminescence, which
corresponds to the amount of ATP remaining, was measured at every 15 second
intervals. For each time point, individually prepared, separate samples were measured in

triplicate.

3.4 RESULTS AND DISCUSSION

In this work we described two approaches for evaluation of the effect of ATP on
P2X1 function. PRP is a very complex mixture of biomolecules.22 There are number of
plasma ectonucleoside triphosphate diphosphohydrolases (E-NTPDases, plasma
apyrases) that hydrolyze ATP and ADP present in the PRP to keep purinergic receptors
in the sensitized state.”25 Therefore, the first approach consists of the assessment of
PRP aggregation studies where more functional P2X1 receptors exist on platelets. The
results of this experiment are shown in Figure 3.4 (representative curves) and Figure 3.5

(averaged % transmittances).When treated with ADP, PRP shows high aggregation

110

(~37%), which is shown in Figure 3.4a. Although ATP is not able to induce the same
potency, ATP also induces significantly higher aggregation (~23%) shown in Figure

3.4b. One might argue that a greater fraction of ATP induced platelet aggregation in
PRP is due to the break down of ATP by platelet and plasma E-NTPDases to ADP?”24
It is hypothesized that this aggregation could partially be due to ATP hydrolysis to
ADP, but ATP still should contribute to a greater extent. .

When PRP is treated with 0t,[3—methylene ATP, there is no aggregation although
oc,B—methylene ATP induced Ca2+ influx and platelet shape change has been reported
(Figure 3.4c).8’19’20 Chemical structures of ATP and or,B— methylene ATP differ by one
CH2- group in onfi—methylene ATP (Figure 3.3). It is possible that both ATP and
0L,B—methylene ATP open the P2X1 trimer. After initial desensitization E-NTPDase
should be able to clean up the ATP binding pocket and sensitize the receptor for a fresh
cycle. The stable ATP analog a,B—methylene ATP cannot be broken down by NTPDase
and therefore, after initial binding, P2X1 receptors may permanently stay in the
desensitized state. Though, the initial Ca2+ influx due to oc,B-methyleneATP binding is
enough to phosphorilate myosin light chains inducing the platelet shape change,7’26'28
lack of continuous receptor function may have prevented the subsequent platelet

aggregation.

111

50-

 

 

40 -
(a)
8 30 -
g I {i A 0%
AMA ,A . a“ h, 343,1"? , . ,fijb’ “y
E .~. yu'V‘fiv'fi‘vAW;Nfi‘pifihjmlf‘ffi'l Wig“) AUX, U J“ b ‘1' \I J will" V V (b)
"e’ 20 ‘ W” l‘ ‘
a M“
i— /".”l
o\° 10 - In; “
1
ill" “ ‘l‘ ’ V r (c)
1’? .i' I]. 1" \ v 4 I. ' W i ‘ ‘ L ‘ .
0 q 1 "Hit 4“,,” y! («(11va 13’ ’ “ow ‘H "or (d)
'10 I I I T ‘1
300 400 _ 500 600 70
Time (s)

Figure 3.4 Representative aggregation curves for agonist induced PRP aggregation.
Note that (d) PRP and (0) PRP treated with 0t,B—methyleneATP do not
show any significant aggregation compared to (b) ATP and (a) ADP
treated PRP.

112

40-

20-

% Transmittanoe

  

 

 

 

PRP PRP+ATP PRP+ADP PRP+M&ATP

Figure 3.5 Average % transmittance propotional to the PRP aggregation. Note that
PRP and PRP treated with a,B—methyleneATP do not show any significant
aggregation compared to ATP and ADP treated PRP. n=3 *p<0.01

113

Pbosphoenolpyruvate-private kinase system and P2Y] behavior

Pyruvate kinase (PK) or pyruvate 2-O-phosphotransferase catalyses the transfer
of a phosphate group from phosphoenolpyruvate (PEP) to ADP to form ATP and
pyruvate in the presence of Mg2+ and K+ (Figure 3.6).29 There is a large energy drop
(AG = -61.9 K] mol'l) associated with the forward reaction and therefore this reaction is
irreversible.30 Application of the PEP-PK system to PRP is helpful in order to
distinguish the effect of P2X1 from P2Y type receptors.

Typical aggregation curves were observed for PRP and ATP-treated PRP
(Figure 3.7). The addition of PEP-PK in to PRP led to an unusual aggregation behavior
in ATP induced PRP aggregation (7b). A rapid increase in aggregation followed by a
100% recovery is typical when PEP-PK is in the system. In PRP, P2X1 receptors are in
the sensitized state and therefore a continuous Ca2+ flux is expected. ADP can aid ATP
mediated cytosolic Ca2+ increase in two ways. ADP binding to P2Y1 activates Gaq and
PLCB pathways, ultimately mobilizing dense granular Ca2+.31 On the other hand, ADP
activated P2Y12 inhibits the adenylyl cyclase catalyzed cAMP formation.32 In this way,
ADP is switching off the CAMP mediated Ca2+ granulation and assisting maintenance of
higher concentration of Ca2+i .33 Furthermore, the lack of the basal ADP signaling may
be accelerating the granulation of Ca2+i, accumulated as a result of P2X1 mediated
Ca2+ influx.

In the aggregation curve corresponding to the PRP-PEP-PK-ATP (7b), initial rapid Ca2+
influx may be substantial and the most effective towards (IIIBIII activation.”35 ADP

derived as the result of the ATP hydrolysis in this system rapidly converts back to ATP

114

HO
PEP

0H2

 

AG=-61.9 KJmol-1 Mg

 

K+

2

/ N

”k ' \>

i? <3 P. ‘N N

Ho-P-o-P-o-P-o ATP

-0 -0 HO \(0
2Na+ H

OH OH

Figure 3.6 Pyruvate kinase reaction, in the presence of Mg2+ and K+, pyruvate kinase
(PK) transfers a phosphate from phosphoenolpyruvate (PEP) to ADP
results in ATP production.

115

by PEP-PK. Under such conditions, although platelets have continuous Ca2+ flux, the
system is lacking enough support for a high Ca2+ environment due to increased adenylyl
cyclase activity. Despite the fact that the initial Ca2+ flux can induces OLIIBIII activation,

it seems, the system needs to have high and persistent Ca2+i to make it irreversible.

Regulated Ca2+ studies-the P2X1 response

MRS 2179 (MRS) has been used as a competitive and selective P2Y]
antagonist. Structurally MRS resembles ADP and ATP (the MRS IUPAC name is 2'-
Deoxy-N6-methyladenosine 3',5'-bisphosphate). Although MRS is a P2Y] selective
inhibitor, at higher concentrations, it can also inhibit P2X1 (IC50 = 1.15 uM). One of the
difficult issues when handling MRS as a P2Y] antagonist is its high concentration
requirement. Therefore, it is very difficult to selectively inhibit P2Y] without inhibiting
P2X1 to a certain extent. The first aim was to evaluate the minimum MRS
concentration, near its IC50 value for P2X1 that can completely inhibit the cytosolic

Ca2+ mobilizing effect of a known concentration of ADP.

Inhibition of P2Y] with MRS 2179

In order to achieve 100% inhibition of 1.25 uM ADP induced Ca2+i increase, at
least 2.5 uM MRS has to be used (Figure 3.8). Also it is interesting to see the inability
of MRS to entirely inhibit the effect of equimolar ADP on platelet Ca2+i mobilizations.

However, equimolar MRS is sufficient enough to inhibit approximately 97% of the

ADP induced Ca2+z' mobilizations in the presence of PEP-PK (680 U/ml PK in 1.44 mM

116

  
 

 

 

 

 

40 -
(a)
30 -
d)
O
C
(U
SE: 20 -
2 (b)
a
'—
e\° 10 -
C
_- Monti“)
0 _.
0 50 100 150 200 250 300

Time (s)

Figure 3.7 Platelet aggregation is measured in PRP, 5 uM ATP was able to induce a
sustained aggregation (a) while PRP alone did not show any aggregation
(c). In the presence of PEP-PK in PRP, although ATP was able induce its
full aggregation potential at the beginning, it seems ATP along cannot have
a sustainable aggregation, (b) without its break down product, ADP.

117

PEP). This trend has been shown for both 1.25 and 5 uM ADP. In general, doubling the
MRS concentration compared to ADP is able to achieve 100 % inhibition of the ADP
induced Ca2+i mobilization. To avoid deleterious effects by using higher concentrations
of MRS, 2.5 uM MRS in combination with PEP-PK has been used in studies reported
here. However, 2.5 uM MRS can only completely block ADP concentrations _>_1.25
.uM; therefore, the maximum ADP concentration that can be generated in the system

should be lower than 1.25 uM.

Determination of P2Y] independent ATP induced P2X1 response

In the presence of 2.5 uM MRS and PEP-PK, a 100 % inhibition of 1.25 uM ADP
induced Ca2+i increase was detected (figure 3.9 and 3.13). A considerable increase in
Ca2+i under the same inhibitory conditions has been observed upon stimulation of
platelets with 5 uM ATP (Figure 3.90). Compared to the 100 % increase in Ca2+i
observed with 5 uM ADP (Figure 3.9a and Figure 3.13a), 5 uM ATP has shown a 24 %
increase (Figure 3.13d). This ATP associated Ca2+ influx is unique and only due to the
P2X1 activation. However, before a conclusion can be drawn, it is necessary to validate
that the amount of ADP present in the system is below the 1.25 uM concentration limit
that can be completely inhibited by the 2.5 uM MRS. In the presence of 0.5 U/ml
apyrase in the platelet suspension, it is possible that ATP hydrolysis generates a
substantial concentration of ADP. Therefore, it is very important to evaluate this

particular ADP concentration.

