$.11? ‘ 1:1:
#23.“

‘ 12.345312. ‘
«at:
"C v‘,

-~°£~:m.,‘:*?€

I. '
. {'4 2s M
1;:

 

. A. N .
"L‘a .'

u i; .
:52»: N“,
, . "£33 ‘

A.

, '0 ~§> 1 "- 7-: ‘ I § .

1' '1‘ ' mé - ‘ ‘ Jr's. ..~

1 .‘ r

' 3g» ‘25:“ .Lu, , aim-h
‘r‘xw 1's

tr, 1-. . ,

EflfiL .3,» \ur‘v: . '31:. ‘ .».'m?j; ‘ .» ‘ ~
‘ ' ' ' ‘ ' n ‘_
.7}; ~ ., ‘ v.1 4‘, ‘ R": .. :5 7' W “i . v _ "13 .. I ~ , ‘ . .fi.

"a ."‘ I." 1" I, W , \ I. . :5 M '5.

" ‘ ' ‘ ‘ , . - ' {.3233 .

' r! 29.". 3'93;

 

30

WNW“

This is to certify that the

dissertation entitled

SIMMENTAL HEREDITARY THROMBOPATHY:

A COMPARATIVE ANALYSIS OF AFFECTED
AND CONTROL BOVINE PLATELETS

presented by

Barbara A. Steficek "

has been accepted towards fulfillment
of the requirements for

PhD degree in PatriOJIDgy

mQfiQfi

Major professor

 

 

Date July 25, 1994

 

MSU is an Affirmative Action/Equal Opportunity Institution 0- 12771

 

LIBRARY
Mlchlgan Stale
Unlversity

 

 

 

PLACE II RETURN BOX to mnovothb chockoutfrom your record.
To AVOID FINES Mum on or befor- ddo duo.

DATE DUE DATE DUE DATE DUE

I‘Anm a

 

 

 

 

 

We

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

’—
MSU!=.‘.H..”‘ “ ‘ ' " ‘n, ‘ ',.. "‘"

 

SMNIENT AL HEREDITARY THROIVIBOPATHY:

A CONIPARATIVE ANALYSIS OF AFFECTED

AND CONTROL BOVINE PLATELETS

By

Barbara A. Steficek

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements

for the degree of

DOCTOR OF PHILOSOPHY

Department of Pathology

1994

 

ABSTRACT

SIMMENTAL HEREDITARY THROMBOPATHY:
A COMPARATIVE ANALYSIS OF AFFECTED
AND CONTROL BOVINE PLATELETS
by

Barbara A. Steficek

A severe bleeding disorder in Simmental eattle has been described in wide—
spread loeations in the United States and Canada. The clinieal findings are consistent
with a hereditary hemorrhagic diathesis, and include spontaneous epistaxis, hematuria
and excessive blwding associated with trauma or standard management procedures.
In our preliminary investigation of this defect, the platelet numbers and coagulation
profiles of affected cattle were normal. Plasma von Willebrand factor levels were
also within normal limits. Affected animals have a marked dysfunction of platelets
which we have termed Simmental Hereditary Thrombopathy. Measurement of m
m platelet aggregation following activation by various agonists revealed a
pronounced lack of response of affected platelets to ADP, A23187 and collagen.
Aggregations were decreased or delayed in response to platelet activating factor and
thrombin but were normal in response to phorbol myristate acetate. Dense granule
release of ATP in response to ADP, platelet activating factor and thrombin was

normal. Transmission electron microscopy of resting and post-activation platelet

samples revealed no ultrastructural abnormalities. In addition, no differences were
noted in structural proteins using l-D sodium dodecyl sulfide polyacrylamide gel
electrophoresis. Total resting platelet ealcium was normal as assessed by inductively
coupled plasma argon emission spectrophotometry. Clot retraction kinetics were
abnormal in affected animals. Studies in which the comparative aggregation response
to variations in the external calcium concentration were assessed demonstmted a
dependence of the maximum % aggregation achieved in thrombin-activated platelets
from normal but not affected animals. Measurement of cytosolic ionized ealcium in
resting and agonist stimulated platelets was evaluated utilizing the luminescent
photoprotein, aequorin and the fluorescent calcium indieator dye, fura-2/AM. Results
obtained from aequorin studies revealed no differences in cytosolic calcium levels
between control and affected animals. The fura-2/AM laboratory procedure required
modification of standard methodology for use in the bovine platelet. These
modifieations included a decrease in the loading time of the dye to 15 minutes and the
use of the organic anion transport system blocker, sulfinpyrazone. The results
presented in this dissertation describe the platelet function disorder in purebred
Simmental cattle and demonstrate the unique properties of calcium assessment in the

bovine platelet.

 

Copyright by

Barbara A. Steficek

1994

ACKNOWLEDGEMENTS

I would like to express my sincere thanks and appreciation to the members of
my graduate guidance committee, Drs. Thomas G. Bell, John C. Baker, Douglas W.
Estry, William S. Spielman, and Ben Yamini. Additional thanks are owed to Dr. Bell

for his patience, advice and support throughout this research endeavor.

In addition, I would like to express my gratitude to the members of the
Departments of Pathology and Large Animal Clinieal Sciences, and the Animal Health
Diagnostic laboratory for their support and consultation in the completion of my

research.

To Mr. John Allen, Veterinary Research Farm, special thanks is owed for the

outstanding eare he has provided in maintaining our research eattle.

Finally, I would like to thank those who provided financial support for my
graduate work. My funding was derived from a postdoctoral fellowship,

F32HL07560, from the National Institutes of Health.

 

TABLE OF CONTENTS

LIST OF TABLES .
LIST OF FIGURES
LIST OF ABBREVIATIONS

INTRODUCTION .

CHAPTER 1: LITERATURE REVIEW

Platelet Structure
Thrombopoiesis .
The Platelet Response .

Adhesion

Shape Change

Aggregation

Secretion
Agonists and Platelet Aggregation
Platelet Membrane Receptors
Second Messenger Systems .

Calcium

vi

ll
11
11
ll
12
13
17
18

Phosphoinositides
Cyclic AMP .
Platelet Pathology

Characterization of the Bovine Platelet .

CHAPTER H: STATEMENT OF THE PROBLEM .

CHAPTER III: METHODS DEVELOPMENT AND RESULTS .

Platelet Aggregation and Secretion Studies .
Introduction
Materials and Methods
Platelet Aggregation
Platelet Dense Granule Secretion .
Results

Transmission Electron Microscopy
Introduction
Materials and Methods
Results

Citrate Concentration Study .
Introduction
Materials and Methods
Results

External Calcium Concentration Study

vii

22
22
24

26

29

33
33
33

36
37
37
42
42
42
43
51
5 l
51
52

54

Innoducfion
Materials and Methods
Remus

Von Willebrand Factor Assay
Introduction
Materials and Methods
Results

Clot Retraction Study .
Inuoducfion
Materials and Methods
Results

I.C.P. Evaluation of Platelets
Innoducfion
hflmnhkzmthMKES
Remus

l-D SDS Polyacrylamide Gel Electrophoresis .
Inuoducfion
Materials and Methods

Results

CHAPTER IV: JOURNAL ARTICLE .

 

viii

54
54
55
58
58
58
59
61
61
61
62

67-

70

70

70

75

 

CHAPTER V: JOURNAL ARTICLE .

 

. 101

CHAPTER VI: MEASUREMENT OF RESTING AND POST-STIMULATION

PLATELET CYTOSOLIC CA++ CONCENTRATION

Introduction

Aequorin Studies
Results

Fura-2/AM Studies .

Results

CHAPTER VII: SUMMARY AND CONCLUSIONS .

LIST OF REFERENCES:

ix

. 119
. 119
. 120
. 122
. 122

.127

.140

. 145

Table

LIST OF TABLES

Platelet dense granule ATP secretion. Comparison of time to maximum
ATP secretion and concentration of ATP between control and affected
bovine platelets. . . . . . .

Aequorin assessment of [Ca++],. Comparison of peak [Caf‘], (am
following stimulation with various agonists in control and affected
aequorin-loaded bovine platelets. .

Page

41

123

LIST OF FIGURES

Figure Page

1 Comparison of the effects of various agonists on maximum percent
aggregation in normal (series 1) and affected (series 2) platelets. [Agonist
concentrations: ADP= 10. OuM; A23187= 40.0}LM; collagen 2.0ug/ml;
PAF= 10. 0M; thrombin= 1.0 U/III]; PMA=3.75IIM; vertieal
bars= Standard Error]. . . . . . . . . . 40

2 Paired transmission electron micrographs of control (a) and affected (b)
resting bovine platelets showing similar ultrastructural features. . . 46

3 Paired transmission electron micrographs of control (a) and affected (b)
bovine platelets 5 minutes following the addition of 1.0;IM PAF, an
agonist which induces an aggregation response in both groups. . . 48

4 Paired transmission electron micrographs of control (A) and affected (B)
bovine platelets 5 min following the addition of 0.6 U/ml thrombin,
showing the response to an agonist which induces aggregation in both
groups. . 50

5 The effect of citrate concentration on lpM PAP-induced aggregation in
normal bovine platelets (o) and in Simmental Hereditary Thrombopathy
C“) . 53

6 Aggregation index for control bovine (striped bars) and affected SHT
(solid bars) platelets su'mulated by 0.1 U/ml thrombin In the presence of
varying concentrations of external Ca“. . 57

7 Comparison of the %clot and clot retraction index between control and
affected bovine samples [time = 24 hours]. Clot retraction index= 96
serum/(IOO-PCV). . . . . . . . . . . 65

8 Multi-element analysis of resting bovine platelets by Inductively Coupled
Argon Plasma Atomic Emission Spectroscopy (ICP-AES). [Normal= S];
Affected= $2] . 69

10

11

12

13

l-D SDS polyacrylamide gel electrophoresis. Resting platelets.
(A=affected bovine platelets; B=affected bovine platelets; C=control
bovine platelets; D=low molecular weight standard; =canine
platelets). . . . . . . . . . . .

Comparison of the intensity of fluorescence in counts per second (cps) of
eanine vs bovine fura-2 loaded platelets following the addition of 1.0 U/ml
thrombin. [A=canine 340nm; B=canine 380nm; C=bovine 340nm;
D=bovine 380nm. Addition of agonist=60 sec; Fmax=120 sec;
Fminm=180 sec; FW=240 sec]. . . . . . .

Fura-2/AM loading assessment in resting normal bovine platelet samples.
Comparison of the effect of plasma (Pls), buffer (But), and buffer
containing 300/,IM sulfinpyrazone (sz) on intensity of fluorescence over
time. .

Comparison of the effect of varying concentrations of sulfinpyrazone on
the uptake (intensity of fluorescence) of fura-2/AM over time in resting
normal bovine platelet samples. . . . . .

Comparison of the intensity of fluorescence in counts per second (cps),
following the addition of 1.0 U/ml thrombin, of control bovine platelet
samples loaded with 4uM fura-2 in plasma (A, B) or in buffer containing
300p.M sulfinpyrazone (C, D). [A= 340nm; B= 380nm, C= 340nm;
D= 380nm. Addition of agonist= —60 sec; Fmax= 120 sec; Fminhm=l90
sec; Fminpm=240 sec]. .

xii

74

129

134

136

139

ATP
Ca++ .
cAMP
DAG .
DMS .
DMSO
DTS
EDTA
EGTA
fura-ZIAM
GDP
GOC .
GFP
GP .
GTP
IP, .

1P3.

LIST OF ABBREVIATIONS

arachidonic acid
adenosine diphosphate

. adenosine triphosphate
calcium

. cyclic adenosine monophosphate

. 1,2—diaeylglycerol

demarcation membrane system

. dimethyl sulfoxide
. dense tubular system

ethylene diaminetetraacetate

ethylene glycol tetraacetic acid

xiii

. fura-2 acetoxymethyl ester
. guanosine diphosphate

. G-protein operated channel
. gel filtered platelets

- glycoprotein

. guanosine triphosphate
inositol 1,4-bisphosphate

. inositol 1,4,5-trisphosphate

 

1P. . . . . . . . . . . . . . . . . . .inositol 1,3,4,5-tetra]dsphosphate
MLCK . . . . . . . . . . . . . . . . . . myosin light chain kinase
OCS . . . . . . . . . . . . . . . . . . . . open eanalicular system
PA . . . . . . . . . . . . . . . . . . . . . . . phosphatidic acid
PAF . . . . . . . . . . . . . . . . . . . . platelet activating factor
PDGF . . . . . . . . . . . . . . . . . platelet derived growth factor
PI . . . . . . . . . . . . . . . . . . . . . phosphatidylinositol
PIP . . . . . . . . . . . . . . . . . phosphatidylinositol 4-phosphate
PIP, . . . . . . . . . . . . . . . . . phosphatidylinositol biphosphate
PKC . . . . . . . . . . . . . . . . . . . . . . protein kinase C
PLA, . . . . . . . . . . . . . . . . . . . . . . phospholipase A2
PLC . . . . . . . . . . . . . . . . . . . . . . phospholipase C
PMA . . . . . . . . . . . . . . . . . . . phorbol myristate acetate
PPP . . . . . . . . . . . . . . . . . . . . . platelet poor plasma
PRP . . . . . . . . . . . . . . . . . . . . . platelet rich plasma
SHT...............SimmentalHereditaryThrombopathy
SMOC . . . . . . . . . . . . . . . second messenger operated channel
TEM . . . . . . . . . . . . . . . . .transmission electron microscopy
TXA, . . . . . . . . . . . . . . . . . . . . . . thromboxaneA,
'I'XB2 . . . . . . . . . . . . . . . . . . . . . . thromboxane B2
VOC . . . . . . . . . . . . . . . . . . . voltage operated channel
vWD . . . . . . . . . . . . . . . . . . . von Willebrand’s disease

vWF....................vonWillebrandfactor

 

INTRODUCTION

A purebred Simmental cow was presented to the Veterinary Teaching Hospital,
Michigan State University in November of 1989 with a history of recurrent epistaxis
and hematoma formation associated with minor trauma. Following a difficult but
successful parturition, severe anemia, massive internal hematoma formation and shock
occurred. Circulating platelet numbers and size distribution were normal. A blood
coagulation profile, including prothrombin time, activated partial thromboplastin time,
fibrin degradation products and fibrinogen, was also within normal limits. Due to the
severity of the clinical disease, the owner elected to donate the dam to Michigan State
University. At that time, a member of our Comparative Hematology Laboratory, Dr.
Jennifer S. Thomas, was approached with the request to evaluate platelet function in

Soon after this encounter, we beeame aware of a 1990 report from Canada
describing a potentially fatal bleeding disorder in eight Simmental eattle. Platelet
numbers and ultrastructural morphology, and coagulation screening tests were normal
in these animals. In whole blood aggregation studies, platelets from affected animals

did not aggregate following stimulation with ADP.1

In order to assess any alteration in normal hemostasis, a systematic approach
must be taken which takes into account the possible dysfunctions which might produce

a particular hemorrhagic diathesis. The major eategories to consider include

2
alterations of primary or secondary hemostasis and disorders of the vessel wall. If, as

was the case with the hemorrhagic diathesis of Simmental eattle, the coagulation
profile and circulating platelet number are assessed to be normal, then a detailed

analysis of the platelet structure and function is warranted.

Platelet disorders may be categorized as qualitative or quantitative and primary
or secondary. In addition, the representative disorders in each of these categories
may be further classified as heritable or acquired.2 Examples of these combined
pathogeneses include: Glanzsmann’s Thrombasthenia, a primary hereditary platelet
defect; Von Willebrand’s Disease, a secondary hereditary platelet defect; drug
interaction with excess platelet surface antigen-antibody complex formation, a primary
acquired function defect resulting in subsequent thrombocytopenia; and, disseminated
intravascular coagulation, a secondary acquired disorder. Other factors that have
been reported to contribute to an acquired alteration in the normal platelet response

include autoimmunity, endotoxemia, parasitism, and infectious disease.

The initial approach taken to assess the bleeding disorder in this Simmental
cow involved evaluation of the platelet rich plasma aggregation response following
stimulation by various agonists. This allowed assessment of different pathways in
platelet activation. The results of these laboratory studies revealed a marked
abnormality in the aggregation response to the agonists ADP, collagen, and A23187.

These findings, in conjunction with the clinieal history and physical findings, were

 

3
judged to be consistent with a severe primary, inherited platelet defect. Preliminary

laboratory studies, in which the effect of changes in the concentration of external
ealcium on thrombin-stimulated platelet aggregation was assessed, revealed that
aggregation in control bovine samples was enhanced by increasing the concentration
of external calcium; however, affected bovine samples were insensitive to similar
increases in calcium concentration. This finding, in combination with the aggregation
studies suggested that a ealcium mediated mechanism may be involved in this platelet
function defect.“ A second similarly affected purebred Simmental heifer and a
male Simmental ealf were identified in 1990 and 1992 respectively, and were

purchased by the university to enhance the studies of this defect in platelet function.

Although a great deal is known about the mammalian platelet response, very
little specific information has been published on the bovine platelet. The
identifieation of a blwding diathesis in purebred Simmental eattle has evoked intense
interest in multiple laboratories in the United States and Canada, and the investigation
of the structural and functional characteristics of this disorder, or thrombopathy, is the

focus of this dissertation research."5-‘v7-3

CHAPTER 1: LITERATURE REVIEW

Hemostasis has been defined as the stagnation or arrest of blood flow. Normal
hemostasis is a complex phenomenon, involving a fine balance between, and
participation of, a number of elements including constituents of the blood vessel wall,
plasma clotting factors, and platelets. Any alteration or deficit in these elements may
result in a shift from what ideally should be an organized reparation to a life
threatening pathologic process. Depending on the pathogenesis, the outcome may
range from inappropriate thrombosis to atherosclerosis. Conversely, failure of

hemostasis may result in excessive, often fatal hemorrhage.9J0

Platelets are a crucial component of the primary hemostatic process and
extensive investigations have been conducted to evaluate the phenomena of platelet
activation. The platelet response is a sequential process involving platelet adhesion to
the site of vessel injury shape change, recnIitment of other platelets with aggregation,

and release of granule constituents.