118

- Platelets +PEP-PK+ 5 uM ADP
Platelets +PEP-PK+ 1.25 uM ADP

10000 3

 

 

 

n a a +
Fluorescence mtensrty - Cytosolic Ca2
N
O
o
o
o

 

b 075 i {25 2:5 3.}5 é
"° PEP'PK [MRS 21791- (0M)

Figure 3.8 Platelet cytosolic Ca2+ increase was investigated upon addition of 1.25 and
5 uM ADP. Except for the first two bars, all other platelet samples contain
PEP-PK and different concentrations of MRS. MRS blocks the Ca2+i
increase in a concentration dependent manner. Comparing the Ca2+i
concentration in the two series, it can be conclude that equimolar
concentrations of MRS can block the cytosolic Ca2+i increase by 97%.
When the MRS concentration is doubled, 100% inhibition of ADP induced
Ca2+i increase can be achieved

119

ATP (5 uM) decay assay with 0.5 U/ml apyrase

Indirect determination of ADP formation as a result of the action of 0.5 U/ml on
5 uM ATP is the aim of this experiment. ATP-diphosphohydrolase, also known as
apyrase, has an exceptionally high ATPase/ADPase ratio (10) and has been used to

7,36,37

sensitize platelets. Apyrase hydrolyzes nucleoside di- and triphosphates to

monophosphate esters and inorganic phosphate in the presence of a divalent cation like

Ca2+ and Mg2+.38
ATP —> ADP+Pi —+ AMP+2Pi39

A time dependent ATP decay profile in the presence of apyrase was generated by
employing a luciferin/ luciferase bioluminescence assay. In order to maintain the exact
same conditions in Figure 3.9c, a solution of 5 uM ATP with 0.5 U/ml apyrase has been
used. Due to the temporal resolution of the method, a fitted exponential decay curve
(Figure 3.10) has been used to calculate the remaining ATP concentration in the system
within the first 20 seconds, the critical time period for ATP induced Ca2+ influx after the
addition of ATP in to the platelet suspension shown in Figure 3.9c.

Figure 3.11 represents the projected data from Figure 3.10. The concentration of
degraded ATP after 10 seconds is 0.9937 uM. Even if all the degraded ATP remained
as ADP, it is still well below the concentration needed to overcome the P2Yl inhibitory
conditions elicited by 2.5 uM MRS shown in Figure 3.90. Furthermore, apyrase used in
this experiment is able to break down 1 ADP molecule for every 10 ATP molecules. In

addition to that, PEP-PK enzyme system used in the platelet suspension used evaluate

120

2.8e+5 -

 

 

 

2.6e+5 -
.é‘
m
c
.03 2.4e+5e
.E
a)
8
8 2.26+5 - ‘ 4M 1 a
(I) l ‘ W
o ' . ~ b
'5 ‘ W
2 2.0e+5J
u.
1.8e+5 - c
1‘ .
1.66+5 r r l l I
0 20 40 60 80 100

Time (s)

Figure 3.9 Platelet cytosolic Ca2+ increase upon addition of ADP and ATP. a) platelets

with 5 uM ADP, b) platelets with 1.25 uM ADP, c) platelet initially
incubated with 2.5 uM MRS and PEP-PK then with 5 uM ATP. d) platelet
initially incubated with 2.5 uM MRS and then with 1.25 uM ADP.

121

+ Experimental decay curve
—0— Fitted decay curve
4 a
3 a
2
:L
I
'5? 2 -
I.—
s
1 ..
0 i

 

 

I I I I I I

0 20 40 60 80 1 0(

Time (s)

Figure 3.10 ATP breakdown profile of 5 uM ATP treated with 0.5 U/ml apyrase. Curve
has been extrapolated to find the ATP decay within the first 20 seconds.
The first data point at time zero is the chemiluminescence signal of 5 uM
ATP without apyrase. A theoretical exponential decay curve (white circles)
has been constructed using MATLAB®, which describes the decay
behavior within first 20 seconds and the curve equation is f(t)=1.476e(-
0.0245t). '

122

the Ca2+ influx should convert the majority of ADP formed back to ATP. Therefore the
actual ADP concentration in the platelet suspension should be substantially lower than
0.9937 uM. With these evidences, we can strongly conclude that the 24% increase in
platelet Ca2+z' compared to the 5 uM ADP induced Ca2+i increase (Figure 3.9a and
3.13a) is solely due to ATP gated P2X1 Ca2+ influx (Figure 3.9c and 3.13c).

In this particular experiment, the MRS concentration is 2.17 times higher than
its IC50 value for P2X1, which is 1.15 uM. Therefore, the 24 % ATP induced Ca2+
influx should be due to the activation of less than 50% of the P2X1 receptors.
Therefore, 5 uM ATP should be able to induce a Ca2+ influx greater than the 1.25 uM
ADP induced Ca2+z' increase. There is a ~42 % increase in Ca2+i corresponding to 1.25
uM ADP without inhibitors. If we assume that, due to MRS inhibition, only 50% of the
P2X1 receptor function was lost, then the Ca2+ influx due to ATP should be ~ 48%
compared to the 5 uM ADP induced Ca2+i increase. We have shown that 1.25 uM ADP
is sufficient enough to induce a sustained aggregation in PRP (Figure 3.4). Therefore,
under P2X1 functioning conditions, extracellular ATP along can induce platelet

aggregation through P2X1 Ca2+ channel gating.

123

20 ........-----.-,.._.--.-..- --.--- --..-- .. - ---“.a . . r -, _ 5.---..

1 l .
6 4M~mgv~nvw -..... 7......— ._ ~.. ~- -s-.— -‘—- -.- -«~.-.-_---~. ~e_.~«~_-. .-. - Nx-‘w‘ m-q—qv -..-.--u-.~ .. «_V .‘g\!—r.'~'v‘*-~\-r'.‘- -- ---- .—‘,-'--.~..- ....-...-..-..—. .. - .. . .... A- .
i
- l

1 4 ...F.aaw.-...._.._..-_..__----..--.--...--.--.-..____..---..______-N..-.-aa.-.._......-.”-..,_..,.._-_._...-.--.... --..-. “a- W.-.
U

‘I Z _i..-__.-.VM-..M-.._.__.-_._V.....- .... .-..--.........-,..-.v7.4, a_2._ ---—..1. -.-.._._ --..... _................a........._. _,_.-.a‘ .-. .... u. H.
' .
.

 

l
0 8 -... .-.- .Wue -_ .---.._..---,- -...-..._..._.___. .-....-.,, a, _. , ,
.

Amount of ATP decayed- uM

.
.

0.0 e 4 . i

 

 

 

Figure 3.11 ATP breakdown profile of 5 uM ATP treated with 0.5 U/ml apyrase within
first 20 seconds. The curve has been constructed using the fitted decay

curve described in Figure 3.9. Note the 0.99 pM ATP decay (arrows) at
10 seconds.

124

230

225

x103

220

Fluorescence ~ Ca2+ f,

205

200

210 -

P + 2.5 uM MRS + PEP-PK + 5 uM ATP

.1

‘

 

 

    

     

 

 

Agonist addition P + Me P (b)
8 13 18 23
Time (s)

Figure 3.12 Differentiation of the magnitude of ATP and a,B—methyleneATP induced,

P2X1 mediated cytosolic Ca2+ influx. Both platelets suspensions
contained 0.5 U/ml apyrase. ATP treated sample additionally contained
2.5 uM MRS and PEP-PK. Note the 3.5 times higher Ca2+ influx in ATP
induced platelet sample. Curve a) is the smoothed data and the curve b)
the raw data of the experiment

125

Reasons behind the underestimation of the role of extracellular ATP and P2X1

Continuous function of P2X1 receptor is a result of the ectoapyrase’s ability to
recycle and sensitize the receptor after ATP binding. Apyrases and ATPases both are
ATP hydrolyzing enzymes. ATPases are identified as substrate specific enzymes while
apyrases can hydrolyze both tri and diphospho nucleotides.4O Thus, it is important to be
aware of the possible ways by which apyrases sensitize P2X1 receptors.
P2X1 receptors are rapidly desensitized immediately after ligand binding.l4’15
Addition of apyrase in washed platelets with desensitized P2X] receptors (no Ca2+
influx response is observed for 0L,B—methylene ATP under this condition) brings them
back to the sensitized state. It seems apyrase is able to cleave off phosphate group(s) of
ATP bound to the binding pocket of the extracellular domain of P2X1. Some reports
suggest that ADP binding to P2X1 can desensitize the receptor.24’41 It is not clear
whether or not initial ATP binding is associated with a cleavage of a phosphate group
from ATP due to the P2X1 activity leaving ADP in the P2X1 binding pocket, which
makes the receptor desensitized. If this is true, desensitized receptor nucleotide binding
site should be occupied by ADP. The assumption about ADP mediated P2X1
desensitization could be a result of this behavior. Under these assumptions, we can now
explain a possible way of apyrase mediated P2X1 sensitization.

When an apyrase interacts with ADP bound P2X1 receptor it would cleave off a
phosphate group which may help the remaining AMP to leave the binding pocket. This
may bring the receptor to the sensitized state which is ready for new ATP binding cycle.

When a,B—methylene ATP, a non hydrolysable analog of ATP, is used to activate

P2X1,

126

 

 

 

0000

8000

6000

4000

Fluorescence intensity-Caz?

2000

    

P+ATP P+MeATP

Figure 2.13 a) Averaged normalized Ca2+i increase in ADP, ATP and a,B-MeATP
treated platelets in the presence of 0.5 U/ml apyrase. a—platelets with 5 nM
ADP, b-platelets with 1.25 uM ADP, c-platelet initially incubated with
2.5 pM MRS and then with 1.25 M ADP, d—platelet initially incubated
with 2.5 pM MRS and PEP-PK then with 5 pM ATP, e-platelets with 0L,[3-
MeATP, b) Raw data indicating the Ca2+i increase in ATP and 0:43-
MeATP treated platelets, n=3 rabbits, P<0.01

127

the initial receptor opening and the desensitization would not be a matter (refer the
structures of cub—methylene ATP and ATP in Figure 3.3. However when it comes to
the sensitization, apyrase or NTPDases, would not be able to cleave any more
phosphate groups on a,B-methylene ATP due to its structure. Therefore, it is not
surprising to see a Ca2+ influx which barely enough to induce a platelet shape change

with a,B—methylene ATP.