Platelet aggregation is separated into two phases: primary and secondary.
Primary aggregation refers to aggregation alone. Secondary aggregation is
aggregation accompanied by granule secretion. In the former, aggregation may be

reversible. In the latter, the response is irreversible and represents consolidation into

5
larger, more dense aggregates. A major platelet function is hemostasis in rapid blood

flow and high shear vascular environments (such as arterioles and post-capillary
venules).“ Three important factors that determine the initiation of thrombus
formation are: l) alteration in blood flow, 2) damage to the vessel wall, and 3)
activation of the coagulation easeade. Certain rheologie alterations, such as the extent
of turbulence at arterial branches or zones of endothelial injury, may potentiate the
margination of platelets. This initiation process results in the head of the thrombus
being composed of platelets (white clot) and the downstream tail of the thrombus
being composed of red blood cells and fibrin (red clot). In contrast, venous
thrombosis is generally associated with areas in which there is relative stagnation of
blood such as within valve pockets. In this instance, the removal of activated
coagulation factors is slowed and the cellular composition of the thrombus is more
random. The resultant thrombus has the appearance of a clot that forms within a test

tube.12

A major increase in research on the platelet has become apparent in both
human and veterinary medicine in recent years. This intensifieation of focus on
platelet function has substantiated the importance that these small, anucleate cells play
in both the maintenance of normal hemostasis as well as in the pathogenesis of
thromboembolic disorders. One area of study which has received a large amount of
attention is the contribution that the platelet makes in the pathogenesis of
atherosclerosis.13 A proposed mechanism associated with this common, serious

vascular alteration centers on progression through the stages of endothelial injury,

6
platelet adhesion, and resultant platelet contribution to the inflammatory mechanism

through secretion of mitogens such as platelet derived growth factor (PDGF),
epidermal growth factor (EGF), and transforming growth factor-B (TGFB). Platelet
derived growth factor (PDGF) stimulates the proliferation and migration of smooth
muscle cells and also has a proposed chemotactic effect on other inflammatory cells
such as neutrophils and monocytes.“ Also, in humans, vascular thrombosis may
result from softening and eventual ulceration of atheromatous plaques with subsequent

activation of platelets.

Much of what currently is known about the biochemistry of the multi-phasic
platelet response resulted from investigation of heritable disorders in both human
beings and animals. These disorders encompass a wide spectrum of cellular
pathology and involve alterations of platelet morphology, physiology or biochemistry.
Examples of well-documented heritable disorders in man include Glanzmann’s
thrombasthenia and Bemard-Soulier syndrome, which are attributable to defects in the
structure or function of membrane glycoproteins IIb-IIIa (am,83) and lb, respectively.
Also described are Chediak-Higashi Syndrome, a defect in dense granule adenine
nucleotide content/ secretion in many animal species, and Basset Hound Hereditary

Thrombopathy, the pathogenesis of which has been incompletely defined.

Platelets, beeause of their role in hemostasis and thrombogenesis, their
interaction with coagulation proteins, their potential to modulate the inflammatory

response, and their association with sub-endothelial components to promote wound

 

7
healing following cellular damage, continue to be a foeal point in the study of both

human and animal disease.

PLATELET STRUCTURE:

The platelet can be divided into 4 basic compartments or structural domains.”
The first is the domain of Membrane System.“ The primary components of this
domain include the dense tubular system and the open eanalicular system. The dense
tubular system (DTS) is composed of residual smooth endoplasmic reticulum from the
megakaryocyte. The DTS functions to sequester ealcium and is a storage site for
enzymes such as Ca+ +-A'I‘Pase, cyclooxygenase, and thromboxane synthetase.“ The
open eanalicular system (OCS) is a series of invaginations of the plasma membrane.
This network serves to increase the potential platelet plasma membrane surface area
and acts as a site of attachment and release of platelet granule contents in most
mammalian species. The lack of an OCS in the bovine platelet is one of the major

comparative structural differences noted in platelets from this species."

The second structural domain is the sol-gel zone. Components of this domain
comprise the platelet cytoskeleton and include the matrix of the cytoplasm. Integral
constituents of this compartment include the actin and myosin filaments, the
membrane skeleton, and the microtubule coil. Actin is the major platelet protein and

40-50% of this protein exists in the filamentous form in the resting platelet. The

8

interaction of actin with myosin is primarily responsible for the contractile force
generated in the activated cell.12 The membrane skeleton consists in large part of
short actin filaments which are cross-linked by actin binding protein. These filaments
form a lining to the plasma membrane resulting in stabilization of the lipid bilayer.
The membrane skeleton is linked to the plasma membrane primarily at the site of
GPIb-IX, GPIa, and GPIIa. The membrane skeleton is ulu'mately disrupted through
the activation of a Ca+ +—dependent protease which hydrolyzes actin binding protein.19
Maintenance of the normal discoid shape is the function of the microtubule coil.
These microtubule forms exist in a circumferential band and are, in addition,
responsible for the organized internal contraction of the platelet which results in the
centralization of organelles.” The primary component of this microtubule coil is
tubulin. In addition to the above described members of the sol-gel zone are a group
of proteins which are associated with the cytoskeleton. This group of proteins
includes profilin which inhibits actin polymerization, gelsolin which binds to actin
filaments resulting in their fragmentation, talin, vinculin, a-actinin, spectrin, and

actin-binding protein.

The third structural domain of the platelet is the organelle zone. The
organelles in this category are the platelet alpha granules, dense granules (dense
bodies), lysozomes, peroxisomes, glycogen granules, and mitochondria. Enzymes
present within certain of these organelles are of importance in the production of ATP
within the resting cell. The systems whereby glycogen or extracellular glucose are

metabolized in the resting platelet are the processes of glycolysis, which yields

 

9
lactate, and the Kreb’s cycle, which results in the production of carbon dioxide. It is

reported that each of these two systems accounts for approximately 50% of the ATP
produced in m conditions.12 The organelles most frequently studied are the
alpha granules and the dense granules. Alpha granules are the most abundant
organelle21 and the contents include platelet specific proteins such as platelet factor 4,
PDGF, and B-thromboglobulin. In addition, these granules contain coagulation
factors (fibrinogen, factor V), von Willebrand factor, thrombospondin, fibronectin,
albumin, a-2 plasmin inhibitor, a-l antitrypsin, and a-2 macroglobulin.22 The
primary function of the dense granule is storage of the adenine nucleotides (ATP and
ADP). The dense granule also stores serotonin, calcium, magnesium, inorganic

phosphates, and catecholamines.

The final main domain of the platelet is the peripheral zone. This
compartment is made up of the submembranous zone (an area in which signals from
the cell’s exterior are translated to chemical messages and physical events), the unit
membrane (includes the phospholipid bilayer and glycoproteins) and the glycocalyx
which includes the exterior components of the plasma membrane such as receptors for

various platelet agonists.

THROMBOPOIESIS:

Megakaryocytes derive from a precursor pluripotent stem cell which is also

common to myeloid and erythroid cells."'” Megakaryocytes undergo

10
endoreduplication during maturation, eventually reaching a size of 15 x 10’ fl. Each

cell gives rise to approximately 1,000 to 1,500 platelets.

The rate of thrombopoiesis is probably controlled by regulatory T-cells at the
stem cell level and by the aZ-globulin, thrombopoietin, at the level of differentiation
and maturation. The maturation of the cytoplasm of megakaryocytes is associated
with the appearance of the demarcation membrane system (DMS). This system is

continuous with the surface membrane of the megakaryocyte.“

Megakaryocytes are transformed in the marrow from a large spherical shape to
an irregular shaped cell with cytoplasmic protrusions. These projections extend out
through the sinusoid lining into the marrow sinusoids. The cytoplasmic protrusions,
proplatelets, are pinched off and released into the circulation as platelet precursors.
There is a relative lack of a readily mobilizable pool of platelets within the bone
marrow. Therefore, alternate sites of thrombopoiesis are present within the body
which function subsequent to migration of both megakaryocytes and proplatelet forms
from the bone marrow. These sites include the pulmonary system, the liver and the

spleen. “

Platelets are the smallest circulating cells, with a volume of approximately 10
fl. These cells circulate for an average of 9-10 days and platelet senescence is

through progressive lipid peroxidation.”

l 1
THE PLATELET RESPONSE:

ADHESION: Adhesion is a three step process. This process involves binding
of vWF to the subendothelium, platelet contact (when vWF binds to GPIb), and
finally, platelet spreading and platelet plug formation. This final step is dependent on

vWF binding to GPIIb-IIIa following collagen binding to GPIa.”""1’7

Fibrinogen, fibronectin, and vWF all contain the peptide sequence arg-gly-asp
(RGD) which is a critical component of cell-to—cell as well as cell-to-subendothelial

interactions.

SHAPE CHANGE: Shape change is a rapid response which is independent of
the external calcium concentration and fibrinogen levels. This response is described
as the loss of the normal discoid shape seen in the resting cell, and transformation to
an irregular form with long, spiky pseudopods becoming prominent. These
pseudopods progress to become dendritic forms which provide surface receptors for
the adhesive platelet-platelet and platelet-endothelial cell reactions.” Following
continued exposure to a stimulus, the platelets are seen to spread, with cytoplasm

subsequently filling the pseudopods.“

AGGREGATION: Aggregation is a calcium dependent process. Two primary
mechanisms important to this process are release of intracellular components from

adherent, activated platelets and very close apposition or collision between

12
surrounding platelets.” Subsequently, the platelet cytoskeleton undergoes structural

changes which are dependent on: a) activation of the Ca+ +-dependent protease which
detaches the membrane skeleton from the plasma membrane; and, b) association of
GPIIb-IIIa with cytoplasmic actin filaments.30 Following these initial stages of
activation, the platelet membrane receptor GPIIb-IIIa is rendered able to bind
circulating fibrinogen which enhances the platelet-to—platelet cohesion and

subsequently leads to the formation of a stable thrombus.

SECRETION: Upon specific stimulatory conditions, the contents of the
platelet organelles (alpha granules and dense granules) are extruded into the platelet’s
external environment without loss of, or affect on, the other intracellular organelles.
This phenomenon is termed secretion or release. This process is very rapid and
generally is completed within 20 seconds to 2 minutes depending on the type of
stimulus involved. Secretion is not specifically induced in the platelet without

involvement of other stages of the platelet response, namely shape change and

aggregation.31

Platelet secretion has historically been reported to be the result of contraction
of the platelets circumferential microtubule coil. This contraction of the microtubule
coil causes a centralization of granules which is followed by attachment of the granule
membrane to the membranes of the OCS. Finally, granule contents are released into

the OCS through the process of exocytosis.’”3

 

13
AGONISTS AND PLATELET ACTIVATION:

Three categories of agonists have been described. The first category are weak
agonists such as adenosine diphosphate (ADP) and epinephrine. These agonists
require release of cyclooxygenase metabolites and the presence of primary aggregation
to induce irreversible aggregation and granule secretion. Members of the second
group, agonists of intermediate strength, include platelet activating factor (PAF),
vasopressin, and prostaglandin endoperoxides/ thromboxane mimetics such as the
compound U46619. Intermediate strength agonists induce secretion independent of
prior aggregation. The third category of agonists utilized in platelet evaluation studies
are the strong agonists. One example of this strong agonist classification is high dose
thrombin, which has the capacity to stimulate aggregation and secretion in a manner

unaffected by cyclooxygenase inhibitors.

Six different agonists (both physiologic and non-physiologic) representing all
three of the above described categories were used in the laboratory investigations for
this project. Collagen is a strong physiologic agonist which aggregates platelets
following a delay. Adherence of platelets to collagen fibrils is involved in the
response. During the adhesion process, granule release occurs; therefore, collagen
can induce release in the absence of aggregation. Similar to thrombin, the collagen

response is accompanied by ADP release and arachidonic acid biotransformation.”

 

14
Adenosine diphosphate is described as a primary aggregating agent.

Depending on the concentration of ADP, the resultant aggregation response may be
reversible or irreversible. Activation of platelets by ADP results in further release of
this compound by release of dense granule contents, which may serve to enhance the
local aggregatory response?“ ADP binding to platelet has two effects: 1) inhibition
of the activation of adenylate cyclase, and 2) stimulation of shape change, fibrinogen
binding site exposure and aggregation.“5 The ADP receptor is intimately associated
with the GPIIb-IlIa receptor, although the exact mechanism of this association has not
been completely or clearly defined.37 However, when ADP is added to
thrombasthenic platelets, no aggregation or release occurs. Although the exact
mechanism of the ADP-platelet interaction and activation is a point of controversy, it
is widely accepted that ADP binds to a 100 kDa platelet surface protein, aggregin?”
Aggregin itself inhibits the fibrinogen binding site on GPIIb—IIIa, therefore, the
binding of ADP to the aggregin receptor is proposed to cause a conformational
change in aggregin which allows exposure of the fibrinogen binding sites and
subsequent binding of fibrinogen to this GP complex. Further, ADP stimulates
release of arachidonic acid by Na”/I-I+ exchange—dependent activation of PLA,,
ultimately resulu'ng in the production of thromboxane which then amplifies the platelet
response through the activation of PLC?” The process of secondary or irreversible

aggregation with ADP is dependent upon this generation of AA metabolites.

A23187 is a calcium ionophore which acts as an agonist of intermediate

strength in platelet activation.40 In general, calcium does not enter the platelet unless

15

it is stimulated by agents such as thrombin. A23187, however, forms a membrane-
penetrating complex with calcium and induces shape change, aggregation, and
granules release. The response generated by stimulation of the cell with A23187 is
assumed to be due to mobilization of platelet calcium from storage depots such as the
dense tubular system (DTS). This resultant elevation of ionized calcium is not
affected by inhibition of cyclooxygenase or by removal of ADP. Therefore, it is a

primary affect of the calcium ionophore itself.29

Platelet activating factor (PAF) is a phospholipid which, though not
manufactured within the platelet, is stored within alpha granules.“ A high
concentration of PAF non-specifically alters cell membranes. PAF binds to its
receptor and mediates the hydrolysis of the membrane phosphoinositides (PI) and
yields production of the second messengers (1,4,5)-inositol trisphosphate [(1,4,5)-IP3]
and 1,2-diacylglycerol (1,2-DAG).‘2 PAF has the ability to mobilize calcium; the
majority of this increase in ionized calcium is believed to be derived from the influx
of external calcium.‘3 PAF-induced platelet activation leads to shape change and
primary aggregation, with these stages of the platelet response occurring
independently of ADP and AA metabolism. However, irreversible platelet
aggregation is strictly dependent upon ADP release. PAF causes inhibition of

adenylate cyclase and this mechanism is Gi-mediated.33

Two mechanisms have been described by which thrombin is able to activate

platelets. The first is receptor-mediated and the second is a proteolytic process.“ In

A

16
the first instance, thrombin binds to a transmembrane, G—protein type receptor with 7

transmembrane domains!” Cleavage of the extracellular N-terminus allows the
binding of thrombin to this receptor setting in motion the signal transduction, effector
system, and second messenger-generating process.‘7 In the second process, thrombin
binds to GPI, and causes the proteolytic cleavage of GPV. Thrombin initiates platelet
activation by stimulating PLC activity, both through a receptor-mediated system in
which adenylate cyclase is inhibited by a mechanism which is receptor-mediated and
dependent on G—protein activation, and by a proteolytie process (through the activation
of calpain, cleavage of aggregin, and exposure of fibrinogen binding sites).

Thrombin stimulates calcium mobilization, most of which is reported to be due to an
influx of external calcium.‘8 Cyclooxygenase inhibition partially blocks only external
calcium influx and does not affect the mobilization of calcium from internal stores.
Thrombin causes fibrinogen receptor exposure irrespective of the presence of external

ADP.

Phorbol myristate acetate (PMA) is a member of the group of phorbol esters
which are classed as tumor promoters. These substances have been shown to activate
platelets by substituting for diacylglycerol, thus having a direct effect on protein
kinase C (PKC).‘9 Phorbol esters are non-physiologic agonists which cause both
aggregation and secretion without initiating shape change.32 These compounds have a
structure similar to 1,2-DAG. Phorbol esters are not metabolized by the cell and

have the capability to cause permanent PKC activation.

17
PLATELET MEMBRANE RECEPTORS:

Numerous glycoprotein moieties have been identified associated with the
platelet plasma membrane. These glycoproteins function as receptors for various
compounds including agonists such as thrombin and collagen, ligands such as
fibrinogen and fibronectin, divalent cations (i.e. Ca“, Mg“), plasma proteins
(vWF) and some hormones?”l Possibly the most frequently studied and well defined
glycoprotein is the glycoprotein (GP) lIbIIIa receptor.‘2 This receptor has more
recently been classified as a member of the family of integrins.53"‘ These compounds
are described as heterodimers of a and B subunits which are products of separate
genes and are mutually interdependent for correct surface expression.” Integrin
expression and function is dependent upon millimolar concentrations of either calcium
or magnesium which may occupy binding sites on the compound itself or on the
associated ligand. Integrins typically have short cytoplasmic domains that are
accessible to intracellular second messenger components and thus may mediate signal
transduction.” GPIIb-IIIa has been termed the and}, integrin, however, for the
purpose of this dissertation, the original GPIIb-Hla designation will be used. GPIIb-
IIIa functions as a receptor for RGD—containing ligands. Once activated, this receptor
can bind to fibrinogen, fibronectin, vitronectin, and vWF, therefore becoming as
instrumental component in the platelet-to—platelet and platelet-to-vessel wall phases of
the platelet response.” GPIIb-IIIa does not associate with the membrane skeleton, but

with the cytoplasmic actin filaments of the cytoskeleton, and then only when

18
aggregation takes place.“5"” In most mammalian platelet systems this association

with the actin filaments appears to be, in part, dependent upon the presence of talin.

A second, well defined receptor is that associated with the protease platelet
agonist thrombin. The thrombin receptor is a member of the G protein-coupled
family and consists of a single polypeptide chain with seven trans-membrane domains.
Thrombin acts upon a cleavage site within the N—tenninus of the receptor. The
intracytoplasmic C-terminus of this receptor is then associated with succeeding events
such as transmission of the receptor signal through the G-protein/effector system

pathway resulting in eventual second messenger activation!”

Other glycoproteins of importance in the platelet include: 1) GPIa which is
reported to function as a receptor for collagen; 2) GPIV (GPIIIb), a receptor for
thrombospondin which may also serve as a receptor for collagen adhesion; and, 3)
GPIb which functions in several different capacities including acting as a receptor for

certain antibodies, vWF, and thrombin."’-"’°"‘l

SECOND MESSENGER SYSTEMS (signal transduction):

Calcium: Calcium plays a complex role in mediating various platelet

reactions. Aggregation by most physiologic agonists has an absolute requirement for

 

l9

calcium.“2"‘3 Contractile and secretory processes are moderated by an increase in
cytosolic ionized calcium concentration.“'“ Calcium regulation of contractile proteins
in platelets is through an actin-linked mechanism mediated by tropomyosin and
troponin. Calcium and the calcium binding protein calmodan are necessary for

activation of myosin light chain kinase.“

Resting intracellular calcium concentration is acn'vely maintained at a
concentration of approximately 100nM and there are approximately 20 nmol of Ca“
per 10' cells. Calcium efflux is dependent on a source of energy and is stimulated by
calmodulin. Membrane Ca+ +-ATPases are responsible for the energy-dependent
transport of calcium across membrane barriers against a concentration gradient?“
There are two distinct forms of calcium ATP-ases; one, a calcium pump located in
the internal membrane system, and the other present within the plasma membrane.“
Inhibition of calcium ATP-ase activity occurs following agonist induced stimulation of
PLC and subsequent hydrolysis of membrane P12, resulting in production of (1,4,5)IP3
and (1,2,cyc1ic 4,5)IP3 which are the major stimuli for the receptor-mediated release
of calcium from the DTS. Calcium sequestration in the DTS is stimulated by 1)
calmodulin, 2) increased levels of cytosolic AMP, and 3) by PKC activation.69

Calmodulin functions to stimulate the uptake of calcium into the DTS.

Platelet storage sites of calcium include the non-exchangeable pool, most of
which is contained within dense granules, and the exchangeable pool. The

exchangeable platelet calcium pool is further subdivided into 2 intracellular pools; the

 

20
rapidly exchangeable pool present within the cytosol (independent of the external
calcium concentration) and the slowly exchangeable pools. These slowly
exchangeable pools include the calcium associated with mitochondria and the dense
tubular system (DTS). The DTS is responsible for the majority of calcium present
within this pool. The calcium concentration of this slowly exchangeable intracellular
pool varies with the external calcium concentration. An additional member of the
exchangeable calcium pool classification is that calcium which is found bound to, or
near the platelet surface. In this category are high- and low-affinity binding sites
which are in part associated with the GPIIb-IIIa receptors. Calcium is typically found

to be most concentrated near the plasma membrane.

Mobilization of Ca++ from the external environment into the platelet cytosol is
via a series of calcium channels?“ Two basic types of channels are recognized:
voltage operated channels (V CC) and receptor operated channels (ROC)."2 Voltage
operated channels are felt to play only a minor role in relation to calcium
homeostasis. Receptor operated channels may be divided into second messenger
operated channels (SMOC) or G-protein operated channels (GOC).73'7‘ SMOC,
through activation of specific receptors coupled to the hydrolysis of
polyphosphoinositides, activate calcium influx from the extracellular medium. The
phosphoinositides typically involved in this process include 1,2,5- trisphosphate and
l,3,4,5- tetralrisphosphate.7"7°'"'" GOC function through receptor-channel interaction
in the plane of the membrane. This mechanism is mediated by a GTP binding

protein . 72.73.."

21

Calcium mobilization may vary in response to platelet stimulation by various
agonists."""°"l For example, ADP action upon the platelet is partially independent of
the release of AA metabolites.“2"‘3 This therefore suggests that this phenomena is not
dependent on a receptor—mediated product or a diffusible second mediator to induce
calcium influx, but instead, is associated with a direct coupling between the receptor
and the plasma membrane calcium channel, possibly via a G—protein mediated
mechanism."'“"5'“'"'“ In addition, platelet calcium flux is relatively independent of

thromboxane production in some species such as the bovine.

Intracellular calcium also plays an important role in the organization of
cytoskeletal proteins.”-’° It has been proposed that it is the binding of fibrinogen to
the GPIIb-HIa complex or the incorporation of the GPIIb—IIIa complex into the
cytoskeleton which opens a pathway for calcium influx.90 Conversely, steriochemical
calcium-dependent changes in platelet membrane receptors, such as GPIIb-IIIa, allows
the high-affinity binding of fibrinogen.91 Calcium affects fibrinogen binding in 2
ways: 1) through regulation of expression of fibrinogen-receptor function (this
requires Inicromolar concentrations of calcium), and, 2) through mediation of binding
of fibrinogen to its receptor. The latter mechanism requires millimolar, and therefore
extracellular, concentrations of calcium. Calcium does not cause the
heterodimerization of the GPIIb—IIIa complex, but instead, affects the conformation of

the preexisting complex.91

22
Secretion is a response, due in part, to an increase in cytoplasmic free
calcium?”2 This release of the contents of storage granules through fusion of the
granule membrane with the OCS (or plasma membrane) requires calcium as a
fusogen. Platelet secretion is thought to be associated with conversion of globular
actin to filamentous actin and the subsequent interaction with myosin, as well as with
phosphorylation of a 47 kDa protein by way of PKC. Both of these processes are

calcium regulated."

PHOSPHOINOSITIDES: One of the major second messenger systems
involves the hydrolysis of membrane phosphoinositides by phospholipase C (PLC) to
yield the products 1,2 diacylglycerol (DAG) and 1,4,5-inositol trisphosphate (IP,).”
Phosphoinositides are anionic glycerophospholipids present in the inner layer of cell
membranes. The parent compound is phosphatidylinositol (PI), which, through the
action of specific kinases, is hydrolyzed to form phosphatidylinositol 4-
monophosphate (PIP) or phosphatidylinositol 4-5, bisphosphate (PIP2)."
Phospholipase C is a phosphodiesterase that is stimulated by a GTP-binding regulatory
protein (G-protein).” This G-protein causes dissociation of GDP from its alpha
subunit thus permitting the binding of GTP and subsequent activation of the effector,

PLC."'96

A further member of the signal transduction/second messenger family is the

cyclic AMP (cAMP) system. Receptor-induced stimulation or inhibition of adenylate

 

23
cyclase controls intracellular levels of cAMP, which in turn acts to monitor platelet

inactivation or activation, respectively. Receptors responsible for the mediation of the
adenylate cyclase levels are the adrenergic receptors on the platelet membrane.
Typically, B-adrenergic receptors act to stimulate adenylate cyclase production, and a-
adrenergic receptors function in an inhibitory role. Cyclic AMP removal from the
cell is regulated by the enzyme cAMP phosphodiesterase. Cyclic AMP decreases the
stimulatory effect on the platelet by inhibiting the agonist-induwd rise in ionized
calcium through limitation of the influx from the external environment and by
suppressing release of calcium from internal stores.”98 Activators of adenylate

cyclase include prostacyclin, PGDZ, PGE1, and adenosine.

Important to the generation of the platelet aggregatory response is the
production of various prostaglandins through the hydrolysis of AA from membrane
phospholipidsiz'33 This mechanism procwds by a calmodan mediated process
through hydrolysis by PLA2 resulting in the generation of PGG2 and PGH2, both of
which are potent platelet aggregating agents. PGHZ is converted, via the
cyclooxygenase pathway, to the stable prostaglandins PGE, and PGFh, which do not
directly aggregate platelets.” In addition, PGD; is generated; this is a compound with
inhibitory properties on aggregation. An integral component of the above described
pathway is thromboxane A2. This product is the most potent aggregating agent

derived from AA.

 

24
PLATELET PATHOLOGY:

Pathologic conditions involving the platelet may be classified as qualitative vs
quantitative and inherited vs acquired. Quantitative disorders are perhaps the easiest
to detect; however, their pathogenesis may be more elusive. ‘°°"°‘ Acquired defects
may stem from a wide variety of causes in which the absolute number of platelets is
affected or platelet function may be compromised. Examples of acquired platelet
defects include the inhibition of the platelet response of aggregation by non-steroidal
anti-inflammatory drugs (NSAID) such as aspirin, indomethacin, phenylbutazone,
ibuprofen, naproxen, and fenoprofen. This inhibitory action is mediated through

cyclooxygenase which catalyzes the conversion of AA to PGsz

Additional examples of acquired defects in platelets include: ineffective
thrombopoiesis seen in calves with trypanosorniasis; thrombocytopenia, a consistent
finding in acute equine infectious anemia; and, the thrombocytopenia associated with
some cases of non-cytopathic BVD in sew-negative immunocompetent calves.
Thrombocytosis with small platelets is characteristic of iron and copper deficiency,
and may also be a sequelae to splenectomy, polycythemia vera or megakaryocytic

myelosis.

Systemic lupus erythematosus (SLE), certain drugs/medication, or infectious
processes may cause an acquired thrombocytopenia resulting from excess agzab

complexes which attach through the Fc fragment to the Fc receptors present on the

25

platelet membrane. Recognition of these complexes then causes premature uptake of

affected cells by the reticuloendothelial system, primarily of the spleen.

Inherited platelet disorders encompass a wide spectrum of platelet pathology
and include syndromes such as Gray Platelet Syndrome which is an absence of alpha
granule contents;‘°3-‘°‘"°‘ Chediak Higashi Syndrome (CHS),‘°‘"°”"°‘"°’ Hermansky
Pudlak Syndrome, and Wiskott—Aldrich Syndrome, all of which are characterized by a
deficiency of dense granules with associated defects in skin pigmentation; Bernard
Soulier Syndrome depicted by a severe deficiency of the GPIb-IX complex and GPV;
and, Glanzmann’s Thrombasthenia, a complete lack or abnormal function of GPIIb-

IIIa receptors.

A further example of an inherited platelet disorder is Basset hound hereditary
thrombopathy (BHT). Extensive analysis of this defect have revealed the mode of
inheritance of BHT to be autosomal recessive. It has been hypothesized that the BHT
defect involves a dysfunction in the signal transduction system and/or the generation
of second messengers. Platelets from BHT affected animals produce excessive
amounts of the prostaglandin, thromboxane A2 (TXAz) when activated with certain
agonists. In addition, platelets from affected Basset hounds exhibit a markedly
decreased phosphorylation of a 65-67 kDa protein on autoradiography when compared
with controls."°"“"‘2’“3 Continued interest in the mechanisms of various
hemorrhagic diatheses has led to the identification of many new familial disorders of

platelet structure and function.“"“5

26
Bleeding disorders in which there is an abnormality associated with platelet

number or function must be differentiated from disorders of the coagulation system.
These disorders, too, may be acquired (moldy sweet clover or warfarin ingestion and

toxicity; or, may be inherited (Factor VIII or Factor X deficiency).

CHARACTERIZATION OF THE BOVINE PLATELET:

When compared to human platelets, less in known about the structure and
function of bovine platelets. Several studies comparing the bovine platelet
ultrastructure and response to those seen in platelets from other mammalian species

have revealed several unusual features of these cells.“‘"mmeM‘"

Bovine platelets are unique in that they exhibit a lack of an open canalicular
system (OCS).“-“"“‘ This system of tortuous interconnected invaginations of the
plasma membrane can be seen transversing the platelet cytosol in most mammalian
species and serves as one mechanism by which cytoplasmic granules are released to
the external environment during secretion. The OCS is also a route for the
internalization of substances from the plasma. It appears that in bovine platelets, the
granules are located toward the periphery and release their contents by fusing directly
with the external membrane during activation.“ This discharge of granule contents in
the example of thrombin-activated platelets follows two routes. Most of the granules
close to the surface fuse with the membrane and secrete directly to the outside

medium (associated with internal contraction leading to the formation of dense spots

27

of contractile gel).121m However, development of a primitive channel system (short

invaginations) has also been reported in activated cells.122

It is reported that bovine platelets are more sensitive to PAF stimulation than
to ADP.“‘-m-"" There appears to be no correlation between thromboxane A,
production and release and the extent of platelet aggregation in bovine
platelets.“7"’"‘” The dependence, therefore, of bovine platelet aggregation on a
phospholipid pathway and calcium mobilization is indicated. Bovine platelets, in
general are less receptive to stimulation than most other mammalian platelets (except

in the case of thrombin).

Surface-activated bovine platelets do not spread, they "unfold". Human
platelets react to surfaces by losing their discoid shape, extending pseudopods,
converting to dendritic forms, and finally spreading into thin films resembling
pancakes. Bovine platelets are unable to fill in spaces between pseudopods, a step

required for spreading.126

It has been shown that circumferential microtubules are more resistant to
disassembly in surface-activated bovine than human cells; therefore, bovine platelets
are more resistant to deformation.“ Bovine platelets are significantly smaller than

human cells, have larger alpha granules, and circulate in larger numbers.

28
Finally, aggregation studies involving bovine platelets have demonstrated less

inhibition following treatment with acetylsalicylic acid (ASA). Thus, it is suspected
that the aggregation response in bovine platelets is much less dependent upon the
cyclooxygenase pathway generation of thromboxane from arachidonic acid than other
mammalian species, in which ASA produces a marked dampening effect on the

maximum percent aggregation."“-“7

The distinctive differences in platelets seen in diverse mammalian species
afford a unique opportunity to more clearly understand the various biochemical and
physiologic processes involved with the phenomenon of the platelet response.
Further, the recognition of an inherited disorder of platelet function in purebred

Simmental cattle provides a model by which comparisons between normal and

abnormal bovine platelet systems may be made.

 

 

Chapter H: STATENIENT OF THE PROBLENI

The blwding diathesis in purebred Simmental cattle is obvious to the observer,
whether livestock breeder, herd manager, or veterinary practitioner. Affected cattle
display overt blwding tendencies which range from mild recurrent epistaxis to life
threatening hemorrhage occurring at surgery or parturition. The presence of this
bleeding trait in purebred cattle began to be documented in the late 1970’s, with

subsequent sporadic anecdotal reports in the literature?"

The next report was a retrospective clinico-pathologic publication
encompassing the physical, hematologic, and preliminary platelet aggregation findings
noted in Canadian Simmental cattle seen at the Western College of Veterinary
Medicine, University of Saskatchewan, Saskatoon, Saskatchewan."7 Concurrently,
with the identification of similarly affected cattle in the midwest, the study at
Michigan State University evolved around our hypothesis that there was a hereditary
thrombocytopathy in these cattle?" Although preliminary groundwork on this
hereditary disorder had been done, clarification of the genetic basis, detailed
definition of the platelet responses, and eventual identification of the biochemical
nature of the platelet function defect in what we have termed Simmental Hereditary
Thrombopathy (SHT) needed to be addressed. These points and considerations, then,

have become the focus of this research endeavor.

29

 

 

30

To begin the investigation of the problem, affected Simmental cattle were
screened for hematologic values, coagulation profiles, clinical history and basic
physical attributes. All laboratory criteria were compared with control animals (both
Simmental and Holstein breeds) as well as with the Michigan State University Clinical
Pathology laboratory reference values, and were judged to be within normal ranges.
Plasma samples were submitted to the New York State Department of Health,
Comparative Hematology Laboratory for von Willebrand factor evaluation. All
samples submitted, including those from normal, affected, and carrier animals were

within normal range for cattle.

Initially, this project was focused on the evaluation of platelet function
following agonist stimulation. Previous reports had addressed only the response to
adenosine diphosphate (ADP) in platelet activation studies.” It was therefore
necessary to select a broader range of both physiologic and non-physiologic agonists
to more clearly categorize the stimulatory response of the platelets from affected,
carrier and control cattle. Agonists chosen for use in this study included ADP,
platelet activating factor (PAF), the calcium ionophore A23187, thrombin, collagen
and phorbol myristate acetate (PMA). These compounds afforded the opportunity to
compare results with those previously reported (i.e. ADP) as well as to evaluate
stimulation of the platelet response through specific and diverse pathways such as
receptor-mediated activation of the phosphoinositide second messenger system (PAF,

thrombin), membrane-association and subsequent mobilization of internal calcium

 

31
stores from the dense tubular system (A23187), and direct activation of protein kinase

C (PMA).

Platelet aggregation and dense granule secretion studies utilizing the above
described agonists were coupled with transmission electron microscopy to compare
the activation results with changes in platelet ultrastructure and morphology.
Dramatic differences in platelet aggregation responses were seen between normal
control and affected cattle. Affected cattle showed minimal to no response to ADP,
A23187, or collagen, and had mildly to moderately dampened responses to PAP and
thrombin} Only with PMA were the aggregation responses in affected animals

indistinguishable from controls.

Additional studies utilizing variations in the concentration of calcium within
the external medium revealed affected platelets to be much less sensitive to increases
in concentration of this divalent cation. Evaluation of the dynamics of clot retraction
revealed a marked difference between samples from control and affected cattle.
Whole blood from normal cattle displayed prominent clot retraction with development
of a stable clot within one hour. However, whole blood samples from affected
animals had very poor clot retraction with formation of a soft, friable clot at one
hour. The supposition was made that it may be a defect in calcium mobilization!
utilization (either from internal calcium stores or from influx of external calcium) that
is the basis of the attenuated platelet responses in affected animals. This dreary led to

the next phase of the research which centered around the investigation of cytosolic

32

ionized calcium concentration ([Ca+ +1» in platelets at rest and following agonist-
stimulation, utilizing the fluorescent calcium indicator dye, fura-2/acetoxymethyl ester

(fura-2/AM).3""

Incorporating knowledge gained from reports on quantitation of fibrinogen
binding sites and rate of fibrinogen binding in platelets from Canadian Simmental
cattle with this defect,8 in concert with the aggregation, secretion, clot retraction and
[Ca+ *J, studies reported in our cattle, it was hypothesized that affected platelets have
abnormal utilization of calcium in respect to the requirement for calcium in the
expression of the GPIIb-IIIa heterodimer and binding of fibrinogen to this receptor.
Future studies proposed would include “Ca” labelled calcium binding analysis to
assess the number of high- and low-affinity calcium binding sites available on the
platelet plasma membrane surface, and, isolation/characterization of the individual

GPIIb and GPIIIa membrane structures.