Utilization of optimized P2Y] inhibitory conditions to compare the effect of ATP

and (LB—methylene ATP on P2X1 mediated platelet Ca2+ influx

Previously described platelet aggregation studies, performed with PRP reveals
the inability of (LB-methylene ATP to induce any aggregation (Figure 3.4), which is
consistent with the literature. Figure 3.12 and 3.13 b) a shows the relative magnitudes
of 5 uM ATP and 5 uM a,B— methylene ATP induced Ca2+ influxes. Here we have
employed 2.5 uM MRS and PEP-PK enzyme substrate system only with ATP study, but
not with a,B—methylene ATP induced Ca2+ influx study. During these experiments,
platelets were suspended in 0.5 U/ml apyrase. Even with greater than 50% P2X1
inhibition, ATP was able to induce two times as much Ca2+ influx as a,B—methylene

ATP.

128

3.5 CONCLUSIONS AND OTHER CONSIDERATIONS

In this work, we hypothesized that the ATP receptor, P2X1 plays a critical role
in platelet function. Based on our previous investigations in chapter 2 on ATP behavior
as an agonist and antagonist, depending on the concentration, we predicted that previous
findings in the literature conducted with cull—methylene ATP have underestimated the
P2X1 function and the potency of extracellular bioavailable ATP.

Primarily, we have demonstrated that oc,B—methylene ATP cannot induce
platelet activation and subsequent platelet aggregation. However, this is not due to the
lack of P2X1 receptor potential, but rather the unusual behavior of 0L,B—methylene ATP
in the receptor gating. We initially demonstrated that a,B—methyleneATP cannot induce
platelet aggregation in PRP, which is consistent with the literature. Although a similar
study with ATP demonstrated a substantial and sustained platelet aggregation, it was
challenging to distinguish this behavior from ADP, which is the immediate hydrolyzed
product of ATP due to plasma or membrane apyrases. This goal was successfully
achieved by inhibiting P2Y] receptor function with MRS while preventing ADP
accumulation in the system with the use of PEP-PK enzyme substrate system. Although
1.25 uM ADP was unable to induce any Ca2+i increase under this conditions, 5 uM
ATP was able to induce a considerable Ca2+z' increase which is purely due to the P2X1
mediated Ca2+ influx. It is very important to mention that, the P2Y1 inhibitory condition
we used here was two times higher than the IC50 value of the inhibitor for P2X1.
Therefore, this considerable Ca2+z' increase is a result of the function of less than the 50

“/0 P2X1 receptor population.

129

The P2Yl inhibitory conditions applied here was enough to inhibit the effect of
ADP concentrations less than 1.25 nM. To ensure that, the application of 5 uM ATP, in
the presence of 0.5 U/ml apyrase, would not generate ADP concentration over 1.25 nM,
we have conducted a kinetic study to assess the ATP degradation under same
experimental conditions. Results from this experiment suggest that, under these
experimental conditions, the ADP accumulation in the system should be lower than the

1.25 uM limit.

Similar studies conducted with a,B—methylene ATP (without P2Y] inhibition or
PEP-PK ADP degrading reaction) has demonstrated that it can only induce a P2X1
mediated Ca2+ influx that has a half magnitude of the parallel ATP stimulation
conducted with over 50% P2X1 inhibitory conditions. Although a,B—methylene ATP
has a higher binding affinity towards P2X1 than ATP“, this binding is not reflected in
its capacity for overall P2X1 function regulation.

Therefore we conclude that the ATP gated P2X1 receptor is capable of inducing
platelet aggregation depending on the extracellular ATP concentration. This also
explains the platelet hyperactivity in disorders where RBCs can release unusually high
amounts of ATP into the circulation and conditions where RBCs are more prone to lysis
and create localized high concentration ATP environments in the circulation.““15 In this
study, it has also be shown that the use 0c,[3—methylene ATP to demonstrate the P2X]
receptor function in the literature has underestimated the P2X1 receptor potential and

the significance of bioavailable ATP in platelet hyperactivity and aggregation.

130

10.

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Protein Kinases Blood Cell Funct. 1993:245-298.

Kovacsovics TJ, Hartwig JH. Thrombin-induced GPIb-IX centralization on the
platelet surface requires actin assembly and myosin II activation. Blood.
1996;87:618-629.

Ainsworth S, Kinderlerer J, Gregory RB. The regulatory properties of rabbit
muscle pyruvate kinase. The influence of substrate concentrations. Biochem J.
1983;209:401-411.

Ambasht PK, Kayastha AM. Plant pyruvate kinase. Biol Plant. 2002;45:1-10.
Purvis JE, Chatteijee MS, Brass LF, Diamond SL. A molecular signaling model
of platelet phosphoinositide and calcium regulation during homeostasis and
P2Y1 activation. Blood. 2008;112:4069-4079.

Ferreira IA, Mocking AIM, Feijge MAH, et al. Platelet Inhibition by Insulin Is
Absent in Type 2 Diabetes Mellitus. Arterioscler, Thromb, Vasc Biol.
2006;26:417-422.

Chang T-S, Lee K-S, Lee G-Y, et al. NQ-Y15 inhibits the calcium mobilization
by elevation of cyclic AMP in rat platelets. Biol Pharm Bull. 2001;24:480-483.

Cosemans JMEM, Iserbyt BF, Deckmyn H, Heemskerk J WM. Multiple ways to
switch platelet integrins on and off. J Thromb Haemostasis. 2008;6:1253-1261.

133

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

Leisner TM, Yuan W, DeNofn'o JC, Liu J, Parise LV. Tickling the tails:
cytoplasmic domain proteins that regulate integrin alpha IIbbeta 3 activation.
Curr Opin Hematol. 2007;14:255-261.

Ardlie NG, Perry DW, Packham MA, Mustard JF. Influence of apyrase on
stability of suspensions of washed rabbit platelets. Proc Soc Exp Biol Med.
1971;136:1021-1023.

Hechler B, Lenain N, Marchese P, et al. A role of the fast ATP-gated P2X1
cation channel in thrombosis of small arteries in vivo. J Exp Med.
2003;198:661-667.

Alvarez A, Chayet L, Galleguillos M, et al. Characterization of ATP-

diphosphohydrolase from rat mammary gland. Int J Biochem Cell Biol.
1996;28:75-79.

Laliberte JF, Beaudoin AR. Sequential hydrolysis of the gamma - and beta -
phosphate groups of ATP by the ATP diphosphohydrolase from pig pancreas.
Biochim Biophys Acta, Protein Struct Mol Enzymol. 1983;742:9-15.

Komoszynski M, Wojtczak A. Apyrases (ATP diphosphohydrolases, EC
3.6.1.5): function and relationship to ATPases. Biochim Biophys Acta, Mol Cell
Res. 1996;1310z233-241.

Murugappan S, Kunapuli SP. The role of ADP receptors in platelet function.
Front Biosci. 2006;11:1977-1986.

Bo X, Burnstock G. Characterization and autoradiographic localization of
[3H]alpha ,beta -methylene adenosine 5'-triphosphate binding sites in human
urinary bladder. Br J Urol. 1995;76:297-302.

Papadimitriou CA, Travlou A, Kalos A, Douratsos D, Lali P. Study of platelet
function in patients with sickle cell anemia during steady state and vaso-
occlusive crisis. Acta Haematol. 1993;89:180-183.

Kenny MW, George AJ, Stuart J. Platelet hyperactivity in sickle-cell disease: a
consequence of hyposplenism. J Clin Pathol. 1980;33:622-625.

Amer J, Zelig O, F ibach E. Oxidative status of red blood cells, neutrophils, and

platelets in paroxysmal nocturnal hemoglobinuria. Exp Hematol. 2008;36:369-
377.

134

CHAPTER 4

4.1 THE EFFECTS OF CFTR DYSFUNCTION IN ALVOLAR

MACROPHAGES ON CYSTIC FIBROSIS LUNG DISEASE

CF is a genetic disorder mainly characterized by chronic lung infections and
deteriorating lung function caused by the mutations in CFTR gene and its product
CFTR, which functions as a chloride channel in epithelial membranes."3 Some of the
proposed mechanisms related to the mutated CFTR focus on its chloride conductance
function such as an altered airway surface liquid (ASL) composition and volume,
decreased lysosomal acidification in phagocytes, defective airway sub-mucosa] gland
function and abnormal immune cell response are prominent as described in section
1.4.1.3'7 Although these mechanisms provide some explanation for CF pathogenesis,
the major unanswered question in CF biology, with considerable importance in
development of new therapies, is how CFTR mutations cause lung disease.8

As a result of decreased mucus clearance, CF airways become fertile ground for
bacteria and viruses.6 These conditions have shown to inactivate the pulmonary
epithelial defensins limiting the bacteria identification and destruction.9 Defensins are
small cysteine-rich cationic peptides on cell membranes specified for pathogen
sensing.lo An accelerated bacterial colonization can be expected when defensins are
defective in CF airways.11 These infections are the prime cause of death of people with

CF, although advancements in CF therapy has significantly increased the life span of

the patients from 15-20 years to 35-40 years.12

135

Numerous CF TR mutations are associated with CF. Efforts to relate the mutated
CFTR phenotype with the severity of the CF lung disease have not found much
success.13 The most widely exhibited phenotype is caused by deletion of phenylalanine
at position 508 commonly abbreviated as AF 508.l4 CFTR mutations can be broadly
categorized in six classes.2 (1) defective CFTR synthesis, (2) defective processing, (3)
malfunction in the channel regulation, (4) interferences with channel conductance, (5)
partially defective CFTR production or processing, (6) defective regulation of other
channels. CF TR functionality is determined by evaluating the mean channel open time
(MCOT) and the closed time (MCCT). Wild type and AF508 mutant both show a
MCOT of 220 ms but the MCCT showed a variation of 230 ms from wild type to 7.8
ms in AF 50815 . There are several different ways that CFTR may elicit its effects on
cellular functions. Being a chloride channel, it has direct regulatory role on
transmembrane pH gradient and associated mechanisms 16’”. CFTR is also involved in
the regulation of growing list of many other cellular transport processes as summarized
in Table 1CFTR.‘8"9

When investigating the mutated CFTR on CF lung disease, a considerable
attention has to be given to the lung defense system. Although the ASL secretion,
composition, .and the mucus clearance in the lung epithelia play a role in lung
protection, ingestion of pathogens by alveolar macrophages is the one of the foundation
mechanisms of pulmonary innate immunity against pathogen invasion. Therefore it is
very important to find the role of CFTR on macrophages function. Despite the fact that

CFTR has been characterized as a receptor for P. aeruginosa, allowing for recognition,

136

Membrane Membrane

 

 

 

 

 

spinning domain 1-MSD‘I spinning domain 2-MSDQ
ATP ATP
rains.“ _
ADP ; . carboxyl terminal-TRL
AF508 mutafion/ ' (threonine, arginine,

N l t' d leucine)

Nucleotide 892.80 lde . 2 NDBZ

binding domain 1-NDB1 '" mg omain '
Regulatory domain

Figure 4.1 Proposed structure of CFTR protein which contains 1480 amino acids.
CFTR activation depends on the phosphorylation of regulatory (R) domain
mainly through protein kinase A (PKA) but other kinases like protein kinase
C (PKC) are also involve in the R domain phosphorylation. The channel
pore which conducts chloride ions is controlled by the conformation changes
of the two nucleotide-binding domains (NDB) linked to the two membrane-
spanning domains (MSD) contains six membrane-spanning alpha helixes.
Although it is not certain the carboxyl terminal consisting of threonine,
arginine, and leucine (TRL) of CFTR is proposed to involve in the
regulation of other channels, signal transduction, and localization at the
apical plasma membrane. Each, portions of which form a chloride-
conductance pore. The common AF 508 mutation in CF occurs on the surface
of NDBl.

137

internalization and death of the pathogen,20 evidence in the literature is conflicting
about the effect of CFTR on macrOphage bacterial killing ability and no information
available about its phagocytosis. For example, one such study showed the requirement
of CFTR for phagosomal acidification and subsequent bacterial killing,“ while
opposing studies performed later denied the involvement of CFTR in macrOphage
phagosomal associated bacterial killing.”18 In summary, the role of defective CFTR in
alveolar macrophages on their function as well as the effect of such macrophage

dysfunction on CF pathogenesis have not been thoroughly investigated.

4.1.1 Macrophages in brief

Macrophages are the major differentiated cells in the mononuclear phagocyte
system as shown in Figure 4.2. They are widely distributed in the body and
characterized by great structural and functional heterogeneity.19 Differentiated
macrophages are involved in numerous functions other than the phagocytosis such as
bactericidal activity, antigen presentation, tumor cytotoxicity, removal of aged or
damaged cells, injured tissue repairing, bone resorption, and special lipid metabolism.20
Macrophages originate in the bone marrow where the resident macrOphages co-exist
with their monocytic precursors such as monocytes, promonocyte, and monoblasts.
Monocytes spend less than 24 in bone marrow hours before entering the circulation.
Once monocytes undergo extravasations (leaving the circulation), they never return,

instead remain in the tissues as macrophages.19’21’22 As 1.5 X 106 monocytes are

138

     

Neutrophil

Eosinophil

 

Basophil

Figure 4.2.

 
 
  

Hemopoitic stem cell

Fibroblast Myeloid progenitor

 

Macrophage

Macroglia Pre-ostioclasts

Dendritic cell

Ostioclast

 

Myelinating oligodendrocyles

Differentiation of myeloid progenitor cells into different mononuclear cell
types. GM-CSF, granulocyte—macrophage colony-stimulating factor; IL-4,

interleukin-4; MCSF, macrophage colony-stimulating factor; TREM,
triggering receptors expressed by myeloid cells.

139

produced in mouse bone marrow per day, it is predicted that there should be
substantial macrophage consumption in order to protect the body from infectionsnThe
discovery of macrophage specific growth factors, mostly secreted by macrophages such
as macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony
stimulating factor (GM-CSF) resolved the mystery of mononuclear phagocyte such as
macrophages, differentiation from monocytes.23 Macrophage exposure to external
stimuli such as phagocytosable particles and endotoxins stimulates the synthesis and the
secretion of M-CSF and GM-CSF.24’25 Also macrophage derived cytokines such as
interleukin-1 (IL-1) and tumor necrosis factor (TNF) have been shown to induce the
production of M-CSF and GM-CSF in fibroblasts (cells that synthesize extracellular

26'” At the same time GM-CSF can stimulate

matrix) and in endothelial cells.
macrophage TNF synthesis,29 explaining the substantial production of macrophages

during infections.

Macrophages are one of the longest lived cells in the body derived from the
circulation. Earlier it was reported that macrophages have a half life of two weeks to
one month,”32 but recent reports suggested a half life up to four months.33Although
they are derived from a common bone marrow progenitor, macrophages display a
remarkable heterogeneity depending on their ultimate habitat.“35 Depending on the
tissue micro environment, macrophages are further transformed into subpopulations.
Different phenotypes of macrophages with regard to their morphology, cell surface
antigen expression, and function are differentiated from monocytes depending on the
physio-chemical environment of the tissue. Macrophages are named on the basis of

their habitat: alveolar macrophages in the lungs, peritoneal macrophages in the

140

 

 

 

 

 

 

 

 

 

 

 

 

 

Name of the channel Function
ENaC sodium channel Na+ absorption
KVLQTI K+ channel
HCO3'/Cl' exchanger (pancreas) Gap-junction channels
ICOR Cl'channels Mucus secretion
Ca2+ and swelling-activated Cl' channels ATP-transport
ROMK2, Kir6.l (K channel) Glutathione transport
KCNN4 (SK4, ik) K channel
AQP3,7 water clearance

18,19,36

Table 4.1 CFTR regulated the key transport processes

14]

peritoneum, Kupffer cells in the liver, microglia cells in the brain, and osteoclasts in the

bones.

4.1.2 Pulmonary alveolar macrophages

Lungs have two distinct phenotypes of macrophages: alveolar macrophages
(AM) and interstitial macrophages (1M).37 AMs are frequently found protruding from
alveolar epithelia to the alveolar lumen. The habitat of interstitial macrophages is the
interstitial space between alveoli, much closer to the circulation. Because of this micro
environment, interstitial macrophages play a significant role in specific immune
responses, regulatory and proliferative functions while alveolar macrophages are critical
in primary defense against inhaled particulate matter, environmental toxins and more
importantly, microorganisms.‘5’35’38 It has been shown that alveolar macrophage damage
result in enhanced host susceptibility to microbial infection and chemotoxins.37

Investigations described in this chapter are focused on CFTR dysfunction in pulmonary

alveolar macrophages on their function.

4.2 ALVEOLAR MACROPHAGE FUNCTION, ENERGY METABOLISM

AND CFTR

Macrophage glucose transport is mainly occurs through facilitative glucose

transporter GLUTl , in addition, it has been shown that P. aeruginosa ingestion by AM

142

  
    
 

 

C

0)

Na" Cl g

Almay P2 receptors Ligand-gated ;.
surface . ---- 0 chloride channels; g
quuld '5;

(ll
=5
10
‘Do
on
0m

..... .. .J .,._.. _

lnterstitium

a Cl' Q 9 Epithelial sodiqu

channel (ENaC)

 

Figure 4.3. CFTR involved Airway surface liquid (ASL) volume regulation in airway
epithelium. (A) In healthy airway epithelia, CFTR mediated airway
epithelial cells release of ATP onto the airway surface and hydrolyzed
products of ATP induced purinergic receptor activation, retards the
epithelial sodium ion channel, ENaC. Moreover, CFTR directly inhibits
ENaC and regulate Na+ absorption. Normal airway epithelia balance Na+
absorption against Cl_ secretion to maintain ASL homeostasis, through
CFTR mediated regulatory effects. (B) CF airway epithelia do not express
functional CFTR protein in the apical membrane. However, P2 receptor
signaling regulates the ENaC up to certain extent. Therefore CF airway
epithelia have a tendency to absorb more salt and water than normal
because of the absence of CFTR inhibition of ENaC and CFTR Cl—
secretion. This phenomenon results in unusually dehydrated airway
epithelia with decreased ASL volume. The scenario is further worsening
by elevated activity of mucus glands.3'4’39’40

143

is glucose dependent.“42

Although there is no available direct evidence for defective
macrophage phagocytosis in CF, a study on CF pulmonary neutrophils showed that
investigators were unable correlate the effect of CFTR mutation as they were unable to
detect CFTR on neutrophils.43 However, later investigations on the same issue
suggested the involvement of defective CFTR in CF neutrophil malfunction.44 In
general, it seems that defective CFTR plays a role in CF macrophage function. An
active phagocytic cell such as the macrophage should have intensive energy

consumption when activated and therefore it is likely to have a direct correlation

between macrophage phagocytosis and its energy metabolism and CFTR function.

4.2.1 Dependency of Macrophage energy metabolism on the CFTR function

Energy generation pathways for different macrophages are unique. For example,
the energy metabolism of alveolar macrophages relies primarily on aerobic respiration;
whereas peritoneal macrophages are generally dependent on glycolysis, though they
have mitochondria. This may be a result of the lungs being an oxygen rich organ and
the requirement for an intense energy source to permit instantaneous phagocytosis, as
the lungs are continually exposed to infectious materials in the air.20 Away from
abundant energy sources like plasma, macrophages are able to produce ATP and
function under adverse conditions by utilizing a number of energy sources such as lipids
other than the main energy source, glucose.”46 Energy intensive activities such as,
phagocytosis and phagosomal bacterial killings require macrophages to mostly depend

. . . . 47
on energy from glucose whether eondltlons are aerobic or anaeroble.