Finally, knowledge gained through these studies will be employed in attempt to
identify carrier animals and, eventually, to develop a method of testing potential
brwding animals for this unique hereditary defect. The bleeding diathesis in
Simmental cattle is a problem which needs to be clarified, not only for the future
well-being of the breed but also for the sake of the scientific enigma that the disease

presents.

CHAPTER HI: NIETHODS DEVELOPIVIENT AND RESULTS

AGGREGATION AND SECRETION STUDIES

Introduction:

Measurement of platelet aggregation and dense granule secretion is performed
as a means of evaluating the response of the platelet to stimulation by a variety of
physiologic (ADP, collagen, PAF, thrombin) and non-physiologic agonists (PMA and
A23187).

Two distinct measurements are obtained from aggregation studies. The first is
the maximum percent aggregation, which is an assessment of the degree or extent of
activation of the platelet and is reported as a percentage. The second measurement is
the slope of the aggregation curve. This value is an indication of the rate of

aggregation and is reported in arbitrary units of slope.

In addition to evaluating the rate and extent of aggregation, platelet sample
stimulation by the above described agonists can be utilized to examine dense granule
ATP secretion. This assessment allows the investigator to appraise, by an additional
means, the overall platelet response. The release of the dense granule adenine

nucleotide constituents, which ultimately serves to enhance the continued platelet

33

34
activation process, also is directly related to the degree of irreversibility of

aggregation.

All studies were run in triplicate on each animal tested. The individual
aggregation and secretion study results were averaged to obtain a mean and standard

error for each group.

Materials and Methods:

For all agonists except thrombin, aggregations were measured in platelet rich
plasma (PRP). Whole blood was collected from the jugular vein through an 18-gauge
needle into plastic syringes containing 3.2% w/v trisodium citrate at a ratio of 9 parts
blood to 1 part citrate. If the collection was interrupted in any way, the sample was
discarded and a second collection made. Polyethylene or polypropylene labware was
used in all platelet isolation procedures. Platelet rich plasma was prepared by pooling
the supernatants from two centrifugations at 1324 x g (90 and 60 seconds). Following
removal of the platelet rich plasma, the blood was centrifuged for 13 minutes at 1324
x g to obtain platelet poor plasma (PPP). The platelets were counted manually using
the Unopette system and a Neubauer hemocytometer. Autologous platelet poor
plasma was used to dilute the platelet rich plasma to a concentration of 3.0 x 10‘

platelets/ ml. Platelets were aliquoted into glass aggregometer cuvettes (0.5ml),

covered with parafilm, and allowed to rest for 30 minutes at room temperature. In

 

35
m platelet aggregation was measured using either of two aggregometers described

below. Agonists were added to stirred platelet suspensions and the changes in light

transmission recorded.

Thrombin-induced platelet aggregations were measured in gel-filtered platelet
preparations. Platelet rich plasma was collected as described above and prostaglandin
E, was added from a lmM stock in ethanol to give a final concentration of IM.
This was done to minimize platelet activation during the subsequent centrifugation and
gel-filtration. After a 5 minute incubation at room temperature, the platelet rich
plasma was centrifuged at 800 x g (20°C), the supernatant discarded and the platelet
pellet resuspended in 1 III] of HEPES—Tyrode’s-albumin buffer (HTA) containing
130mM NaCl, 2.6mM KC], 0.42mM NaHzPO“ 5.5mM dextrose, 0.01mM HEPFS
and 3.0 mg/ml bovine serum albumin, pH 7.2. The platelet suspension was
transferred to a polystyrene column (10 m] bed volume) filled with Sepharose 4B and
equilibrated with HTA at room temperature. Gel filtered platelets were collected in
the void volume, manually counted (U nopette and Neubauer hemocytometer), and the
eluate diluted to a concentration of 3.0 x 10' platelets/ml with HTA. CaCl, and
MgClz were added to give a final concentration of 1.0mM each. [Note: The MgCl,
was omitted when the platelets were to be used for ATP secretion studies as the
Chrono-Lume reagent contains IOmM MgSO, which resulted in a final concentration
of lmM MgSO, after addition to the aggregation cuvette]. The gel filtered platelets

were then handled as described above and allowed to rest at room temperature for 30

minutes in preparation for aggregation and secretion studies.

 

36

Platelet Aggregation:

Platelet aggregation studies were carried out using one of two laboratory
aggregometers, a Lumi-Aggregometer (Chronolog Corporation, Havertown, PA) or a
Platelet Ionized Calcium Analyzer (PICA, Chronolog Corporation, Havertown, PA).
Platelet samples were maintained within the aggregometers at 37°C and all trials were
run within 5 hours of collection of blood samples. Platelet samples (PRP or GFP)
were allowed to equilibrate within the aggregometer block warmer for 5 minutes
before evaluation began or agonists were added. A teflon-coated magnetic stir bar
was added to the sample and the cuvette was then placed into the aggregometer. The
sample was allowed to stir at 900 rpm for approximately 30 seconds at which time
20rd of an agonist was added. Aggregation results were recorded on a Houston
Omniscribe Chart recorder (Houston Instrument Division of Bausch and Lombe, Inc. ,
Austin, TX). Two measurements were made on each trial run. The first was the
maximum percent aggregation which was a value depicting the change in light
transmission detected through the sample. The second measurement taken was the
slope of the aggregation curve. This measurement is an indication of the rate at
which change in light transmittance is occurring, and therefore, is an indicator of the
rate of platelet aggregation within an individual sample. Aggregation measurement
results were not recorded in the presence of Chrono-Lume reagent as this compound
is noted to have somewhat of a potentiating effect on platelet aggregation. Agonists

(final concentration) used for aggregation studies included: ADP (3.5-10.0p.M), PAF

37
(0.1-10.0uM), A23187 (10.0-40.0flM), collagen (2.0-20.0ug/ml), thrombin (0.1-

1.0U/ml), and PMA (3-0‘3-75Il-M)-

Platelet Dense Granule Secretion:

For the investigation of dense granule ATP secretion, aggregometer cuvettes
containing 0.47 III] of platelet rich plasma or gel filtered platelets equilibrated to 37°C
were placed in the aggregometer and stirred at 900 rpm. Chrono-Lume luciferin-
luciferase reagent (Chronolog Corporation, Havertown, PA) was added in the amount
of 30rd to the cuvette immediately prior to addition of 20m of agonist. Immediately
after the ATP secretion tracing on the chart recorder had peabd, a standard solution
of ATP, final concentration 4.0pM, was added to quantitate the ATP secretion. Two
measurements were again taken to evaluate dense granule ATP secretion. Dense
granule secretion was described both by the amount of ATP in M measured at the
peak of the secretion curve, and, the time in seconds taken to reach that peak.
Agonists (final concentration) utilized in secretion studies were ADP (10.0pM) and

PAF (0.1, 0.5, 1.0 and 10.0,IM).

The aggregation responses, designated as slope and maximum 96 aggregation,
. of affected SHT platelets were absent (ADP, A23187, collagen) or decreased (PAF,

38
thrombin) when compared to the responses recorded in platelets from normal cattle.

In addition, the threshold concentration of PAP and thrombin required for aggregation
was increased in affected platelets. Only when PMA was used as the agonist was the
aggregation response seen to be comparable between control and affected samples.
These results were highly reproducible in all 5 affected animals tested. The results of
aggregation trials run on the carrier cow utilized in this study were indistinguishable
from those of control cattle. Results of the aggregation studies are summarized in
Figure 1 and within chapters IV and V (Journal of Veterinary Diagnostic Investigation

and Thrombosis Research articles).

The studies which compared platelet dense granule release of ATP in response
to ADP and PAP revealed that both the amount of ATP released as well as time to
maximum secretion were similar in the affected SHT and control platelet samples.
Results for the secretion trials are summarized in Table l and in Chapter V

(Thrombosis Research article).

39

Figure 1. Comparison of the effects of various agonists on maximum percent
aggregation in normal (series 1) and affected (series 2) platelets. [Agonist
concentrations: ADP=10.0uM; A23187=40.0uM; collagen=2.0ug/ml;

PAF=10.0uM; thrombin= 1.0 U/ml; PMA=3.75;IM; vertical bars=Standard Error].

40

 

 

41

Table 1. Platelet dense granule ATP secretion. Comparison of time to maximum ATP
secretion and concentration of ATP between control and affected bovine platelets.

 

 

AGONIST ATP (pM) TIME (sec)
Control Affected Control Affected
(n=4) (n=3) (n=4) (n=3)
ADP mm 0.19 :1; 0.01 0.24 i 0.06 8.00 1; 1.27 12.67 :1: 2.62
PAF mm 3.82 :t 0.20 3.87 d; 0.57 19.50 :1; 0.20 16.50 i 1.50
PAF LOpM 3.54 :t 0.24 4.52 :1; 0.79 18.00 1; 0.00 18.00 i 0.00
PAF 0.5uM 3.44 :1; 0.21 3.11 :1; 0.64 22.00 :I: 2.00 18.00 1; 0.00
PAF 0.1uM 1.18 :1: 0.17 0.55 3; 0.05 22.50 :l: 1.50 18.00 :l: 0. 00

 

Values = mean :1; SE; n = number of animals tested; ATP (pM) = maximum ATP

released; TIME (sec) = time to maximum ATP release.

TRANSMISSION ELECTRON MICROSCOPY

Introduction:

Transmission electron microscopy (T.E.M.) of control and affected platelet
preparations were examined for evidence of any ultrastructural differences between
the two groups. The electron Inicrographic evaluation of resting and agonist-
stimulated platelets was also used to confirm the aggregation defect seen on the
corresponding aggregometer tracings. Agonists selected for these evaluations were

determined by the results of the aggregation studies. .

Materials and Methods:

Platelets were prepared for transmission electron microscopy as described by
Mattson et a].128 Whole blood was collected in 3.8% trisodium citrate and gel
filtered platelet samples prepared as previously described in the section on aggregation
and secretion studies. Gel filtered platelet aliquots (0.5 ml) were covered with
parafilm and allowed to rest at room temperature for 30 minutes. Five minutes prior
to placement within the aggregometer, the samples were warmed to 37°C and a teflon-
coated magnetic stir bar added. Samples identified as resting platelets were allowed
to equilibrate in the aggregometer at a stir spwd of 900 rpm for 30 seconds. At that
time, 0.5 ml of 0.1% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) was

42

43
added and the sample allowed to continue stirring for one minute. For post-

stimulation samples, 20):] of agonist was added to the platelet sample within the
aggregometer and the response stopped at 1 or 5 minutes by the addition of 0.5m] of
0.1% gluteraldehyde in 0.1 M cacodylate buffer. Agonists used for these studies
included ADP (10.0uM), PAF (1.0uM) and thrombin (0.6 and 0.05 U/ml). The
contents of each cuvette were transferred to an Eppendorf tube and placed in an
Eppendorf centrifuge for 20 seconds at 12,800 x g. The supernatant was discarded
and 1.0 ml of 3.0% glutaraldehyde in 0.1 M cacodylate buffer was added. The
platelet pellet was not resuspended in this solution. The sample was then allowed to
fix for 3-6 hours, the supernatant discarded, and 1.0 III] of 0.1 M cacodylate buffer

added. The samples were then refrigerated until processing for transmission electron

microscopy.

Results:

On T.E.M. , there no consistent differences noted in ultrastructural morphology
of resting (non—stimulated) platelets between affected and control samples. Occasional
prominent electron dense crystalline-like band structures were noted within alpha
granules, and, in some samples, these band forms appeared more pronounced within
affected platelets. These structures were judged to be consistent with the large
molecular weight protein, von Willebrand factor (personal communication, Drs. Ken

Meyers and Michele Menard).

44
Transmission electron microscopic evaluation of post-stimulation platelet

samples confirmed the results of the corresponding aggregation studies. At one
minute post-activation with ADP, PAF or thrombin, evidence of shape change was
noted in both control and affected platelets, with resultant loss of normal discoid
shape and formation of pseudopods. There was then no indication of platelet
aggregate formation with ADP-stimulation in the affected (SHT) samples; prominent
aggregate forms were seen in micrographs from the paired control samples. Both the
PAF- and 0.6 u/ ml thrombin-stimulated platelet samples exhibited prominent
aggregate formation and granule secretion at 5 minutes post-activation. However,
where 0.05 U/ III] thrombin was used as the agonist, no evidence of aggregation or
granule secretion was noted in either control or affected samples. These findings
were in agreement with the aggregation studies performed. Results of T.E.M. are

further illustrated in Figures 2, 3, and 4.

45

Figure 2. Paired transmission electron micrographs of control (a) and affected (b) resting

bovine platelets showing similar ultrastructural features.

 

Figure 2b.

47

Figure 3. Paired transmission electron micrographs of control (a) and affected (b) bovine

platelets 5 minutes following the addition of 1.0;IM PAF, an agonist which induces an

aggregation response in both groups.

48

 

49

Figure 4. Paired transmission electron micrographs of control (a) and affected (b) bovine
platelets 5 minutes following the addition of 0.6 U/ml thrombin, showing the response

to an agonist which induces aggregation in both groups.

50

 

Figure 4a.

 

Figure 4b.

CITRATE CONCENTRATION STUDY

Introduction:

Previous platelet aggregation research studies in Hereford cattle revealed that
extrapolation of trisodium citrate concentrations used for anticoagulant in human
platelet studies may yield misleading results in bovine platelet studies. Therefore, it
was decided that the effect of varying the citrate concentration on the process of

platelet aggregation should be investigated.

Materials and Methods:

A 60 ml blood sample was collected by jugular venipuncture in 3.8%
trisodium citrate at a ratio of 1 part citrate to 19 parts blood. PRP was isolated by
repeated centrifugations as previously described. A manual platelet count was
performed and the PRP diluted to 300,000 cells/pl with autologous PPP. The platelet
suspension was then separated into 0.5 ml aliquots and the samples allowed to rest at
room temperature for 30 minutes. Phosphate buffered saline (PBS) was used to make
dilutions in 3.8% citrate resulting in a range of citrate concentrations between 1.9%
and 3.8% . Five minutes prior to aggregation testing, a teflon coated magnetic stir bar
and trisodium citrate were added to the aggregation cuvette. The citrate (or PBS) was
added in a quantity that would result in a final concentration of 0.19 to 0.38% within

51

52
the sample. Agonists used for this study were ADP (1.0, 3.5, and 10.0uM) and PAF

(0.1, 1.0, l0.0p.M). In addition, in some trials using PAF as agonist, the platelets
were also treated with acetylsalicylic acid (final concentration lmM). The maximum

% aggregation and slope of the aggregation curve were calculated for each trial.

Results:

The inhibitory citrate concentration, which is the point at which chelation
reduced the external Ca“ * to below that required to support aggregation (s 40uM),
was shifted to the right in SHT platelets (from 0.38% to between 0.4% and 0.80%).
These preliminary results suggested that affected platelets may be less sensitive to the
external CaM concentration than control bovine platelets. As expected, changes in
the citrate concentration had no effect on slope or maximum percent aggregation in
affected platelet samples stimulated with ADP. These samples, as seen in the
aggregation trials described previously, showed no response to any concentration of
ADP. Results of the paired citrate concentration trial using 1.0uM PAF are
illustrated in Figure 5. Of note, the addition of lmM acetylsalicylic acid (ASA) to
samples of PAF-stimulated platelet preparations did not cause any noticeable alteration

in the resultant values of slope of the aggregation curve of maximum percent

aggregation.

53

80-1 0\

0
70- \o\o\ o
o o\ /o——o/
6 0- *—""* \O/ O \

A

G

G 50-

R \/\*

E

G /

A 40- * *-—*

'1" \/

I 0

O

N 30-

(’6)
20- a:
lo-

 

o = =

.18 .20 .22 .24 .26 .28 .30 .32 .34 .36 .38 .40 .80 1.2

 

CITRATE CONCENTRATION ( %)

Figure 5. The effect of citrate concentration on luM PAF-induced aggregation in normal

bovine platelets (o) and in Simmental Hereditary Thrombopathy (*).

EXTERNAL CALCIUM CONCENTRATION STUDY

Introduction:

To more directly assess the aggregation defect, platelets were transferred by
gel-filtration into buffer containing physiologic concentrations of Ca“ to examine the
contribution, if any, of plasma factor(s) to the platelet defect. In order to express the
change in aggregation curves seen with increasing concentration of ealcium in the
resuspension buffer, an aggregation index was ealculated. This aggregation index is
described as the slope of the line extending from the aggregation tracing at the
addition of agonist to the intercept of the lines representing the maximum percent
aggregation and the slope of the aggregation curve. This index was meant to better
standardize the results men between the control and affected samples and to factor out

the delayed onset of aggregation seen in the affected platelets.

Materials and Methods:

To perform the external calcium concentration study, 40 ml of blood was
drawn in 3.8 96 trisodium citrate by jugular venipuncture. Platelet rich plasma was
collected by multiple centrifugations as previously described. Prostaglandin E, (lmM
stock in ethanol) was added at a concentration of lpl PGE, to 1 ml of PRP and the
sample allowed to rests at room temperature for 5 minutes. The PRP was then
centrifuged at 800 x g (20°C) for 15 minutes, the supernatant discarded, and the

54

55
platelet pellet resuspended in 1 ml of Hepes-Tyrode-albumin (HTA) buffer containing

130mM NaCl, 2.6mM KC], 0.42mM NaH2P04, 5.5mM dextrose, 0.01mM HEPES
and 3.0 mg/ml bovine serum albumin, pH 7.2. The sample was gel-filtered, the
eluate collected and a platelet count performed. The sample was then diluted to
300,000 cells/pl and l M MgCl, was added to a final concentration of lmM. The
platelet sample was then aliquoted into aggregometer cuvettes (500 pl each) and the
samples allowed to rest for 30 minutes at room temperature. Five minutes prior to
aggregation, the platelet sample was warmed to 37°C in the aggregometer, a teflon
coated magnetic stir bar added, and CaCl2 at a final concentration of 0.0, 0.5, 1.0 or
1.5mM added. Aggregation trials were run in triplicate on control and affected
animals with PAF (0.1, 0.5 or 1.0 uM) or thrombin (0.01, 0.1, 0.6, 1.0 U/ml) used
as agonist. The maximum % aggregation, slope of the aggregation curve and

aggregation index were calculated for each tracing.