144

If conditions are aerobic, macrophages generate most of their energy by
metabolizing glucose through glycolysis.48’49 An increased macrophage half life has
been achieved by increasing macrophage glucose uptake through a phosphoinositide 3-
kinase (PIBK) and phospholiphase C (PLC) mediated mechanism (Figure 45).”52
Despite the fact that the exact mechanism is unknown, the PI3K pathway has emerged
as a key regulator for both glucose transporter expression and trafficking glucose in
response to extrinsic signals.51’53’54 There are studies which demonstrate an important
link between energy metabolism and the macrophage nitric oxide productionss’56 Also,
increased glucose uptake is associated with increased NADPH generation, an energy
source for superoxide and other reactive oxygen species generation, which is required
for the bactericidal role of macrophages. Moreover, there is an evidence for the insulin
independent glucose transporter, GLUTl to be the basic glucose transporter expressed
in activated macrophages.57 In order to attenuate macrophage oxidative ‘stress and
related disorders associated with enhanced glucose uptake in diabetes, measures to

decrease the glucose uptake have been suggested.58

Depletion of cellular ATP triggers the up-regulation of GLUTl and this process
has been shown to regulate the glucose uptake ability of endothelial cells.”’60 The
inhibition of GLUTl by cytoplasmic ATP accumulation has been reported,61 suggesting
a concentration dependent regulation of GLUTl by ATP. Before this very interesting
finding, ATP driven GLUTl glucose transport inhibition triggered by AMP and ADP
was reported.62 In other words, ATP breakdown products stimulate glucose uptake. It
has also been suggested that the similar mechanisms may govern the glucose uptake

process in cell types where GLUTl is the major glucose transporter.61 Therefore

145

 

Figure 4.462 Carruthers model of ATP controlled GLUTl modulation in RBCs. The

figure to the left shows a section through the catalytic region of one
dimer of tetrameric GluTl embedded in a lipid bilayer and viewed from
the plane of the bilayer. In the absence of ATP or when AMP is bound to
GluTl, tetrameric GluTl contains only one sugar binding site per
monomer and newly imported sugar has easy access to bulk cytosolic
water. Upon ATP binding (right), a conformational change occurs that
restricts diffusion of sugar to and from the cytosol (a water-filled cage is
formed) and exposes another sugar binding site per monomer. Under
these conditions, a newly imported sugar has high probability of being
exported back out of the cell or reacting with the sugar binding site.
When tetrameric GluTl is reduced to dimeric GluTl, the ATP binding
site is lost, the cage relaxes, and a sugar binding site is lost.62

146

macrophage glucose transport appears to be an ATP sensitive mechanism. Depending
on the ratios of AMP: ATP and ADP: ATP, the macrophage may sense the energy
demand and determine their energy requirements, subsequently controlling glucose

transport.

CFTR assisted conductive ATP transport from the cytosol to the extracellular
matrix is well established.”68 Correlation between increased glucose uptake and higher
cytosolic ATP concentration is also profound.”71 Higher ATP concentrations in the
cytosol result in higher cAMP concentrations, leading to increased PKA activation.72'74
Subsequent R domain phosphorylation and ATP hydrolysis in CFTR opens the chloride
channel activating CFTR (Figure 4.1). CFTR assisted conductive ATP release may
induce more ATP production, demanding more glucose uptake. The resulting increased
rate of glycolysis generates higher cytosolic concentration of glucose-6-phosphate for
the citric acid cycle and the pentose phosphate pathway.48 Also extracellular ATP
stimulated purinergic activation of glucose transport has been shown for other cell
types.75 Therefore increased glucose uptakes in macrophages generate more energy as
well as reducing power which are essential for active phagocytosis and bactericidal

activities .37

Considering this, it would be valuable to analyze the behavior of CFTR and
determine the abundance of the mutation AF508 in CFTR on the apical membrane
compared to the wild type. This will give us the opportunity to rule out the contribution
from abnormal CF TR residence time on the apical membrane and the contribution from
defective performance of the each CFTR for the overall CFTR functionality. Wild type
CF TR is docked in posts Golgi -membrane in close proximity to the apical membrane

147

and has an average life of 16-24 hour depending on the cell type. It has been shown that
AF508 CFTR rapidly disappears from the apical membrane and does not reappear.76
Overall, in cystic fibrosis, the number of CFTR‘on apical membrane at a given time is
less compared to the wild type and the chloride channel open time is also significantly
less. Therefore CFTR inhibition would be an effective way to evaluate the effect of

mutated CFTR in cystic fibrosis.

In an attempt to reconcile these diverse findings and mimic the RAM CFTR
dysfunction in CF lung disease, experiments were performed to investigate the effect of
CFTR inhibition on RAM phagocytosis and glucose transport. We have previously
shown that the Zn2+ activated C-peptide can enhance the RBC glucose uptake through a
GLUTl mediated mechanism and increase ATP release.” Therefore here we also
investigated the effect of Zn activated C-peptide on RAM glucose uptake and IgG

opsonized bacteria particle ingestion.

148

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149

4.3 EXPERIMENTAL

4.3.1 Isolation and purification of rabbit alveolar macrophages:

Rabbits were anesthetized with ketamine (8 ml/kg, im) and xylazine (1 mg/kg,
im) followed by pentobarbital sodium (15 mg/kg iv). A cannula was placed in the
trachea, and the animals were ventilated with room air. A catheter was then placed into
the carotid artery for administration of heparin and for phlebotomy. After heparin (500
units, iv), animals were exsanguinated and the whole blood collected for other uses.
With the continuing ventilation, a sterile piece of tygon tubing is inserted 1 cm below
the cannulated point in to the trachea and inserted until a resistance is felt. A 10 ml
portion of sterile phosphate buffered saline (PBS) was injected in to the lungs through
the tygon tubing over the period of 5 minutes and the bronchoalveolar lavarge (BAL)
was retrieved by applying negative pressure. This procedure was repeated until 100 m1
of BAL is collected. BAL fluid was centrifuged at 300g for 10 minutes at 6°C. The
combined cell pellets were resuspended in RPMI-1640 contained L-glutamine and
sodium bicarbonate (Sigma, St. Louis, MO) supplemented with 1% fetal calf serum and
antibiotics (penicillin-100 U/ml, streptomycin-100 ug/ml, gentamycin- 4 jig/m1,
amphotericin- 25 ug/ml). All studies involving animal use were approved by the

Animal Investigation Committee at Michigan State University.

150

4.3.2 Liquid scintillation determination of macrophage l“C labeled glucose

uptake

Whether any correlation exists between macrophage glucose uptake and CFTR
inhibition is critical for understanding our hypothesis. Incubation of macrophages with
or without glybenclamide, a selective CFTR inhibitor and then with the B emitter, l4C
tagged glucose, would allow the determination of the dynamic flow of glucose in to the
macrophages. It was essential here to avoid the use of 2-deoxy-D-glucose, a glucose
analogue that cannot undergo further glycolysis. Although 2-deoxy-D-glucose facilitates
the glucose accumulation inside the cytosol enhancing sensitivity, it may prevent or
disturb the metabolic energy flow which is one of the criteria hypothesized here to

govern the glucose uptake.

Aliquots of 500 pl RAM suspension were incubated in a 24-well plate with a
flat bottom lid for-24 hours at 37°C with 5% C02. Incubation was continued another
hour after addition of 40 uL buffer or buffered glybenclamide (100 uM). Cells were
exposed to FITC tagged E. Coli bacteria particles opsonized with rabbit polyclonal
antibodies (ratio of macrophage to bacteria particle~100) and further incubated for 30
minutes. Next, macrophages were treated with 5 ul of 1 uM l4C labeled glucose and the
incubation was continued for three more hours at 37°C with 5% C02. Cells were
detached from the wells using a rubber policeman and centrifuged at 500g for 10 min

and washed three times to remove excess bacteria particles and 1"(C labeled glucose.

Supernatant of the third washing was analyzed with the scintillation counter to ensure

151

the complete removal of the extracellular radiolabeled glucose. The washed RAMs were
resuspended in 70 pl of HBSS. A 10 p1 aliquot of this suspension is counted with the
hemocytometer. Finally 15 p1 of AM suspension in HBSS was mixed with 185 pl of
cell lysis solution and 100 pl. of scintillation cocktail in the wells of scintillation
microplates. 14C radioactivity of the lysed samples was measured in a Wallac 1450
PLUS microbeta liquid scintillation counter and counts per minute (CPM) were

normalized to the corresponding concentration of macrophages.

4.3.3 Fluorescence spectroscopy determination of opsonized bacteria particle

ingestion

Establishing the relationship between CFTR inhibition and the changes in
macrOphage opsonized bacteria particle ingestion is the second half of the hypothesis to
link CFTR in regulating macrophage function through the regulation of glucose uptake.
RAMs (500 p1) were pre-incubated for 24 hours in 24-well plate with a flat bottom at
37°C with 5% C02. Incubation was continued another an hour after addition of 40 pL
buffer or buffered glybenclamide (100 pM). Cells were exposed to FITC tagged E. Coli
opsonized with rabbit polyclonal antibodies (ratio of macrophage to bacteria particle
concentration was 100) and filrther incubated for 30 minutes. Cells were washed two
times to remove free bacteria particles and immersed in 100 p1 HBSS and cells were
detached with rubber policeman. Fluorescence from any remaining extracellular

bacteria particles were quenched by incubating 5 min with 60 pl of 0.08% tryphan blue.

152

Each suspension was further diluted up to 500 pl with HBSS and the fluorescence
emission of ingested FITC tagged bacteria was detected at 518 nm with 494 nm

excitation with a Fluoromax-4 spectrofluorometer (HORIBA J obin Yvon, Edison, NJ).

4.3.4 Single cell fluorescence imaging for RAM phagocytosis:

As macrophages are large cells with a diameter ranging from 15-20 pM,
confocal fluorescence imaging with a lOOX oil immersion objective can be performed.
This investigation is conducted in order to visualize the ingested bacterial particles and
to verify that the fluorescence emission is exclusively coming from bacterial particle
inside the macrophage. Cells were prepared in the same manner as the fluorescence
experiment in the section 4.2.3 and a drop of the each suspension allowed settling down
on cover glasses coated with fibronectin for 20 minutes. Macrophages were carefully
observed with Olympus confocal fluorescence microscope under oil immersion

objectives and fluorescence imaging of ingested bacterial particle was conducted.

4.3.5 Macro fluorescence microscopy evaluation of C-peptide mediated recovery

of the inhibitory effect of glybenclamide on RAM phagocytosis

Expansion of limited but interesting information gathered from fluorescence

imaging with limited number of macrophages to a large macrophage population was the

153

primary goal of this experiment. Additionally, errors associated with imaging single
cells such as incubation timing, photo bleaching, macrophage size distribution and
issues related to the precision also encourage this particular experiment using macro

fluorescence microscopy.