Results:

The SHT platelets had lower aggregation index values which were affected
relatively little by the concentration of external calcium present. The control platelets
had a much higher index which increased as the external calcium concentration was
increased from no added calcium to 1.5mM Ca”. Findings were consistent with all
concentrations of PAP and thrombin used. Representative paired data from these
trials are shown for 0.1 U/ ml thrombin in Figure 6. The control platelet samples

showed a consistent increase in the aggregation index value as the external calcium

56
concentration was increased. In contrast, samples from affected animals exhibited a

maximum aggregation index value at 0.5mM Ca“; further increases in Ca++
concentration had no additional positive effect on this value. The results from these
investigations suggest that aggregation of affected SHT platelets, in response to
varying concentrations of PAF and thrombin, was relatively insensitive to changes in

the external Ca” concentration when compared to control bovine platelet samples.

[Ca++] Study

We

@-

 

   

 

W/fl////////////u

 

 

 

qqqqqqqqqqqqqq

5432J183355A321.
00000000000000

0.0
cudum Garcons-aim

m

Figure 6. Aggregation index for control bovine (striped bars) and affected SHT (solid

bars) platelets stimulated by 0.1 U/ml thrombin in the presence of varying

concentrations of external Ca+ +.

VON WILLEBRAND FACTOR ASSAY:

Introduction:

Examination of control and affected platelet ultrastructure by transmission
electron microscopy revealed occasional electron-dense, bar-like crystalline arrays in
alpha granules of affected platelets. These structures were thought to possibly
represent von Willebrand factor. This finding, in combination with the obvious need
to evaluate plasma von Willebrand factor levels in any unusual bleeding diathesis,
initiated the study of samples from all affected and carrier SHT animals, as well as

representative clinieally normal Simmental and Holstein cows.

Materials and Methods:

Blood was drawn and prepared using jugular venipuncture and following
procedures as outlined by the New York State Department of Health, Comparative
Hematology Laboratory. Briefly, venous blood was drawn into a plastic syringe
containing 3.8% trisodium citrate using a dilution of one part citrate to 9 parts blood.
The sample was transfered to a plastic test tube and centrifuged at 1384 x g for 15
minutes. The supernatant plasma was aspirated, and 1 ml was placed in a plastic

stoppered tube and frozen at -20°C for storage prior to shipment.

58

59
Results:

Samples were submitted to the Comparative Hematology Laboratory from

seven animals; two affected Simmental cows, one affected Simmental bull, one

crossbred carrier cow, one control Holstein cow, and two control Simmental cows.

The results were based on an ELISA assay for von Willebrand factor antigen

(vWF:Ag) utilizing pooled Holstein cow plasma as the 100% standard. The results of

the samples submitted from our laboratory are as follows:

l'lD ..

affected cow

affected cow

affected bull

carrier cow

normal Holstein cow
normal Simmental cow

normal Simmental cow

W

88
69
74
92
133
145

96

The mean value 1; Standard Error of the mean (S.E.M) for the affected and

control groups were: affected = 77.0% d; 5.7; control = 124.7% :I: 14.7.

Interpretive comments from the Comparative Hematology Laboratory stated that they

do not have an established normal range for bovine samples; however, the comments

also stated that the canine ranges are probably appropriate. For canine samples, the

60
normal range = 70-180% vWF:Ag. Therefore, the Laboratory reported that the

results for the bovine samples submitted from the SHT study do not indieate a
deficiency of plasma vWF, in any of the animals tested, that would be likely to eause
a clinically demonstrable defect in primary hemostasis (written clinical report from

Marjory B. Brooks, DVM, Dip.ACVIM).

Although a difference existed between the man % value of vWF in the
affected group and the control group, it is difficult to make a judgement as to the
exact significance of this difference based on the number of animals tested (trials
run). All animals tested had vWF:ag values which were judged to be within the
normal range, except one affected cow which had a vWF:ag value one percent below

the low normal range.

Possible explanations for this difference between the affected and control
animals may be that the affected animals, in general, are less reactive to stimulation
after vessel injury and, therefore, the process of venipuncture to obtain the blood
sample may have resulted in less platelet granule secretion of von Willebrand factor
into plasma than in normal animals. Alternatively, the affected eattle may utilize
more von Willebrand factor in the process of thrombosis in response to vessel injury
during collection, as a compensatory mechanism for their defective platelet-to-platelet
aggregatory response. Further comparative quantitative analysis of the von
Willebrand factor content of affected and control platelets (alpha granules) and plasma

is warranted when larger numbers of affected animals are available.

CLOT RETRACTION STUDY

Introduction:

The phenomenon of clot retraction has been described as being dependent on
three factors: 1) platelet number in the sample, 2) amount of fibrinogen in the
sample, and 3) concentration of ionized ealcium within the sample. From the initial
clinical evaluation of animals with Simmental Hereditary Thrombopathy it was
already known that there are adequate platelet numbers and fibrinogen levels. This
study; therefore, could afford an opportunity to evaluate the third factor, ealcium
concentration, as it relates to the dynamics of clot retraction. Although clot retraction
it typically considered to be a qualitative means of evaluation, careful comparison of
test results between control and affected animals may further the understanding of the

part that calcium plays in the platelet function defect in SHT.

Materials and Methods:

Clot retraction was evaluated using a modifieation of the procedure described
by Miale.'” Whole blood samples were obtained from control and affected animals
by clean jugular venipuncture with 18 gauge needles and glass tubes containing no
anticoagulant. The samples were allowed to clot. These samples were then placed in
a water bath at 37°C and were examined for the presence of clot retraction and

61

62
separation of the clot from serum at l, 2, and 24 hours. At 24 hours, the clot was

removed using a wooden applicator stick. The remaining serum and cell sediment
was centrifuged at 1384 x g for 90 seconds and the serum removed. The weights of
the empty tube prior to blood collection, the tube containing the blood sample, the
removed clot, the tube with sediment and the serum were recorded and relative
percentages calculated. In addition, the clot retraction index was determined. This
value was incorporated in the quantitative evaluation of clot retraction in an attempt to
standardize the results in relation to animal-to—animal and day-to-day variations in the
packed cell volume of the whole blood samples. The clot retraction index was
determined by dividing the value for % serum of a sample (following the 24 hour clot
retraction study) by [lOO-PCV]. Samples were run in duplieate on each animal tested

and results were averaged to obtain means and standard errors for each group.

Results:

All samples from both the control and affected cattle were men to form a
uniform clot within the glass tube within minutes of collection. Evaluation for
evidence of clot retraction in the samples from control animals revealed progressive
and prominent separation of the clot from the sides and base of the glass tubes. Clot
retraction was most noticeable at 2 and 24 hours. The resultant serum was observed
to be clear and free of cellular sediment. Samples from affected cattle, however,
displayed disintegration or dissolution of the clot, commencing at the 1 hour
examination interval and progressing over the 24 hour test period. The remaining

clot was small and friable in consistency and was surrounded by dark red serum.

63
Samples from affected animals had markedly higher percent sediment and clot

retraction index scores than those from control and carrier eattle. Results are further

described in Chapter V (Thrombosis Research article) and in Figure 7.

Figure 7. Comparison of the %clot and clot retraction index between control and

affected bovine samples [time = 24 hours]. Clot retraction index = % serum/(100-

PCV).

.hnflsmwu

Dmhommm<

5.6.x. mmEm<o

4015.200
7 If
.82. mo

1 MHHM\n//IHHHWHHUMIo

\TOP

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

WOO?

ZOHBOENK ROAD

PROCEDURE FOR ICP EVALUATION OF PLATELEI‘S
Introduction:

Inductively coupled argon plasma atomic emission spectroscopy (ICP-ABS) is
an instrumental technique that allows quantitative analysis of as many as 40 elements
in solution simultaneously. The technique involves nebulization of the liquified
sample into an argon plasma that is sustained by a high frequency oscillating magnetic
field. ‘3" Elements are atomized and elevated to excited states in the high temperatures
(8,000 to 10,000°K) of the plasma, and emit characteristic photons as they deeay
back to lower energy states. Wavelengths are dispersed by a diffraction grating and
simultaneously measured in a direct reading polychromator or sequentially in a
seanning monochromator. Because of the nature of the argon plasma, chemieal
interferences are almost nonexistent, self-absorption at high analyte concentrations is
greatly reduced and most spectral interferences are removed by high resolution optics.
These factors greatly simplify sample preparation procedures and permit analyses to
be carried out over much broader concentration ranges. The Thermo-Jarrell Ash
Polyscan 61F. instrument available for this project is equipped with a 24 element
polychromator as well as a scanning monochromator to allow measurement of
additional elements or selection of alternate wavelengths to those fixed on the

polychromator.

67

ICP evaluation is an important independent method of assessment, not only of
calcium, but also of multiple other cellular elements at detection levels of parts per
million (ppm). It was therefore considered that the ICP technique may provide an
additional method of evaluating relative ealcium concentrations within the platelet as
well as within the plasma. All ICP samples were run in the Toxicology laboratory
section of the Animal Health Diagnostic Laboratory, Michigan State University, under

the supervision of Dr. Emmet Braselton.

Materials and Methods:

Blood was collected in 3.2 % trisodium citrate at a concentration of 1 m1 citrate
to 9 ml blood. The sample was separated into 12 ml polystyrene centrifuge tubes and
PRP was collected as previously described in the aggregation studies. Prostaglandin
E, (PGEl) was added from lmM stock in ethanol to a final concentration of IM to
minimize platelet activation. The sample was then separated into 1 ml aliquots in
Eppendorf micro centrifuge tubes. Resting platelet samples were centrifuged in an
Eppendorf centrifuge for 20 seconds at 12,800 x g. The supernatant was poured ofi'
and pooled for ICP analysis. The pooled supernatant and the resultant platelet pellets
were frozen at -20°C until submitted for analysis. It was subsequently decided that
the ICP evaluation would be performed on quantities of 2 million platelets (four

Eppendorf tubes per animal).

68

Results:

ICP evaluation was performed on both the pooled supernatant sample and the
appropriate platelet pellet from each animal. Results of ICP evaluation of resting
control and affected bovine platelets is summarized in Figure 8. Levels of elements
including ealcium (Ca), copper (Cu), iron (Fe), magnesium (Mg), phosphorus (P),
zinc (Zn), sodium (Na), potassium (K), and sulfur (S) are presented in ppm. No
appreciable differences were noted in these elements between the control and affected
platelet or plasma samples. If further ICP evaluations were to be conducted, it would
be important to incorporate studies on agonist-stimulated platelet and plasma samples

to investigate the relative increase, if any, in ealcium following activation.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 8. Multi-element analysis of resting bovine platelets by Inductively Coupled

Argon Plasma Atomic Emission Spectroscopy (ICP-ABS). [Normal =S 1; Affected =82]

l-D SDS POLYACRYLAMIDE GEL ELECTROI’HORESIS

Introduction:

By utilizing electrophoresis techniques, potential abnormalities related to
structural or Ca++ transport proteins may be detected. In l-D reduced SDS-
polyacrylamide gel electrophoresis (SDS-PAGE) native proteins are separated in the
first dimension by molecular weight. Results of these studies on affected (SHT)
platelet samples may then be compared to similar samples from normal (control)

bovine platelets.

Materials and Methods:

Whole blood samples were collected by jugular venipuncture into 3.8%
trisodium citrate (9 parts blood to 1 part citrate) and PRP was collected as previously
described. PGE, from lmM stock in ethanol was then added to the PRP sample to a
final concentration of 1.0p.M and the sample allowed to rest at room temperature for
5 minutes. The sample was then centrifuged at 800 x g for 15 minutes (20°C) to
sediment the platelets and the platelet pellet washed by resuspension in the original
plasma volume of a buffer containing l20mM NaCl, l3mM Na, citrate, and 30mM

dextrose, pH 7.0 (Buffer #1). This step was repeated twice with a manual platelet

70

71
count performed between each wash. The resultant platelet sample was then

resuspended to a final count of 3.0 x 10‘ platelets/ml in Buffer #3 containing 138mM
NaCl, 2.9mM KCl, 12mM NaHCO,, 0.36mM NaHzPO4, 5.5mM glucose, and
1.0mM EDTA, pH 7.4. Protein content was then assayed using the Markwell protein
assay procedure which is a modification of the Lowry method that is not affected by
SDS or Triton X-100 in samples. An aliquot of the platelet suspension sample was
solubilized by the addition of an equal volume of 4% SDS. Duplicate samples of 50
and 100 pl and standards of 0 to 200 pg of BSA were used. Briefly, 100 ml of
Markwell A solution (2% NaZCO3, 0.4% NaOI-l, 0.16% Na tartrate, and 1% SDS)
was combined with 1 ml of Markwell B solution (4% CuSO, -5H20). Three ml of
the Markwell A & B combination was added to each sample tube. The sarnple was
vortexed and incubated at room temperature for 30 minutes. Folin reagent was mixed
1:1 with distilled water and 0.3 ml of this reagent was added to each tube. The
sample was then vortexed and incubated at room temperature for 45 minutes. The
absorbance of each tube was read at 660nm. Standards were used to construct a
standard curve through linear regression and these values subsequently used to

determine the protein content of the unknown samples.

The platelet suspension was solubilized by the addition of an equal volume of a
2 x concentrated buffer containing 4% SDS (w/v), 10% 2-mercaptoethanol (v/v), 20%
glycerol (w/v), 0.004% bromphenol blue (w/v), and l25mM Tris HCl, pH 6.8. The
samples were incubated at 100°C for 3 minutes in a boiling water bath or at 37°C for

one hour. Samples were the used immediately or were aliquoted and frozen at -70°C.

72
Solubilized platelet samples were then loaded in the amount of 50 to 80 pg of protein

onto slab acrylamide gels (10% acrylamide resolving and 3% acrylamide stacking gel)
and electrophoresed according to the method of Laemmli.131 Gels were stained with
Coomassie brilliant blue and then de-stained. Following the de—stain procedure, gels

were dried between two layers of porous cellophane.

Results:

The l-D reduced SDS-polyacrylamide gel electrophoresis studies were
conducted utilizing paired control and affected platelet protein samples. In addition,
samples of normal canine platelet proteins were used as a mammalian species

comparison. No differences in protein bands were noted between affected and control

bovine platelet samples upon inspection of the resultant gels (Figure 9).

73

Figure 9. 1-D SDS polyacrylamide gel electrophoresis. Resting platelets. (A=affected
bovine platelets; B =affected bovine platelets; C =control bovine platelets; D =low

molecular weight standard; B=canine platelets)

74

-. $5..
_ 2...... .

     

 

Figure 9.

CHAPTER IV: JOURNAL ARTICLE:

Steficek BA, Thomas IS, Baker JC, Bell TG. Hemorrhagic diathesis
associated with a hereditary platelet disorder in Simmental cattle. J Vet Diag

Invest 5:202-207, 1993.

(Reprinted from a wpyrighted article in Iaumalntlctefirmmagnasds
Investigation, with kind permission of Harvey S. Gosser, DVM, PhD,

Secretary/Treasurer, American Association of Veterinary Laboratory Diagnosticians.)

75

HEMORRHAGIC DIATHESIS ASSOCIATED WITH A HEREDITARY

PLATELET DISORDER IN SIMNIENTAL CATTLE

Barbara A. Steficek, Jennifer S. Thomas,

John C. Baker, Thomas G. Bell

Running Title: Platelet disorder in Simmental cattle.

From the Department of Pathology and the Animal Health Diagnostic Laboratory
(Steficek, Thomas, Bell), and the Department of Large Animal Clinieal Sciences (Baker),
College of Veterinary Medicine, Michigan State University, East Lansing, Michigan
48824. Current Address (Thomas): Department of Veterinary Pathobiology, College

of Veterinary Medicine, Texas A & M University, College Station, Texas 77843

Received for publieation July 13, 1992.

Galley proofs to be sent to: Dr. Barbara A. Steficek/Dr. Thomas G. Bell
Comparative Pathology Research Laboratory; Animal Health Diagnostic Laboratory,
P.O. Box 30076, Lansing, MI 48909

76

A severe blwding disorder in Simmental cattle has been described in wide-spread
locations in the United States and Canada. The clinical findings are consistent with a
’hemophilia’-1ike disease, or more precisely, a hereditary hemorrhagic diathesis, and
include spontaneous epistaxis, hematuria and excessive bleeding associated with
trauma or standard management procedures such as tattooing, ear tagging and
eastration. In our preliminary investigation of this defect, the blood-platelet numbers
and coagulation profiles of affected cattle were normal. Affected animals have a
marked dysfunction of platelets (thrombopathy) which we have termed Simmental
Hereditary Thrombopathy. The defect is very similar or identical to that described in

the same breed by two other laboratories.

78
A bleeding disorder of Simmental cattle was first described in 1977 and

1980.19 This apparently autosomal recessive defect was associated with a mild to
severe bleeding tendency that was exacerbated by trauma. Clinical findings included
spontaneous epistaxis, hematuria, and excessive blood loss associated with injury, all
of which are consistent with defective platelet function. Platelets from affected
animals exhibited impaired aggregation when stimulated with adenosine diphosphate
(ADP) or collagen. Platelet dense body granule serotonin and nucleotide

concentrations were normal.

A 1990 report" from Canada described a similar, potentially fatal bleeding
disorder in eight Simmental eattle. Platelet numbers and ultrastructural morphology,
and coagulation screening tests were normal in these animals. In whole blood
aggregau'on studies, platelets from affected animals did not aggregate following

stimulation with ADP.

A purebred Simmental cow was presented to the Veterinary Teaching Hospital,
Michigan State University in November of 1989 with a history of recurrent epistaxis
and hematoma formation associated with minor trauma. Following a difficult but
successful ealving, severe anemia, massive internal hematoma formation and shock
occurred. Circulating platelet numbers and size distribution were normal. A blood

coagulation profile, including prothrombin time, activated partial thromboplastin time,

79
fibrin degradation products and fibrinogen, was also within normal limits. Due to the

severity of the clinical disease, the owner elected to donate the dam to Michigan State
University. A second similarly affected purebred Simmental heifer and a male
Simmental calf were identified in 1990 and 1992 respectively, and were purchased by

the university to enhance the studies of this defect in platelet function.

In order to further characterize the defect present in Simmental Hereditary
Thrombopathy, we measured mm aggregation of affected platelets in response to a
variety of agonists which activate platelets by different mechanisms. Samples of
resting and post-stimulation platelets were collected for transmission electron
microscopy studies to evaluate platelet morphology and to confirm the results of the
aggregation studies. Finally, gel-filtered platelets were maintained in a range of
concentrations of external calcium and were stimulated by thrombin to evaluate the
effect of external calcium on the aggregation response. This paper reports specific
clinical diagnostic characteristics of the platelet function defect in purebred Simmental

cattle.

80

CASE HISTORIES

ANIMAL #1: A two year old purebred Simmental heifer was presented to the
Michigan State University, Large Animal Veterinary Teaching Hospital in November,
1989 with a history of post-parturient anemia and hematoma formation. Previous
records indicated that the animal had a history of ealfhood pneumonia and repeated
episodes of epistaxis. The heifer had calved one day previously and the single female
ealf appeared clinieally normal. On physical examination, the dam was depressed,
was in sternal recumbency, had markedly elevated pulse and respiratory rates, and
exhibited epistaxis. There were multiple large swellings over the posterior'hip and
thigh region, principally involving the right semitendinosus area, as well as the vulva.
In addition, multiple prominent masses were palpated within the soft tissue structures
of the pelvic canal. Cytologic examination results of needle aspirates from the
external masses were consistent with hematomas. Attempts to parenterally treat the
heifer or to obtain blood samples resulted in localized hematoma formation and/or
prolonged bleeding from injection sites. Initial clinical pathologic data revealed the
animal to be anemic (PCV= 14%) with leukocytosis (white blood count=14,500/ul).
The WBC differential breakdown was consistent with physiologic leukocytosis, a

response commonly seen in cattle during parturition. The platelet count was normal

81
(360,000/ul). Results of a coagulation profile, including prothrombin time, activated

partial thromboplastin time (APTI'), fibrinogen (quantitative), and fibrin degradation

products (FDP) were normal.

ANIMAL #2: In June of 1990 a yearling purebred Simmental heifer was
presented to the Michigan State University, Large Animal Teaching Hospital with a
history of sudden formation of a mass over the right dorsolateral pelvic area. The
heifer’s previous health records indicated a tendency toward prolonged bleeding
following minor cuts or abrasions and recurrent epistaxis. On physical examination,
an approximately 10 x 15 cm mass was noted over the right hip area. The skin
surface overlying the mass appeared normal; however, the animal exhibited a mild
pain response when the area was palpated. The heifer was otherwise judged to be in
good condition. A complete blood count was normal. The platelet count and

coagulation profile results were also within normal ranges for our laboratory.

ANIMAL #3: Our laboratory was contacted in regard to a 2 week old male
Simmental ealf which the owner reported appeared to bleed excessively following a
routine ear tagging procedure. The hemorrhage was reported to decrease slightly
when the animal was confined; however, it beeame noticeable again when the ealf
was allowed greater freedom of movement. Additional history included a tendency

for the calf to experience prolonged bleeding from small lacerations over the head and

82
back, and the appearance of scleral bruising. The ealf was moderately anemic

(PCV == 17%); platelet count and coagulation profile results were within normal
ranges. Evaluation of a platelet rich plasma sample from the dam revealed normal

aggregation studies.

Erperimental Subjects

The experimental group consisted of two affected Simmental cows, one
affected male Simmental ealf and three control animals, two of which were Holstein
cows and one, a Simmental steer. All animals were housed at Michigan State

University and were free from clinical signs of disease other than those related to the

thrombopathy.

Collection of Platelets

Blood was collected by jugular venipuncture via an 18 gauge needle into a
plastic syringe containing trisodium citrate at a final concentration of 0.32% .
Platelet-rich plasma (PRP) was obtained by multiple centrifugation of the blood at
1324 x g for 60 seconds, removing a small quantity of PRP between each spin.

Platelet-poor plasma (PPP) was obtained by centrifugation of the remaining blood at

83
1324 x g for 13 minutes. Platelets were also gel-filtered to remove plasma

constituents for measuring the aggregation response to thrombin. Gel-filtered platelets
(GFP) were obtained by adding 1 pM prostaglandin E1 to the PRP prior to
centrifugation at 800 x g for 15 minutes. The platelet pellet was resuspended in 1 ml
HEPES-Tyrode-albumin (HTA) buffer (130 mM NaCl, 2.6 mM KCl, 0.42 mM
NaI-I2P04, 5.5 mM dextrose, 0.01 mM HEPES and 3.0 mg/ml bovine serum albumin,

pH 7.2), passed over a 10 ml Sepharose 4B‘ column and the eluate collected.

Aggregation Studies

Aggregation studies were run in triplicate on each animal tested. The
aggregation response to ADP‘, PAF“ or calcium ionophore A23187“ was measured
using PRP adjusted to a final concentration of 300,000 platelets/pl with autologous
PPP. The platelets were manually counted using the Unopette system and a
hemocytometer. The aggregation response to thrombin‘ was measured using GFP
adjusted to a final concentration of 300,000 platelets] pl with HTA buffer to which
MgCl, (1 mM) and CaClz (1.5, 1.0. 0.5 or 0 mM) were added. The PRP/GFP
suspensions were divided into 0.5 ml aliquots and rested at room temperature for 30

minutes.

84
The PRP/GFP were warmed to 37°C and stirred at 900 rpm. In an

aggregometer“, agonist (20 pl) was added and aggregation profiles were evaluated by
measuring the change in optieal light transmission through the sample when compared
to a standard (PPP for PRP samples and buffer for GFP samples). Light
transmittance through the standard was considered to be 100% while light
transmittance through the resting platelet sample was considered to be 0% . Both
maximum percent aggregation (indicative of the extent of aggregation) and maximum
slope of the aggregation curve (indicative of the rate of aggregation) were measured.
If the sample did not begin to aggregate within five minutes of agonist addition, the

percent aggregation and slope were recorded as 0% .

Preparation of Platelets for Transmission Electron Microscopy (TEM)

The platelets were prepared as described previously. ‘° Briefly, PRP/GFP was
prepared as for the aggregation studies. Following agonist addition, the response was
stopped at l or 5 minutes by the addition of 0.5 ml 0.1% glutaraldehyde in 0.1 M
sodium cacodylate buffer, pH 7 .4. The sample was stirred for 1 minute prior to
transfer into an Eppendorf“ micro centrifuge tube for centrifugation for 20 seconds at
12,800 x g. The supernatant was discarded and 1 ml of 3% glutaraldehyde in 0.1 M

cacodylate buffer added. The sample was allowed to fix for 3-6 hours at which time

85
the supernatant was disearded and 1 ml 0.1 M cacodylate buffer was added to the

sample. Samples were then processed for TEM.

Affected platelets had no aggregation response to ADP and A23187; in the
case of PAP and thrombin, the aggregation response was decreased or took longer to
complete when compared to control bovine platelets under the same conditions (Fig.
1). Platelets from the affected animals changed shape but did not aggregate in
response to any concentration of ADP (1-100 pM) or calcium ionophore A23187 (10-
40 pM) tested. In contrast, the control animal platelets changed shape as well as
aggregated in response to ADP (3.5-10 pM) and calcium ionophore A23187 (20-40
pM) (Fig. 2). Transmission electron microscopy (TEM) confirmed the aggregation
studies. On TEM, there were no differences in morphology of resting (non—

stimulated) platelets from affected or control cattle (Fig. 3).

In response to PAF, affected platelets changed shape and aggregated, though
the extent of aggregation (maximum percent aggregation) was decreased relative to
control platelets. Affected platelets also appeared to be less sensitive to PAF since

control platelets aggregated at concentrations of 0.1 pM or less while affected

86
platelets did not aggregate in response to 0.1 pM PAF or had a weak, primary

aggregation only.

Affected and control platelets responded similarly to higher concentrations of
thrombin (1 or 0.6 U/ml). In response to 0.1 U/ml thrombin, the affected platelets
had a decreased slope of the aggregation curve (data not shown) although the
maximum percent aggregation was similar to the controls. At a concentration of 0.05
U/ml thrombin, control platelets exhibited irreversible aggregation while affected

platelets failed to aggregate (Fig. l).

Platelet rich plasma samples from obligate carriers of this hereditary
thrombopathy (the calf of animal #1 and the dam of animal #2) were evaluated for
response to various agonists. Results were indistinguishable from the control data in

this study.

Simmental Hereditary Thrombopathy (SHT) is a functional primary platelet
disorder in purebred Simmental eattle characterized by epistaxis, hematuria and

excessive hemorrhage following trauma or surgery. 131mg, platelet function is most

87
commonly assessed by measuring the platelet responses of shape change, aggregation

and the release reaction. Shape change, which is the transformation of platelets from
a smooth disc to a sphere with numerous pseudopodal projections, correlates with the
Ca” *-dependent phosphorylation of myosin light chain kinase.13 Aggregation occurs
following exposure of the platelet fibrinogen binding site on membrane glycoprotein
IIb«IIIa, allowing fibrinogen to form a bridge between adjacent platelets?"l3 In the
present study, we characterized the platelet defect in SHT by measuring platelet shape

change and aggregation responses to a variety of agonists.

We report that isolated platelets do not respond to ADP or calcium ionophore
A23187; similar findings in whole blood from affected animals have previously been
described in response to ADP. ‘2 Our studies showed that SHT platelets changed
shape but did not aggregate while control bovine platelets irreversibly aggregated.
The occurrence of shape change indicates that SHT platelets have a functional
receptor for the agonist being tested irrespective of their ability to aggregate. Since
ADP and A23187 cause platelet aggregation by different mechanisms, the defect in
SHT must be located in an activation pathway shared by both agonists. ADP binds to
a specific membrane receptor(s) which transmits the stimulatory signal into the cell.13
ADP activation of human platelets is associated with exposure of the fibrinogen
binding site on the membrane glycoprotein lIb—IIIa complex’, increased cytosolic free

ionized calcium concentrations“ and activation of phospholipase A2 (PLAz). PLA,

88
initiates the release of arachidonic acid metabolites (e.g. thromboxane A1) which

subsequently activate phospholipase C (PLC); ‘5 Phospholipase C plays a central role
in platelet activation by producing a variety of intracellular messengers which help
regulate the platelet response." Although bovine platelets produce thromboxane A2
following ADP stimulation, the use of cyclooxygenase inhibitors does not interfere
with normal bovine platelet aggregation. This suggests that the ADP-induced
aggregation of bovine platelets, unlike human platelets, is independent of arachidonic
acid metabolism.” Calcium ionophore A23187 is a non-physiologic agonist which
does not bind to a specific receptor; instead, A23187 increases membrane
permeability to calcium.“ The subsequently increased concentration of cytosolic free
ionized calcium then activates a number of calcium dependent intracellular enzymes
including myosin light chain kinase, which phosphorylates myosin light chain and is
associated with shape change, and PLAZ, which releases arachidonic acid from

membrane phospholipids.13

Affected SHT platelets changed shape and aggregated in response to PAF or
thrombin though the response was decreased relative to that of the control bovine
platelets. Both PAF and thrombin bind to specific surface receptors13 which then
activate PLC.“ Studies on normal bovine platelets indicate that PLC plays an
important role in mediating aggregation.2 Our findings show that SHT platelets are

able to aggregate when PLC is stimulated directly, though the decreased

89
responsiveness to PAF indicates that there may be an additional pathway(s) which acts

synergistically to amplify the platelet response and which is absent or defective in

affected platelets.

Transmission electron micrographs showed no ultrastructural differences
between control bovine and affected SHT platelets in the resting state, demonstrating
that both groups are morphologically similar. Other than confirming the presence or
absence of aggregation, the transmission electron micrographs did not reveal any

differences in post-stimulation platelet morphology between the two groups.

Preliminary data implicates a defect in calcium mobilization in affected
platelets, possibly secondary to altered calcium transport across the plasma
membrane.“ Cytosolic free ionized calcium plays a pivotal role in platelet activation
by regulating the activity of a number of Ca+ *-dependent enzymes as well as the
organization of the cytoskeleton.13 Normal bovine platelets mobilize ealcium
following stimulation.‘ Affected SHT platelets appear to mobilize calcium, as
indicated by the occurrence of shape change in response to all agonists tested. Shape
change correlates with the release of internal stores of calcium but not with the

external ealcium concentration. ‘3

90
The thrombin-stimulated aggregation in control platelets was enhanced by

increasing the concentration of external calcium, while the aggregation potential of
affected platelets was relatively unresponsive to alterations in the external ealcium.“
This suggests that there may be a defect in the mechanism controlling the influx of
external calcium in affected Simmental cattle and indicates that closer examination of

the process of calcium mobilization is warranted.

Simmental Hereditary Thrombopathy (SHT) platelets exhibit a unique pattern
of dysfunction. This study enables a differentiation between the SHT platelet defect
and the other primary platelet disorders. In cattle, the Chediak-Higashi Syndrome
(CHS) of Hereford cattle has been extensively investigated.‘ This autosomal recessive
disease is characterized by a primary thrombopathy, hypopigmentation, neutrophil
defects, and altered immune functions. SHT is distinguished by the fact that the
platelet response to 5 uM ADP is absent while in CHS it is normal. The CHS
platelet has a markedly decreased response to agents that require dense body granule

release such as collagen.

Diagnostically, Simmental Hereditary Thrombopathy (SHT) platelets present a
unique pattern of dysfunction. Further investigation of the biochemical pathways and
modes of activation of normal and affected bovine platelets will contribute to the

detection of the basis of this defect.

91

W
This work was supported by Biomedical Research Support Grant, #71-0751,

F32HLO7560, and F32HL08564 from the National Institutes of Health.

92

. Sigma Chemical Co., St. Louis, MO.
. Calbiochem Biochemicals, San Diego, CA.
. Lumi-Aggregometer; Chronolog Corporation, Havertown, PA.

. Baxter Healthcare Corp., McGaw Park, IL.

93

w
Bell TG, Meyers KM, Prieur DJ, Fauci AS, Wolfe SM, Padgett GA: 1976,
Decreased nucleotide and serotonin storage associated with defective function
in Chediak-Higashi syndrome cattle and human platelets. Blood 48(2): 175-

184.

Bondy GS, Gentry PA: 1989, Characterization of the normal bovine platelet

aggregation response. Comp Biochem Physiol 92C:67-72.

Colman RW: 1990, Aggregin: a platelet ADP receptor that mediates

activation. FASEB J 4:1425-1435.

Crouch MF, Lapetina EG: 1988, A role for G, in control of the
thrombin receptor-phospholipase C coupling in human platelets. J Biol

Chem 263:3363-3371.

Gentry PA, Trombay RRM, Ross ML: 1989, Failure of aspirin to

impair bovine platelet function. Am J Vet Res 50:919-922.

10.

11.

94
Kitagawa S, Ito Y, Oda Y, Kametani F: 1989, Inhibitory effects of

phenol derivatives on bovine platelet aggregation and their effects on

Ca2+ mobilization. Biochim Biophys Acta 1011:52-57.

Kociba GJ: 1980, Partial characterization of a hemostatic defect in Simmental

eattle. Vet Clin Path 9:446—47.

Lapetina EG: 1982, Platelet-activating factor stimulates the phospha-

tidylinositol cycle. J Biol Chem 257:7314-7317.

Leipold H, Moore W: 1977, Bovine thrombopathia. In: Dodds W.J., First
Registry of Animal Models of Thrombosis and Hemorrhagic Diseases,

ILAR News 21:Al4.

Mattson J C, Borgerding PJ, Craft DL: 1977, Fixation of platelets for
scanning and transmission electron microscopy. Stain Technology

52:151—158.

Sage SO, Rink TJ: 1987, The kinetics of changes in intracellular
ealcium concentration in fura-2-loaded human platelets. J Biol Chem

262:16364-16369.

12.

13.

14.

15.

16.

95
Searcy GP, Petrie L: 1990, Clinical and laboratory findings of a

bleeding disorder in eight Simmental cattle. Can Vet J 31:101-103.

Seiss W: 1989, Molecular mechanisms of platelet activation. Physiol

Rev 69:58-178.

Steficek BA, Thomas 18, McConnell MF, Bell TG: 1992, A primary
platelet disorder of consanguineous simmental cattle. Thromb Res

(accepted for publication).

Sweatt JD, Connolly TM, Baron BM, Limbird LE: 1988, Involvement of
Na*IH+ exchange in human platelet activation. In: Platelet Membrane
Receptors: Molecular Biology, Immunology, Biochemistry and Pathology.

New York, Alan R. Liss, Inc., pp. 523-557.

White JG, Rao GHR, Gerrard JM: 1974, Effects of the ionophore

A23187 on blood platelets. Influence on activation and secretion. Am

J Pathol 77:135-150.

96

Figure 1. Aggregation studies. Response of control (C) and affected (A) platelet
samples to 10 uM ADP (Fig. 1a), and 0.6 U or 0.05 U thrombin (Fig. lb). Arrow

indieates the point of addition of agonist.

Figure 2. The effect of various agonists on aggregation in normal (striped columns)

and SHT (open columns) platelets.

Figure 3. Paired transmission electron micrographs of control and affected platelets;
resting (A=control, B=affected; Bar=0.5 um) and 5 minutes after the addition of 10

uM ADP (C=control, D=affected; Bar=2 um).

97

Figure l. Aggregation studies. Response of control (C) and affected (A) platelet
samples to lOpM ADP (Fig. la), and 0.6 U or 0.05 U thrombin (Fig lb). Arrow

indicates the point of addition of agonist.