Initial suspensions of RAM in RPMI—1640 with antibiotics were stimulated
with 100 ng/ml lipopolysacharade (LPS-a macrophage activator), 300p] aliquots of this
suspension were further incubated in a 96-well plate for 18 hours at 37°C with 5% C02.
Incubation was continued another an hour after addition of 40 pL buffer or buffered
glybenclamide (100 pM). After the incubation, 100 pl Zn2+ activated C-peptide
(prepared according to reference 86 in HBSS) or 100 pl of HBSS was added to the each
well. Following a 30 minute incubation, cells were exposed to FITC tagged E. Coli
opsonized with rabbit polyclonal antibodies (ratio of macrophage to bacteria
particle~100) and further incubated for 3 hours at 37°C with 5% C02. Cells were fixed
with 1% glutaraldehyde and washed twice with 4°C HBSS. Just before the analysis,
buffer was removed from each well and exposed to lOOpl of 0.4% tryphan blue in
normal saline for one minute to quench the fluorescence from all the remaining
extracellular bacteria particles. Tryphan blue was removed by aspiration. Fluorescence
images of ingested bacteria particles in the entire well were observed with an Olympus

Macro-Fluorescence Microscope equipped with FITC filter cube (Chroma technology

Corp. exciter HQ481/40, emitter HQ535/50).

154

0.8 -:

Normalized CPM / AM

0.6 -:

    

 

0.4 - ' '
AM without gly AM with gly

Figure 4.6 Effect of CFTR inhibition on RAM glucose uptake. RAM treated with
FITC tagged E. coli bacteria particles opsonized with rabbit polyclonal
antibodies were investigated for 1“'C labeled glucose uptake. As expected,
the basal glucose uptake significantly decreased by 31.7 % in the presence of
glybenclamide (P < 0.0005). Error bars are standard error of the mean for n
= 3 rabbits.

155

4.4 RESULTS AND DISCUSSION

The information about the effects of mutated AF 508 CFTR in CF macrophages
are limited to a couple of controversial studies related to the macrophage phagosome
acidification.”18 As hypothesized, if there is any relationship exists between CFTR
function and macrophage glucose uptake, it should reflect in the glucose uptake
measurements. Based on Carruthers’ group’s finding on cytoplasmic ATP mediated
inhibition of GLUTl,61 Speert’s group’s demonstration about the dependency in
phagocytosis of Pseudomonas aeruginosa on facilitative glucose transport,42 and CFTR

63,65,66,78

being a conductive ATP transporter, a reduced glucose uptake in macrophages

incubated with glybenclamide was expected.

Data in Figure 4.6 represents the involvement of CF TR in rabbit alveolar
macrophage (RAM) glucose uptake as measure of 14C labeled glucose intake, in the
presence and absence of glybenclamide evaluated with liquid scintillation counting. The
data is normalized with respect to the basal glucose uptake of macrophages without any
CF TR inhibition. The percentage decrease over basal glucose uptake in CF TR inhibited
RAM is 33.3 :1: 3.8 %, a value representing a significant decrease in of glucose uptake
after inhibiting CFTR (p < 0.0005). However, the up taken glucose by macrophages are
continually metabolized and the therefore this difference in glucose uptake may be

greater, if the glucose consumption could be abolished.

156

0.08

0.06

0.04

0.02

Fluorescence from injested bacteria /AM

      

0.00 ~ . ..

AM without gly AM with gly

Figure 4.7 Influence of CFTR inhibition on opsonized bacteria particle phagocytosis.
RAM treated with FITC tagged E. coli bacteria particles opsonized with
rabbit polyclonal antibodies were investigated for bacteria particle uptake
with and without CFTR inhibition. After washing to remove the excess
bacteria and quenching the fluorescence from un-ingested bacteria with
tryphan blue, RAM were resuspended investigated for the fluorescence
from ingested bacteria. Inhibition of CFTR significantly reduced bacteria
particle uptake (P < 0.005). . Error bars are standard error of the mean for
n = 3 rabbits.

157

MacrOphages may use the metabolic energy from glucose to compensate the
intense energy requirement of the phagocytosis as well as to generate the reducing
power in the form of NADPH for bactericidal activities. Results shown in Figure 4.6
clearly demonstrate that the macrophage glucose uptake mechanism either directly or
indirectly depends on CFTR function. Therefore experiments were performed to
examine the effect of CFTR inhibition on Opsonized bacterial particle phagocytosis by
RAM. The data in the Figure 4.7 demonstrate the ingestion of FITC tagged E. coli
bacteria particles by RAM in the presence and the absence of glybenclamide. The
phagocytosis is decreased by 38.1 i 8.5 % in CFTR inhibited macrophages compared to
macrophages with functioning CFTR. This reduction is not only proportional to the
reduction in glucose uptake elicited by CFTR inhibition shown in Figure 4.6 but also in

the same percentage.

An appraisal of the results shown in Figures 4.6 and 4.7 demonstrate the
correlation of glucose intake and phagocytosis as well as the possible effect of CFTR
inhibition on CF macrophage functionality. By analyzing the overall possible outcome
of AF508, the most prominent CFTR mutation in CF, explained in detail in the
introduction of this chapter, mutated CFTR gene results in overall decrease in entire
CFTR function.2 Therefore, the inhibition of CFTR is a reasonable way to mimic the
clinical situation exists in the CF macrophages. Another way to conduct this experiment
is to use CFTR”/" macrophages. However, the deletion CFTR entirely eliminates the
CFTR function which cannot be expected in CF. Therefore glybenclamide mediated

CFTR inhibition would be a better way to mimic mutated CF TR in CF.

158

P <0.001

  

     

P < 0.001

 

Normalized CPM /AM - glucose uptake
0

Figure 4.8 Effect of CFTR inhibition on RAM glucose uptake and the enhancement of
glucose transport by Zn activated c-peptide. RAM treated with FITC
tagged E. coli bacteria particles opsonized with rabbit polyclonal
antibodies were investigated for 14C labeled glucose uptake. As expected,
the basal glucose uptake (a), was significantly decreased in the presence of
glybenclamide (b). Incubation of CFTR inhibited RAM with Zn activated
C-peptide was not only able to completely overcome, but also enhance the
glucose uptake (c), the effect of Zn activated C-peptide alone on RAM (d)
is not statistically diflerent from (c) (P < 0.001). Error bars are standard
error of the mean for n = 3 rabbits.

159

Bright field Fluorescence

 

RAM without glybenclamide

 

RAM incubated with glybenclamide

Figure 4.9 Confocal fluorescence microscopy images captured with IOOX oil
immersion objectives. A dramatic reduction in phagocytosis was observed
after CFTR inhibition

160

In order to determine whether there are ways to reverse the CF TR inhibition
and/or the ways to improve the glucose transport, Zn2+ activated C-peptide, a known red

blood cell glucose transport enhancer77

was evaluated with RAM in terms of glucose
uptake and phagocytosis. We have previously shown that Zn2+ activated C-peptide has
the ability increase the glucose uptake in to RBCs through GLUTl and promote ATP
release from RBCs. However, at this point, it is not clear whether C-peptide improve
glucose transport directly through a GLUTI or a CF TR mediated mechanism, or both.
Similar to the determination of the effect of CFTR inhibition on RAM glucose uptake,
the effect of Zn2+ activated C-peptide, in the presence and absence of glybenclamide, on
RAM glucose uptake was determined and is shown in Figure 4.8. Incubation of
glybenclamide treated RAM with Zn2+ activated C-peptide increased the RAM glucose
uptake by 60.8 :t 6.8 % compared to the normal RAM glucose uptake, and a 1.35 fold
increase when compared to the glybenclamide treated RAM. Interestingly RAM
incubated with Zn2+ activated C-peptide increased the RAM glucose uptake by 69.3 i
5.3 % compared to the RAM alone. Therefore, while the process through which Zn2+
activated C-peptide acts on RAM GLUTl up-regulation may be independent of CFTR,
it can compensate the CFTR mediated inhibitory effect of glucose uptake.

The next step of this analytical process was linking the Zn2+ activated RAM

glucose uptake with its phagocytosis. FITC tagged opsonized bacteria particle ingestion

by RAM in the presence and absence of glybenclamide and the effect of Zn2+ activated

161

120 -

80*

   

P < 0.005

  

60-

4o-

20-

 

Fluorescence from ingested bacteria particles

 

Figure 4.10 Confocal macro fluorescence microscopy determination of RAM-
opsonized bacteria particle intake. Mean gray values were calculated from
the fluorescence images of RAM plated 96 well plate after the background
subtraction, a) and (b) are opsonized bacteria particle treated RAM without
and with CFTR inhibition respectively, P < 0.005. (c) Incubation of CFTR
inhibited RAM with Zn activated C-peptide was able to entirely overcome
the inhibitory effect of glybenclamide on CFTR, P < 0.05. Treating RAM
with Zn activated C-peptide may or may not enhance the opsonized
bacteria particle intake (d) as it is statistically not different from (c). Error
bars are standard error of the mean for n = 3 rabbits.

162

C-peptide on this process has been evaluated using macro fluorescence microscopy. The
experiment has been conducted using 24 hour cultured RAM on 96 well plates. An
advantage of using the Olympus macro fluorescence imaging system with Olympus
MicroSuite software is its capability of not only providing qualitative bright field and
fluorescence images but also the quantitative data calculated from the mean gray value
intensities. Data obtained from this experiment is shown Figure 4.10 and in this
experiment glybenclamide induced CFTR inhibition was able to reduce RAM bacteria
particle uptake by 77.2 :1: 3.6 % compared to untreated RAM (Figure 4.10a and b). This
reduction is two times higher than the reduction observed in glybenclamide treated
macrophages in section 4.2.3 which was determined via fluorescence spectroscopy.
When treated with Zn activated C-peptide, CFTR inhibited RAM was able to show a
21.8 i 7.5 % increase in phagocytosis compared to the normal RAM Phagocytosis, and
five fold increase when compared to the glybenclamide treated RAM. Although RAM
incubated with Zn activated C-peptide increased the RAM phagocytosis by 83.9 :i: 5.7
% (Figure 4.10c) compared to the RAM alone (Figure 4.10a), this value is not

statistically different (P=0.4) from RAM with C-peptide and glybenclamide together.