Figure 2. The effect of various agonists on aggregation in normal (striped columns)

and SHT (open columns) platelets.

98

   
  
 

' C" 3 Th:::;;.-.

C.“ c 23:03:“:

A 10 an AD!

0
‘ .05 U Thro-ht:
L... A a ._A._k
v " W7 T .-

Figure 1a. Figure 1b.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T] W o
l ’1
.7”
60' [new
A " 1
G
G 50' -——-1
R
E T
G H T
A 40'- R H
T O R
I H O D A
O P B H 2
N 30' A I B 3
F P N I 1
(8) A N 8
I" 7
20' O
A
2
. 3
10- A 1
D B
P 7
o c: m
1 UH 0.6 U/HL 10 “H 20 UN

COICH'I'RNI‘I“ OP MIS‘I‘S

’no mamas-roe useoass to non r-roo an we
’uo seams-rm: nape-as to non 540 an aura?

Figure 2.

99

Figure 3. Paired transmission electron micrographs of control and affected platelets;
resting (A=control, B=affected) and 5 minutes after the addition of lOpM ADP

(C=control, D=affected).

100

 

 

Figure 3B

Figure 3A.

 

Figure 3D.

Figure 3C.

CHAPTER V: JOURNAL ARTICLE:

Steficek BA, Thomas J S, McConnell MF, Bell TG. A primary platelet

disorder in consanguineous Simmental cattle. Thromb Res 72:145-153, 1993.

(Reprinted from a copyrighted article in W, with kind permission
from Elsevier Science Ltd, The Boulevard, Langford Lane, Kidlington 0X5 lGB,

UK).

101

A PRIMARY PLATELET DISORDER

OF CONSANGUINEOUS SIMMENTAL CATTLE

Barbara A. Steficek, Jennifer S. Thomas, Mary F. McConnell, and Thomas G. Bell
Department of Pathology and Animal Health Diagnostic Laboratory

Michigan State University, E. Lansing, MI 48824, USA

Abstract: A severe bleeding disorder of Simmental cattle has been described in wide-
spread locations in North America with clinical and historical findings
consistent with a hereditary hemorrhagic diathesis. Platelet numbers and
coagulation profiles of affected cattle are normal. We have characterized
a severe dysfunction of platelets (thrombopathy) which is very similar to
that described in the same breed by researchers at two other laboratories.
Preliminary data implicate an alteration in calcium (Ca+ +) sensitivity,
possibly involving Ca++ transport across the platelet plasma membrane.
Platelets from cattle affected with Simmental Hereditary Thrombopathy
(SHT) appear to be a valuable model for studying the role of Ca++
mobilization in cell activation which is currently the object of intense

investigation in biology and medicine.

 

Key Words: platelet, bovine, thrombopathy, aggregation, electron microscopy
Corresponding author: Dr. B.A. Steficeler. T.G. Bell, Animal Health Diagnostic
Laboratory, P.O. Box 30076, Lansing, MI 48909

102

103
The investigation of mammalian hereditary platelet disorders has contributed important

information to our understanding of normal platelet function. This report describes the
platelet aggregation and secretion responses associated with a unique hereditary platelet
dysfunction in purebred Simmental cattle; Simmental hereditary thrombopathy (SI-IT).
Affected cattle have a severe hemorrhagic diathesis characterized by spontaneous
epistaxis, hematuria, prolonged bleeding at sites of surgery, injections or trauma, and
episodes of hemorrhage on mucosal surfaces (1,2,3). Circulating platelet number and

size, as well as coagulation profiles are within normal limits (1,2).

A recent investigation of SHT detected a failure of adenosine diphosphate (ADP)
induced aggregation in whole blood samples as determined by impedance measurements
(1). In order to further characterize the defect, we measured ithiEQ aggregation of
affected SHT platelets in response to a variety of agonists which stimulate platelets at
different sites within activation pathways. Release of adenine nucleotides was evaluated
in response to platelet activating factor (PAF), an agonist which induced aggregation in

both normal and SHT platelets.

Samples of resting and post-stimulation platelets were collected for transmission
electron microscopy studies to evaluate platelet morphology and to confirm the results
of the aggregation studies. The aggregation responses of thrombin-stimulated, gel-
filtered platelets exposed to various external calcium concentrations were measured.

Finally, clot retraction was tested.

104

MATERIALS AND METHODS

Experimental Subjects

The experimental group consisted of three SHT affected Simmental animals and
three control animals, two of which were Holstein cows and one, a purebred Simmental
cow. The animals were housed at Michigan State University and were free from clinical

signs of disease other than those related to the thrombopathy.

Reagents

Adenosine diphosphate (ADP), a-thrombin and Sepharose 4B were purchased
from Sigma Chemical Company (St. Louis, MO, USA). Platelet activating factor
(PAF), collagen, and calcium ionophore A23187 were purchased from Sigma Chemical
Co. and Calbiochem Biochemicals (San Diego, CA, USA). Chrono-Lume luciferin
luciferase and ATP standard were purchased from Chronolog Corp. (Havertown, PA,

USA). All other chemicals were of reagent grade.

105
Collection of Platelets

Blood was collected by jugular venipuncture via an 18 gauge needle into a plastic
syringe containing trisodium citrate at a final concentration of 0.32 % . Platelet rich
plasma (PRP) was obtained by multiple centrifugation of the blood at 1324 x g for 60
seconds, removing a small quantity of PRP between each spin. Platelet poor plasma
(PPP) was obtained by centrifugation of the remaining blood at 1324 x g for 13 minutes.
Gel-filtered platelets (GFP) were obtained by adding 1 pM prostaglandin E, to the PRP
prior to centrifugation at 800 x g for 15 minutes. The platelet pellet was resuspended
in 1 ml HEPES-Tyrode-albumin (HTA) buffer (130 mM NaCl, 2.6 mM KCl, 0.42 mM
NaH,PO., 5.5 mM dextrose, 0.01 mM HEPES and 3.0 mg/ml bovine serum albumin,

pH 7.2), passed over a 10 ml Sepharose 4B column and the eluate collected.

Aggregation Studies

The aggregation response to ADP, PAF, collagen or calcium ionophore A23187
was measured using PRP adjusted to a final concentration of 300,000 platelets/pl with
autologous PPP. The platelets were manually counted using the Unopette system and
a hemocytometer. The aggregation response to thrombin was measured using GFP
adjusted to a final concentration of 300,000 platelets/ pl with HTA buffer. For the
thrombin samples only, MgC12 (1 mM) and CaClz (1.5, 1.0. 0.5 or 0 mM) were added
to the platelet suspension. The PRP/GFP suspensions were divided into 0.5 ml aliquots

and rested at room temperature for 30 minutes.

106
Aggregation was measured using optical light transmittance in a Lumi-

Aggregometer (Chronolog Corporation, Havertown, PA, USA). Both maximum percent
aggregation and slope of the aggregation curve were measured. The PRP/GFP were
warmed to 37°C and stirred at 900 rpm. Agonist (20 pl) was added and the response
recorded. If the sample did not begin to aggregate within five minutes of agonist
addition, the percent aggregation and slope were recorded as 0. A comparison of the
effect of the external calcium concentration on the aggregation response to 0.1 U/ml
thrombin was measured. In order to express the change in aggregation curves seen with
increasing calcium concentration ([Ca“ +]) in the resuspension buffer, an aggregation
index was calculated by using the slope of a line extending from the aggregation tracing

at the addition of agonist to the intercept of the lines representing the maximum percent

aggregation and the slope of the aggregation curve.

Preparation of Platelets for Transmission Electron Microscopy (TEM)

The platelets were prepared as described by Mattson et al (4). Briefly, PRP/GFP
was prepared as for the aggregation studies. Following agonist addition, the response
was stopped at 1 or 5 minutes by the addition of 0.5 ml 0.1% glutaraldehyde in 0.1 M
sodium cacodylate buffer, pH 7.4. The sample was stirred for 1 minute prior to transfer
into an Eppendorf cuvette for centrifugation for 20 seconds at 12,800 x g. The
supernatant was discarded and 1 ml of 3 % glutaraldehyde in 0.1 M cacodylate buffer

added. The sample was allowed to fix for 3-6 hours at which time the supernatant was

107
discarded and 1 ml 0.1 M cacodylate buffer was added to the sample. Samples were

then processed for TEM.

ATP Secretion Studies

Storage pool adenine nucleotide secretion was monitored in the Lumi-
aggregometer which simultaneously measures platelet aggregation and ATP secretion as
detected by the firefly-luciferase system. Aggregometer cuvettes containing 0.45 ml of
platelet rich plasma or gel-filtered platelets were allowed to equilibrate at 37 .5°C for at
least four minutes. The Chrono-Lume luciferin-luciferase reagent (50 pl) was added
immediately prior to the addition of 20 pl of agonist to minimize the potentiating effect
of the Chrono—Lume on platelet aggregation. Immediame after the ATP secretion
tracing on the recorder had peaked, a standard solution of ATP, final concentration 4.0
pM, was added to quantitate the ATP secretion. Dense granule secretion is described
by the amount of ATP in pM measured at the peak of the secretion curve and the time

inminutestakentoreachthatpeak.

Clot Retraction Study

Clot retraction was evaluated using a modification of the procedure described by
Miale (5). Whole blood was aliquoted into glass tubes and allowed to clot. The
samples were then placed in a water bath at 37°C and were examined for the presence
of clot retraction at 1, 2 and 24 hours. At 24 hours, the clot was removed using a

wooden applicator stick. The weights of the empty tube, tube with whole blood, clot,

108
sediment, and tube with remaining serum were recorded and relative percentages were

calculated.

Statistical Analysis

All studies were run in triplicate on each animal tested. The individual
aggregation, secretion, and clot retraction study results were averaged to obtain a mean

and standard error for each group.

RESULTS

The aggregations of SHT platelets in response to all agonists tested were
decreased, or, in the case of thrombin, required a greater amount of time to respond
compared to control platelets under the same conditions. Platelets from the affected
SHT cows changed shape did not aggregate in response to ADP (1-100 pM), calcium
ionophore A23187 (10-40 pM), or collagen (2-20 pg/ml). In contrast, the control cows

changed shape as well as aggregated in response to ADP (3.5-10pM),

109

 

Fig. 1.
Transmission electron micrographs of control (A) and affected (B) plate-
lets 5 minutes after addition of 10 pM ADP. Bar=l pm.

 

 

 

 

6 25
5.5 24! I ,
J 23
5 ..
l t 22+ it
4.5 t t . ' i
, 21 t t .
4 , 20 4 : ”L
3.5 l ' ,. I 1 19
L 18
3 .
l ‘ 17
2 ' Phenotype 15 ._
1.5 . _ - Control 14 3“
1 ‘ . . Affect d 13 \
_ ° 12
o r ,, . , 7 7 . . , . , 10‘ w».- , ~44» 7v
0.0 0.1 0.5 1.0 10 0.1 0.5 1.0
Platelet Activating Fm (pM) "W“ “mm FIG" (I‘M)
A B
Fig. 2.

Dense granule ATP secretion (A) and time to maximum ATP release (B)
in control and affected SHT platelets in the presence of varying
concentrations of PAF.

calcium ionophore A23187 (20-40 pM), and collagen (4—20 pg/ml) (Fig.1). In response

to PAF, affected platelets changed shape and aggregated; however, affected platelets

110

were less responsive. The extent of aggregation (maximum percent aggregation) was
reduced and the threshold concentration of agonist required for aggregation was
increased. Control platelets aggregated at concentrations of 0.1 pM PAF or had a weak,
primary aggregation (Table 1). Transmission electron microscopy confirmed the PAP

aggregation studies (results not shown).

Affected and control platelets had similar values for maximum percent
aggregation as well as slope of the aggregation curve in response to higher

concentrations of thrombin (1.0 or 0.6 U/ ml).

TABLE I.

Comparison of maximum percent aggregation and slope of aggregation
curve in response to ADP, A23187, collagen, PAF or thrombin in control

 

 

bovine and affected SHT platelets.
Slope of Aggregation Max. % Aggregation
Agonist Control Affected Control Affected
(n=3) (n=3) (n=3) (n=3)
ADP 10 pM 2.4 :1: 0.3 0.1: 0.0 51.2 :1: 3. 9 0.1:]; 0.4
3.5 pM 1.8 i 0.2 0.1 i 0.1 17.7 i 4. 7 -1.0 :l: 0.7
1 pM 1.0 i 0.1 0.1:l: 0.1 2. 8 i 1.6 0.1:l: 1.0
A23187 40 pM 2.4 :l: 0.7 0.0 :t 0.0 45.8 :I: 4.8 -l.9 :1; 0.6
30 pM 2.6 :l: 0.5 0.0 :l: 0.0 50.7 3: 4.3 -0.5 :l: 0.4
20 pM 2.9 :t 1.2 0.0 :1: 0.0 51.3 :I: 5.1 -l.3 :1: 0.6

(Table 1. cont.)
Collagen 20 pg/ml 2.4 :I: 0.1
10 pg/ml 2.0 :l: 0.2
6 pg/ml 1.2 :l: 0.2
4 pg/ml 0.4 i 0.1
PAF 10 pM 9.1 :l: 2.7
1 pM 13.1 :I: 2.1
0.1 pM 4.1 :1: 1.1
Thrombin 1 Ulml“ 2.5 :l: 0.3
0.6 Ulml“ 3.6 :l: 0.3
0.1 Ulml" 2.6 :i: 0.4
0.01 Ulml" 0.1 :t 0.1

111

0.1 i 0.0
0.1 i 0.0
0.1 i 0.0
0.0 :t 0.0
11.1 :t 1.4
12.1 :1: 0.8
0.8 :t 0.2
2.6 i 0.6
1.7 :1; 0.6
0. 6 j: 0. 4
0. 0 :l: 0. 0

53.9 i 2.
.5
.8
.5

51.3 :I: 2
37.4 i 4
14.8 :i: 3

M
M
O‘NQ

4

ace

0\

9.

:l:

:i: 3.
:i: 6.
.5 :i: 12.3
7 :9; 9.2
64.6 :I: 11.1

3

.4

1
5

2.3 :1: 1.3

 

Values = mean :1: Standard Error (S.E.); n = number of animals tested. * =
Gel filtered platelets (GFP) utilized for aggregation trials.

In response to 0.1 U! ml thrombin, the affected platelets tended to have a decreased slope

of the aggregation curve although the maximum percent aggregation was similar to the

controls. At a concentration of 0.05 U/ ml thrombin, control platelets exhibited irrevers-

ible aggregation; however, affected platelets failed to aggregate. Neither group

aggregated in response to 0.01 Ulml thrombin. Transmission electron micrographs

confirmed the aggregation studies.

Studies comparing the platelet dense granule release of ATP in response to various

concentrations of PAF revealed both similar levels of ATP released as well as time to

maximum secretion betrveen the affected (SHT) platelets and platelets from normal

animals (Fig. 2).

112

A comparison of the effect of the external calcium concentration on the aggregation

response to 0.1 Ulml thrombin was measured.

 

aggregation
C .6 U Thro-bin
1 1
WW
I”? l
s ‘ A .6 Elmo-bin
'7:
.9
E
E
,: t
15 C .05 0 Thrombin
a
D

 

  

t—-t
Tune (1 Min)

A

KHOIH IOHH’Oflfl‘IO”

 

Fig. 3.
Aggregation tracings (A) and aggregation index (B) for control and affected SHT
platelets stimulated by 0.6 U/ml and 0.05 Ulml thrombin (A) and 0.1 Ulml

In order to express the change in

 

0.5 1.0
CALCIUN CONCENTRATION (III)

B

thrombin in the presence of varying concentrations of external Ca++ (B). [A:
A=affected, C=control; B: Solid line=control, Dashed line=affected.]

calcium present. The control pcurves seen with increasing concentration of calcium in

the resuspension buffer, an aggregation index was calculated. The affected platelets had

lower aggregation index values which were affected relatively little by the concentration

of external latelets had a much higher index which increased as the external calcium

concentration was increased from no added calcium to 1.5 mM (Fig. 3).

113
Examination for evidence of clot retraction in the samples from control animals

revealed progressive and prominent separation of the clot from the sides and base of the

glass tube. This change was most noticeable at 2 and 24 hours.

TABLE II.

Clot Retraction Study. Comparison of the clot, serum, and sediment in control
bovine and affected SHT platelets.

 

 

Group # Tested % Clot % Serum % Sediment
Control n=3 57.1 i 1.7 40.61: 1.6 2.2
:l: 0.5
Affected n=3 34.3 i 0.9 53.4 ;|: 0.9 11.9
:i: 0.4

 

Values = mean j; Standard Error (S.E.)

The resultant serum was observed to be clear and free of cellular sediment. In contrast,
dissolution of the clot was evident in affected cattle as early as 1 hour following
collection. This breakdown of the clot progressed over the 24 hour test period resulting
in a small "soft" clot surrounded by dark red serum. Following removal of the clot all
samples were subsequently centrifuged at 1324 x g for 90 sec and the percent (%)
cellular sediment remaining was calculated. Samples from affected animals had a
markedly higher % sediment than control or carrier samples. In affected animals, all

resultant clots were consistently smaller (Table 2).

1 14
DISCUSSION

Few hereditary platelet disorders have been described in cattle. A previous report
by Searcy et a1 discussed clinicopathologic findings and whole blood ADP aggregation
results in purebred Simmental cattle with a bleeding diathesis (1). In Simmental
hereditary thrombopathy (SHT), the severe platelet dysfunction demonstrable in m
mirrors the degree of hemorrhagic diathesis seen clinically (1). Calcium ionophore
A23187 or ADP added to affected platelets at various concentrations did not stimulate
any aggregation response; however, since SHT platelets changed shape after the addition
of ADP, the ADP receptor function is not necessarily suspect in the defect. The initial
action of these two agonists is entirely different. ADP binds to a specific membrane
receptor which transmits the stimulatory signal into the cell (6). ADP activation of
human platelets is associated with exposure of the fibrinogen binding site on the
membrane glycoprotein IIb-IIIa complex ('7), increased cytosolic calcium concentrations
(8) and Na‘fH” antiport-dependent activation of phospholipase A, (PLAZ). Conversely,
A23187 directly initiates release of calcium from internal storage sites as well as
mediating extracellular calcium influx (9). The increased concentration of cytosolic
calcium regulates the activity of a number of calcium dependent intracellular enzymes
including myosin light chain kinase, which phosphorylates myosin light chain and is
associated with shape change (6). Both agonists, however, finally activate normal
platelets though a shared association with phospholipase A2 (PLAz). It may therefore

be suspected that the defect in SHT is located in a segment of the activation pathway

1 15
which is shared by ADP and A23187. Calcium activation of PLA, releases arachidonic

acid metabolites which subsequently activate phospholipase C (PLC) (10).