4.5 CONCLUSIONS AND OTHER CONSIDERATIONS

The work shown here demonstrates that the RAM glucose transport is highly
dependent upon the proper fmetion of CFTR. When CFTR is blocked with

glybenclamide, a significant decrease in glucose intake is measured. This is the same

relationship observed between CF TR inhibition and RAM phagocytosis. However,

163

Fluorescence image Bright field image

Macrophages in clumps

\
/

 

Figure 4.11 Fluorescence imaging for the evaluations of RAM phagocytosis, (Look at
next page for detailed figure caption)

164

d Fluorescence image (1 Bright field image

 

Figure 4.12 Fluorescence imaging evaluations of RAM phagocytosis. Phagocytosis of
IgG-opsonized bacterial particles was diminished by pre-incubation with
glybenclamide and recovered by incubation with Zn activated C-peptide.
Macrophages (2 x105 cells/ well) were incubated in HEPES-supplemented
RPMI 1640 medium 17 hours at 37°C. Then, cells were incubated with
FITC-conjugated IgG-opsonized E. coli for 3 hours at 37°C. Cells were
washed and incubated with tryphan blue to quench any extracellular
fluorescence. The internalized fluorescence signal was determined with
macro fluorescence microscope and normalized by the number of viable
cells in the well as determined with the use of cell tracker red fluorescence
probes. Fluorescence images are at the right hand side of the figure and the
bright field images are on the left. a: RAM without IgG-opsonized
bacterial particles, b: Heat killed RAM with IgG-opsonized bacterial
particles, c: RAM with IgG-opsonized bacterial particles, d: RAM treated
with glybenclamide and then with IgG-opsonized bacterial particles, e:
RAM treated with glybenclamide, incubated Zn activated C-peptide and
then with IgG-opsonized bacterial particles.

165

when treated with Zn2+ activated C-peptide, even in the presence of glybenclamide,
RAM phagocytosis not only completely recovered but increased to a level that is
statistically equivalent to that of the C-peptide treated RAM. In summary, CFTR
inhibition resulted in 33.3 :i: 3.8% (p<0.001) reduction in glucose transport compared to
control RAM. Fluorescence intensity of ingested FITC tagged IgG opsonized bacterial
particles decreased by 38.1 d: 8.5 % (p<0.005) in CFTR inhibited RAM. Incubation of
CFTR inhibited RAM with C-peptide resulted in 60.8 a 6.8 % increase in 1“c labeled
glucose uptake and 77.2 i 3.6 % increase in opsonized bacteria particles ingestion,

compared to the control RAM.

Based on these results, we conclude that the CFTR function is required for
alveolar macrophage glucose uptake and subsequent phagocytosis. Zn2+ activated C-
peptide can enhance alveolar macrophage phagocytic function by up-regulating the
macrophage glucose uptake. Therefore, the macrophage glucose uptake seems to be
regulated indirectly by CFTR, through an ATP mediated mechanism. As we discussed
under section 4.2.1, CFTR inhibition can increase the accumulation of ATP in the
macrophage cytosol. As shown in the signal transduction pathways in Figure 4.5,
increased cytosolic ATP can inhibit the GLUTl activity while acting as a negative
feedback for glycolysis. Therefore, CFTR inhibition (or dysfunction) can hinder the
macrophage glucose uptake. Reduced glucose uptake results in decreased NADPH
production and reactive oxygen species generation, reduces macrophage phagocytosis
and bactericidal activities. As suggested previously, if the application of Zn2+ activated

C-peptide can increase GLUTl activity,77 that would clearly demonstrate the

166

mechanism of Zn2+ activated C-peptide mediated improvement of RAM phagocytic

activity.

This investigation not only reveals the key role of CFTR in RAM glucose
transport and associated phagocytosis but also the avenues to manipulation of alveolar
macrophage glucose transport as a therapeutic strategy in CF lung pathogenesis. From a
therapeutic point of view, stimulation of alveolar macrophage glucose transport would
be an effective way in treating CF lung disease. With further investigations and clinical
trials, Zn2+-activated C-peptide may be able to be used as an aerosol to treat patients

with cystic fibrosis.

167

10.

11.

12.

13.

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174

CHAPTER 5

5.1 OVERALL CONCLUSIONS FROM PLATELET STUDIES

Platelets are the main components of arterial thrombus formation. They are also
involved in all phases of atherosclerosis and when activated, platelets express mediators
of inflammation and smooth-muscle-cell proliferation. Platelets play a central role in the
pathogenesis of cardiovascular diseases mainly by contributing to atherothrombosis, a
condition where sudden disruption of an atherosclerotic plaque leads to platelet activation
and thrombus formation.l Risk factors associated with life style such as high calorie diet,
unhealthy eating habits, mental stress, and lack of exercise substantially increase the risk
of cardiovascular diseases and diabetes. An important feature of platelets in both of these
conditions is their enhanced sensitivity to the activation stimuli. Platelet hyperactivity has
been explained by a gradual build up of platelet activating factors in the cardiovascular
system as a result of atherosclerotic plaque formation and progression. Although platelet
activating factors account for the larger fraction of systemic thrombus formation, they do
not explain the entire spectrum of platelet hyperactivity.

Therefore, here we have investigated the function of platelets beyond the
expression of activating factors. Recent findings by our group concerning RBC-derived
ATP release into the circulation unveiled the importance of the RBCs physiological
behavior on the prOper function of other cell types and tissues. For example, in a
condition such as diabetes, the less deformable RBCs release less ATP while traversing

through the microcirculation and therefore, result in decreased production of endothelial

175

NO, which is essential for vasodilation. In tum, decreased NO in the circulation, results
in increased blood pressure as well as platelet activation. On the other hand, hyperactive
platelets have also been found in the painful crisis of sickle cell disease where RBCs are
subjected to lysis and release large amounts of ATP into the circulaton?’3

Therefore, it was predicted that platelet hyperactivity may be related to the
physiological behavior of RBCs and their ATP release. Furthermore, the potential of ATP
and its primary receptor in platelets, P2X1 has been underestimated as a result of the lack
of available analytical tools and methodologies. Due to the high propensity of P2X1
receptor for desensitization, it is necessary to use the enzyme, apyrase in platelet
investigations to keep the P2X1 receptor sensitive to ATP. Therefore, the evaluations of
the effect of ATP on platelet function described in the literature have been conducted
using a non hydrolysable ATP analogue, or,B-methylene ATP, based on its resistance to
apyrase mediated hydrolysis and high affinity to the P2X1 receptor.

By correlating the conditions where platelets are hyperactive with the
physiological behavior of RBCs, we hypothesized that bioavailable ATP may act as a
concentration dependent agonist and antagonist for platelet function. Also, by carefully
analyzing the literature and the molecular kinetics of ATP and a,B-methylene ATP, we
found that the high affinity of 0L,B-methylene ATP can possibly open the P2X1 channel
one time and allows Ca2+ influx into the cytosol, but prevents the receptor recycling and
continuous operation.

The work shown here demonstrates that ATP mediated activation of P2X1

receptor affects both the NO production and aggregation of platelets, but in a

concentration-dependent manner. When ATP is added to platelets, a significant increase

176

in NO production is observed. However when the P2X1 receptor is blocked with NF 449,
the NO production is decreased to a level equivalent to the un-activated platelets. In these
studies, apyrase was also employed; however, after the addition of apyrase, platelets were
washed twice in apyrase-free buffer. This washing step was performed in order to
guarantee that any authentic ATP added to the platelets would have an opportunity to
bind to the P2X1 receptor before being converted to ADP or other nucleotides form by

the apyrase.

The outcomes of these conducted investigations suggest that the effect of ATP on
platelet behavior is concentration dependent and, moreover, zero to low levels of ATP
result in platelet aggregation. The incremental increase of ATP concentrations reduces
platelet aggregation initially, only to begin increasing with continued increments in ATP.
The essential Ca2+ influx into the cytosol may be mediated by ATP and therefore very
low concentrations of ATP may result in decreased production of the Ca2+-calmodulin
complex and lower production of NO. It seems that a basal level of NO is required to
prevent platelet from activating and aggregating. Our data can most accurately explain
the reduction of platelet aggregation with increasing concentrations of ATP. In one of our
previous publications, we have shown that, at a certain concentration of ATP, platelet NO
production became constant, indicating saturation of eNOS activity with adequate
amounts of Ca2+-calmodulin.4 Beyond this amount of added ATP, the NO production

remained constant.

Collectively, it may be possible that levels of RBC-derived ATP that are
abnormally low (resulting in platelet low Ca2+ influx) may subsequently lead to

insufficient eNOS activation and insufficient production of NO. When the concentration

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of ATP exceeds the amount required for maximum NO production through Ca2+ influx,
the continuing Ca2+-calmodulin formation could activate the myosin light chains and
GPIIb/IIIa integrin leading to platelet shape change and aggregation. Conversely, the
exposure of the platelets to large increments of ATP, such as hemolysis observed in
sickle cell disease, could increase platelet aggregation. Interestingly, another investigator
in the group was able to verify this dual effect of ATP on platelet aggregation by
evaluating platelet adhesion to an immobilized endothelium in a microfluidic device.
Importantly, the effect of ATP on both aggregation and adhesion studies are strikingly

similar and both show a bi-phasic effect of ATP on platelet function.

Our findings on dual role of ATP as a platelet inhibitor and platelet activator are
in good agreement with established findings involving hyperactive platelets and clinical
outcomes in certain types of disease. For example, people with diabetes, cystic fibrosis,
and primary pulmonary hypertension have RBCs that release less ATP than healthy
controlss’6 Furthermore, all these patient groups have platelets that are more hyperactive
than platelets obtained from controls. Also, the increase in platelet adhesion due to
inhibition of RBC-derived ATP does provide a model of “low” ATP release as would
occur with the RBCs from people with diabetes, cystic fibrosis, or primary. pulmonary
hypertension. Interestingly, 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

In the subsequent studies, the hypothesis that the ATP receptor, P2X1, plays a

critical role in platelet function has been investigated and the attention has been directed

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towards the understanding of the true potential of ATP mediated P2X1 function in
platelet cytosolic Ca2+ increase and aggregation. Primarily, we have demonstrated that
a,B—methylene ATP cannot induce platelet activation and subsequent platelet
aggregation, not due to the lack of P2X1 receptor potential, but due to the unusual
behavior of a,B—methylene ATP in the receptor gating. We initially demonstrated that
a,B-—methylene ATP cannot induce platelet aggregation in PRP which is consistent with
the literature. Although a similar study with ATP demonstrated a substantial and
sustained platelet aggregation, it was challenging to distinguish this behavior from ADP,
which is the immediate hydrolyzed product of ATP due to plasma or membrane apyrases.
This goal was achieved by successfully inhibiting P2Y1 receptor and preventing ADP

accumulation in the system.

Similar studies conducted with ct,B—methylene ATP have demonstrated that it can
only induce a P2X1 mediated Ca2+ influx that has only half the magnitude of the parallel
ATP stimulated Ca2+ influx. However, the condition applied in this experiment has
inhibited over 50% of P2X1 population while inhibiting P2Y1 completely. Therefore,
ATP mediated Ca2+ influx would have been several orders of magnitudes larger than
0t,[3—methylene ATP induced Ca2+ influx. In addition, consideration of the extent of ADP
induced PRP aggregation with the ATP induced Ca2+ influx fiirther suggests that ATP
and its receptor, P2X1 can induce platelet aggregation at higher ATP concentrations.
Therefore, the higher binding affinity of a,[3—methylene ATP towards P2X1, which is
greater than that of ATP7, would not essentially reflect the a,B—methylene ATP ability to
govern the P2X1 function. Here we have found strong evidences to conclude the ability

of ATP gated P2X1 receptor to induce platelet aggregation depending on the extracellular

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ATP concentration. In this study, it has also been shown that the use of ocfi—methylene
ATP to demonstrate the P2X1 receptor function described in the literature has

8 underestimated the P2X1 potential in platelet aggregation.

5.2 FUTURE DIRECTIONS FOR PLATELET FUNCTION INVESTIGATIONS

The ultimate goal of the present work is to provide complete understanding of the
ATP gated P2X1 receptor in platelet function regulation and the importance of
bioavailable ATP on platelet activity. The investigation described in chapters 2 and 3 of
this dissertation provides a semi-qualitative description of this receptor function. Here the
authors were able to optimize the methods to prevent interferences from ADP and its
receptor, P2Y] and evaluate the absolute potential of P2X1 in terms of platelet Ca2+
influx and aggregation. Although the P2Y1 interference is avoided, the full P2X1
potential has be extrapolated as the conditions applied in the investigations are partially
inhibitory to the P2X1 receptor too.

Although inhibitors such as NF 449 and MRS 2179 used in this experiments are
selective, they are competitive inhibitors. Therefore, higher inhibitor concentrations have
to be used in order to achieve complete inhibition. Higher concentrations of the inhibitor
usually exceed the IC50 value of that particular inhibitor for the other receptor (P2X1 or
P2Yl). Moreover, receptors cross inhibition complicates the measurements and disturbs
the true value measurements. Therefore, it would be worth to seek synthetic approaches

to develop non-competitive and selective inhibitors for these receptors.

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Another way to avoid P2Y1 interferences is to use P2Y1"’" platelets. The lack of
P2Y] may be able to provide absolute P2X1 mediated Ca2+ influx information. The
disadvantage of P2Yl knockout platelets is the lack of receptor crosstalk, which may play
an important role in the individual receptor function as well. Differential analysis of
P2X1 function with and without P2Y1 would be very usefiil for the understanding of
receptor crosstalk. Genetically modified f P2Y1 or Gaq protein would be another way to
isolate the P2X1 mediated Ca2+ currents from P2Y1. The advantage of this method would
be the possibility to preserve receptor cross talks to a certain extent. Differential analysis
of P2X1 mediated Ca2+ currents after inhibiting phospoliphase C B (PLCB), could be
another useful way to determine the P2X1 function, as ADP elicits its Ca2+ de-
granulation effect through PLCB.

The activation of GPIIb/IIIa upon ATP binding to P2X1 is another way to
measure platelet activation. However, the authors were unable to obtain conclusive data
using flow cytometric analysis for P2X1 activation in terms of GPIIb/IIIa activation. This
was mainly due to the lack of sensitivity of the probe used towards activated GPIIb/Illa.
Although increased GPIIb/IIIa activity was observed upon ATP mediated P2X1
activation, due to the lack of precision, the data has not been included in this dissertation.
Therefore, in the future it would be beneficial to conduct a flow cytometric analysis of

ATP activated platelets with a sensitive probe towards the activated GPIIb/IIIa.

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5.3 OVERALL CONCLUSIONS FROM MACROPHAGE STUDIES

When addressing the RBC deformability in several disease states with its ATP
release, special attention has been paid to the mechanism involved in such conductive
ATP transport. At this date, there is no definite channel/s identified for ATP transport,
however ATP binding cassette family transporters (ABC), especially cystic fibrosis
transmembrane conductance regulator (CFTR) have been shown to participate in this

process.8 Inhibition of CFTR results in the reduction of RBC derived ATP release.9'll

Cystic fibrosis (CF) is a genetic disorder associated with the mutations in CFTR
gene and its protein product, CFTR. Although CF is characterized by the unique CF lung
disease with chronic pulmonary infections and inflammations, the available molecular
level understanding of this disease is not able to explain the failures in the lung defense

against the pathogen invasion, which is the leading cause of the CF lung disease.

Alveolar macrophages are described as the front line defense in the lungs against
particulate matter and pathogen invasion. The primary goal of these investigations is
finding out the relationship between the CFTR dysfunction in alveolar macrophages with
the pathogenesis of CF lung disease. We found that inhibition of CFTR‘on alveolar
macrophages results in reduced glucose uptake. This was expected due to CFTR
involvement in ATP conductive transport. Under inhibitory conditions, ATP may
accumulate in the cytosol where it has been shown to inhibit GLUTl which is the
primary glucose transporter in macrophages. Similarly, CFTR inhibition has resulted in
substantial reduction in opsonized bacterial particle phagocytosis. Here we conclude that

the dysfunction of CFTR prevents macrophage activation, phagocytosis and bacterial

182

killing by reducing the required intense energy production through abolishing the glucose
uptake. Therefore, the investigations described in this dissertation not only unveil the
contribution of the defective alveolar macrophage function in the CF lung disease, but

also provides the molecular basis of this dysfunction to the disease pathogenesis.

We further elaborated our studies to find out whether there are ways to reverse
the effects of CFTR inhibition. Our strategy here was to investigate the effect of RBC
glucose uptake enhancer; Zn activated C-peptide, on CFTR inhibited macrophages. It has
been shown that Zn activated C-peptide enhanced the RBC glucose uptake through a
GLUTl mediated mechanism.12 As RBCs shares GLUTl glucose transporter with
macrophages, it was expected that Zn activated C-peptide may be able to enhance the

macrophage glucose uptake too.

We found that Zn activated C-peptide has been able not only to completely
reverse the reduced glucose uptake due to the CFTR inhibition;, it further enhanced the
uptake to a level even grater than macrophages without any CFTR inhibition. Same
relationship has been observed in terms of opsonized bacterial particle phagocytosis. Zn
activated C-peptide completely overcomes the effect of CFTR inhibition on macrophage
phagocytosis. However, at this point it is not conclusive whether Zn activated C-peptide
drives the glucose uptake through a CFTR mediated mechanism or it is completely

independent of the CFTR.

The investigations described in chapter 4 of this dissertation provides a clear and
more meaningful description for CF lung disease from the standpoint of the failures in

alveolar macrophage function as a result of mutated CFTR. CF lung disease has never

183

before explained as a disorder related to the macrophage dysfunction even though
alveolar macrophages are considered as the front line defense in the lungs. Our
investigations further elaborate the mechanistic detail of CFTR dysfunction and its
correlation with glucose uptake which eventually regulates the phagocytosis and the
bactericidal activities. From a therapeutic point of view, the ability of Zn activated C-
peptide to overcome the CFTR inhibition or enhance the macrophage glucose uptake
would be very important. Furthermore, C-peptide being a natural product produced by
the B cells in the islets of langerhan in the pancreas, it would be an added advantage if

activated C-peptide could improve the CF lung defense due its minimal side effects.

5.4 FUTURE DIRECTIONS FOR MACROPHAGE FUNCTION

INVESTIGATIONS

All investigations described in chapter 4 were conducted using rabbit alveolar
macrophages (RAM) and the defective CFTR was mimicked by inhibition of CFTR with
a selective inhibitor. It is highly advisable to repeat these investigations with real CF
macrophages. Although some investigators prefer CF TR”/ " macrophages, in CF, CFTR is
present and partially functional. Therefore, AF508 mutation transfected macrophages
would be a good choice. Also, correlation of the defective CFTR and alveolar
macrophages glucose uptake and subsequent phagocytosis would have to be evaluated
with human CF macrophages before any trials with activated C-peptide.

Application of radiolabeled 2-deoxy glucose, instead of 14C glucose would be

useful for a more quantitative evaluation of the changes in macrophage glucose uptake.

184

Single cell analysis with trapped individual macrophages in a microfluidic device may be
useful for evaluation of the changes at a single macrophage level. In this way, subtle
changes in the macrophage behavior with the inhibition of CFTR and the effect of
activated C-peptide could be easily obtained. This type of analysis would further assist to
understand the underlying detailed mechanisms, by probing the components of the
macrophage energy metabolism pathways such as NADPH, glucose-6-phosphate and
superoxide. Additionally, the components associated with CFTR signal transduction
pathway such as PKA, PKC, cAMP and adenylyl cyclase could also be evaluated.
Monitoring the up regulation of GLUTl will be useful for the evaluation of Zn2+
activation C-peptide mechanism. It may be an increased expression of GLUTl on the cell

membrane of activation of existing GLUT or both.

185

10.

11.

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