Bovine platelets produce thromboxane following ADP stimulation, but the inhibition
of thromboxane production by cyclooxygenase blockade has been reported to not affect
aggregation in line. The conclusion reached by some investigators has been that the
ADP-induced aggregation of bovine platelets, unlike human platelets, is less critically

dependent on arachidonic acid metabolism (11,12).

Affected SHT platelets changed shape and aggregated in response to PAF or
thrombin; however, the responses were clearly decreased compared to those of the
control platelets. Both PAF and thrombin bind to surface receptors (6) which then, via
a GTP-binding protein, activate phospholipase C (PLC) (13,14). Therefore, the fact that

PAP and thrombin are effective SHT platelet agonists indicates that PLC is activated.

Transmission electron nricrographs revealed no consistent ultrastructural differences
between control and affected SHT bovine platelets. In addition, the electron

micrographs corroborated the results of aggregation studies.

This study clearly differentiates the Simmental hereditary thrombopathy from
previously described defects of the bovine platelet. For example, the storage pool
deficient platelets in bovine Chediak-Higashi Syndrome (CHS) aggregate normally in

response to ADP, but collagen induced aggregation is diminished (15). Also, in CHS,

116

dense granule content of adenine nucleotides and serotonin is greatly reduced. In
contrast, the SHT dense granule adenine nucleotide content is similar to that of normal

cattle, as evidenced by platelet ATP secretion in response to PAF.

In conclusion, we hypothesize that Simmental hereditary thrombopathy may be a
defect in calcium mobilization or utilization. Affected SHT platelets provide a unique
opportunity to investigate the biochemical pathways that regulate the physically
measurable response of aggregation. SHT cattle suffer from a primary dysfunction of
platelets which has characteristics unlike those seen in any known human disorder.
Investigation of the mechanism responsible in Simmental hereditary thrombopathy is

certain to uncover novel information about platelet function.

Acknowledgements

We thank Mr. Ralph Common for his photographic assistance. This work was
supported by Biomedical Research Support Grant, #71-0751, and F32HLO7560 and

F32HL08564 from the National Institutes of Health.

10.

11.

12.

117

REFERENCES

SEARCY, GP. and PETRIE L. Clinical and laboratory findings of a bleeding
disorder in eight Simmental cattle. Can Vet J, 31, 101-103, 1990.

. KOCIBA, 6.]. Partial characterization of a hemostatic defect in Simmental cattle.

Vet Clin Path, 9, 446-47, 1980.

LEIPOLD, H. and MOORE, W. Bovine thrombopathia. In: W
- -4 z D' a.-. .. W..J Dodds(ed)ILAR

 

News,21: A14, 1977.

MATI‘SON, J.C., BORGERDING, P.J., and CRAFT, D.L. Fixation of platelets
for scanning and transmission electron microscopy. Stain Technology, 52, 151-158,
1977.

. Miale, J.B. Appendix-methods and quality control. In:

hematology, Stir ed. St. Louis,MO:C.V. Mosby Co., 1977, pp. 1061-1062.

SEISS, W. Molecular mechanisms of platelet activation. Physiol Rev, 69, 58-178,
1989.

COLMAN, R.W. Aggregin: a platelet ADP receptor that mediates activation.
FASEB J, 4, 1425-1435, 1990.

SAGE, 8.0. and RINK, T.J. The kinetics of changes in intracellular calcium
concentration in fura-2-loaded human platelets. J Biol Chem, 262, 16364-16369,
1987.

WHITE, J.G., RAO, G.H.R. and GERRARD, J.M. Effects of the ionophore
A23187 on blood platelets. Influence on activation and secretion. Am J Pathol, 77,
135-150, 1974.

SWEATI‘, J. D., CONNOLLY, T. M. ,,BARON B. M. and LIMBIRD, L. E. Involve-
ment of Na*/H* exchange in human platelet activation. In: We

tax-1H ' 01'- -_, 30' 110th :0. '11s t a: 10' New

York, Alan R. Liss, Inc. 1988, pp. 523-557.

BONDY, 6.8. and GENTRY, P.A. Characterization of the normal bovine platelet
aggregation response. Comp Biochem Physiol, 92C, 67-72, 1989.

GENTRY, P.A., TROMBAY, R.R.M. and ROSS, M.L. Failure of aspirin to impair
bovine platelet function. Am J Vet Res, 50, 919-922, 1989.

13.

14.

15.

118

LAPETINA, E.G. Platelet-activating factor stimulates the phosphatidylinositol cycle.
J Biol Chem, 257, 7314-7317, 1982.

CROUCH, M.F. and LAPETINA, E.G. A role for G, in control of the thrombin
receptor-phospholipase C coupling in human platelets. J Biol Chem, 263, 3363-
3371, 1988.

BELL, T.G., MEYERS, K.M., PRIEUR, D.J., FAUCI, A.S., WOLFE, SM. and
PADGEI'I‘, G.A. Decreased nucleotide and serotonin storage associated with
defective function in Chediak-Higashi syndrome cattle and human platelets. Blood,
48, 175-184, 1976.

CHAPTER VI: MEASUREMENT OF THE CYTOSOLIC CA”
CONCENTRATION USING AEQUORIN AND FURA-ZIAM

Introduction:

An important element of the preliminary work done on Simmental Hereditary
Thrombopathy was the hypothesis that there is defective or impaired calcium
mobilization/utilization in affected platelets. It was therefore necessary to evaluate
changes in [Ca+ *L in response to platelet stimulation by a variety of agonists. It was
anticipated that the movement of Ca‘ * from the external medium to the cytoplasm
would be inhibited in affected platelets following stimulation by some, if not all,
agonists. Two techniques currently used to measure [Ca+ *]; include the fluorescent
calcium chelator dye, fura-2, and the luminescent photoprotein aequorin.‘”'m"3‘ For
these procedures, [Ca**], is measured in the presence and absence of external Ca“
to determine the relative contributions of internal Ca” release and external Ca++
influx. The change in [Ca+ *1, in the presence of external EGTA represents internal
Ca++ mobilization while that in the presence of external CaM represents both internal

release and external influx.

Aequorin consists of a single polypeptide apoprotein and a tightly bound

chromophore. ‘3‘, Following binding of Ca+ + , the chromophore becomes oxidized.

119

120
The result is the production of a molecule of CO, and a photon. The response of

aequorin to Ca” is exponential over the range of 107-10“.“ The character of the
log-log plot for aequorin suggests that at least three Ca” ions interact with this
photoprotein to generate the luminescence. In the case where there are local Ca” *
gradients within the cell, aequorin does not provide quantitative data about the
average value of the cytoplasmic Ca“ *. The use of aequorin, though well
documented in the literature, involves several additional specific obstacles. 135.136.137.138
These problems include the extreme sensitivity of this compound to the presence of
magnesium; though the light reaction is not initiated by the binding of Mg++ to
aequorin, the Ca+ *- dependence of the aequorin reaction is antagonized by Mg“ +. In

addition, aequorin is markedly temperature sensitive.

Materials and Methods:

Aequorin Studies:

Platelet cytosolic ionized calcium concentration [Ca“ *1, was measured using
modifications of the techniques described by Yamaguchi et a1.” Platelet rich plasma
was prepared as described in the section on platelet aggregation and secretion studies
using 3.8 % trisodium citrate as anticoagulant, and was acidified by the addition of 9pl
of l M citric acid/ml of PRP 5 minutes prior to centrifugation for 15 minutes at 800 x

g (20°C). The supernatant was discarded and the platelet pellet gently resuspended in

121
10 ml of HEPES buffered saline (l40mM NaCl, 2.7mM KCl, 0.1% bovine serum

albumin, 0.1% glucose and 3.8mM HEPES, pH 7.6) containing 1pM PGE, and SmM
EGTA. The sample was incubated for 5 minutes at room temperature prior to
centrifugation again at 800 x g for 15 minutes. The supernatant was discarded and
the pellet gently resuspended in 110 pl of the same buffer. Stock aequorin solution (1
ml in 333 pl distilled water containing 7mM EGTA) was added in the amount of 10
pl. DMSO was then added over a period of 7.5 minutes at a rate of 1 pl DMSO/1.5
minutes to yield a final concentration of 6% by volume. The suspension is then
incubated for 8 minutes at room temperature, diluted to 1 ml using HEPES buffered
saline solution without PGE1 or EGTA and gel filtered by passing the sample over a
10 ml Sepharose 4B column equilibrated with the same buffer. The gel filtered
platelets were counted and diluted to a final concentration of 2.0 x 10‘ plateletsl ml
using the same buffer. MgCl, (final concentration lmM) was added and the sample
divided into 1 ml aliquots. Five minutes prior to the measurement of [Ca+ *], the
cuvettes are warmed to 37.5°C, a teflon-coated magnetic stir bar inserted and either
lmM CaCl, or lmM EGTA added. The cuvettes were placed into a Platelet Ionized
Calcium Analyzer (PICA, Chronolog, Havertown, PA) and stirred at 900 rpm. After
maintaining a steady resting baseline, 40p1 of agonist was added and both aggregation
and peak dye luminescence were recorded on a dual channel Omniscribe chart
recorder. The total platelet aequorin luminescence (L...) was determined by lysis of a
four-fold diluted platelet sample containing 1 mM Ca” by 150pl of Triton X-100.

The [Ca* *], was calculated from the maximum peak of the aequorin luminescence

122
signal as described in the PICA instruction manual, using the calibration curve of log

LIL.m vs log [Ca” *1, which was supplied with each lot of aequorin and was

constructed in a medium containing 150 mM KCl, 5 mM PIPES and 1 mM MgC12.

Results:

Results of studies using the photoprotein aequorin are presented in Table 2.
Agonists utilized in the study included thrombin (0.1 to 1.0 Ulml), PAF (0.5 and
1.0pM), and A23187 (1.0 and 5.0pM). The data accumulated with this form of

[Ca+ *J, determination were similar between the control and affected platelet samples.

hire-21AM Studies:

Evaluation of [Ca2+], has been accomplished successfully in a variety of cell
types through the utilization of a class of internal probes known as fluorescent
calcium (Ca2 +) indicators. Members of this group include quin-2, indo-l , fura-2 and
the more recently developed, fluo-3. 13““ Due to its physiochemical characteristics,
fura-2 has become a valuable tool for the investigation of changes in [Ca”’]; in single

anchored cells or in cell suspensions.

123

Table 2. Aequorin assessment of [Ca“ *L. Comparison of peak [Ca" *1, (pM) following
stimulation with various agonists in control and afl‘ected aequorin-loaded bovine platelets.

 

 

AGONIST CONTROL AFFECTED
(n=4) (n=2)

Thrombin 1.0Ulml 3.08 :l: 0.58 4.25 :1: 0.25
Thrombin 0.6Ulml 4.30 :1; 0.77 4.35 :1: 0.24
Thrombin 0.1U/ml 3.08 :l: 0.67 5.00 :l: 0.00
PAF 1.0pM 3.48 i 0.43 4.33 :t 0.34
PAF 0.5pM 3.38 i 0.68 4.50 i 0.50
A23187 5.0pM 10.37 :1: 3.02 12.85 :1: 4.96
A23187 1.0pM 5.75 :t: 1.09 4.70 :1: 0.60

 

Values = mean 04M) :1: SE; n = number ofanimals tested.

124
Fura-2 is a polycarboxylic fluorescent Ca2+ chelator which changes its

fluorescent properties upon binding Ca“. Advantages of this compound over other
associated indicators include an approximately 30-fold brighter fluorescence, marked
changes in wavelength as well as intensity, and a high selectivity for Ca2+ over other
divalent cations such as magnesium (Mg’*).‘3‘ Fura-2 is loaded into the cell in its
acetoxymethyl ester derivative form, fura-2/ AM . The acetoxymethyl ester groups
modify the fura-2 carboxylic acids, resulting in an uncharged molecule which can
permeate cell membranes. Once in the cell, non-specific esterases cleave the AM

ester groups, allowing the free acid form of the indicator to bind Ca1+.

For fura-2/AM studies, [Ca* *], was to be measured using modifications of
techniques described by Daniel et a1 and Tsien et al.‘“"“ In the original procedural
plan, platelet rich plasma was isolated as described in the platelet
aggregation/secretion study methods section, again using 3.8 % trisodium citrate as
anticoagulant. Platelet poor plasma was obtained as well. The PRP was acidified by
the addition of 9 p1 of 1 M citric acid per 1 ml of PRP. The sample was incubated at
room temperature for 5 minutes and was then centrifuged at 800 x g for 15 minutes
(20°C). The supernatant was discarded and the platelet pellet resuspended in 8 ml of
PPP acidified by the addition of 72pl of 1M citric acid. The platelet sample was
separated into two 50 ml centrifuge tubes for preparation of paired samples. Fura-
2/AM (Molecular Probes, Inc. , Eugene, OR) was added to one of the platelet

suspensions from a stock solution (50 mg fura-2/AM in 50p1 of DMSO) to yield a

125
final concentration of 4pM, and an equal quantity of DMSO was added to the second

platelet suspension sample. This second sample was used to determine background
fluorescence. The two samples were then incubated at 37°C for 45 minutes.
Following this period of incubation the samples were centrifuged again for 15 minutes
at 800 x g, the supernatant discarded and the platelet pellets resuspended in 1 ml
Ca“-Mg”-free HEPES-Tyrode’s buffer solution (137mM NaCl, 2.7mM KCl,
5.5mM glucose, 0.35mM NazHPO“ 10mM HEPES and 0.2% bovine serum albumin,
pH 7 .4). The platelet sample was then gel filtered, the platelets count determined on
the eluate, and the sample diluted to 1.0 x 10‘ cells/ml using the same buffer. MgCl,
(final concentration 1.0mM) was added and the suspension divided into three equal
groups in 12 ml polystyrene centrifuge tubes. One group contained gel filtered
platelets with CaClz added to a final concentration of 1.0mM, the second group
contained EGTA (final concentration 1.0mM), and no additions were made to the
third group. These preparations were made from both of the original PRP samples so
that identical groups were created for fura-2\AM evaluation as well as for the DMSO-
only background determinations. The samples were maintained at room temperature
until 10 minutes prior to the measurement of [Ca+ ’1; at which time they were warmed
to 37°C. A 2 ml quantity of the sample to be tested was transferred into a
polystyrene spectrophotometer cuvette (Sigma Chemical Co. , St. Louis, MO), a
spectrophotometer teflon-coated stir bar was added and the cuvette was placed into a
dual-wavelength spectrofluorometer (SPEX Industries, Edison, NJ) equipped with a

thermally jacketed cuvette holder at 37°C. The emission intensity was monitored at

126
505nm following excitation (450 W, xenon lamp) of the platelet suspension at 340nm

and 380nm. “2 Alternating excitation wavelengths were obtained using a beam
chopper. Ratios and [Ca+ *J; were corrected for intrinsic fluorescence using platelets
treated with DMSO only. The platelet suspension was stirred at a slow stir speed, to
maintain the cells in suspension, and resting fluorescence recorded. Agonist (80 pl)
was then added and the maximum post-stimulation fluorescence recorded. In some
samples, Fm and F“ were determined, where Fm equals the sample fluorescence
following cell lysis subsequent to the addition of Triton-X containing 4mM Ca” *, and
F“ equals the sample fluorescence following addition of 6mM EGTA to the lysed
platelet suspension. In addition, the intensity recordings of fura-2-loaded platelets
were also corrected for leakage of fura-2 from platelets not exposed to agonist.
Manganese (final concentration 40pM) was added to a fura-2-loaded platelet
suspension and the fluorescence intensity remaining 2 see after this addition was
subtracted from the intensity prior to Mn” 1* addition. This difference was due to
extracellular fura-2 and was subtracted from all intensity recordings from that
preparation. The sample used for determination of extracellular fura-2 was not used
for any other experiments. Ratios were determined by dividing the corrected intensity
recording from excitation at 340 nm by that at 380 nm excitation. When appropriate,
[Ca+ 1], was then calculated using the equation from Grynkiewicz et al:‘” [Ca" *l,
=K,,[(R-R,h)/(R._,-R)] X (Salsa); where Rm is the 340/380 nm ratio during Ca" *
saturation, and R... is the 340/380 nm ratio during Ca“-free conditions. Sn and S»

are the emission intensities at 380 nm in Ca” *-free and Ca" *-saturating conditions,

127
respectively. R,m was determined by lysing the platelets with 0.1% (v/v) Triton X-

100 in 4mM Ca“. Ra. was determined on the same sample by adding 40mM

EGTA.

Results:

Attempts to follow established laboratory procedures described for the
fluorescent indicator fura-2 have demonstrated a marked species difference between
bovine and canine platelets, both in the percent of dye which can be loaded into the

cell and the intensity of fluorescence achieved. (Figure 10)

During the course of the investigations of [Ca+ +], using fura-2/AM in bovine
platelets, numerous steps and factors of the previously outlined laboratory procedure
were adjusted because the demonstrable responses in the bovine were not consistent
with results obtained for other mammalian species.143 The following is a compilation

of areas that were evaluated:

128

Figure 10. Comparison of the intensity of fluorescence in counts per second (cps) of
canine vs bovine fura-2 loaded platelets following the addition of 1.0 U/ ml thrombin.
[A=canine 340nm; B=canine 380nm; C=bovine 340nm; D=bovine 380nm. Addition

of agonist=60 sec; Fmax=120 sec; Fminm=180 sec; Fminm=240 sec]

KB

.2 2:5

 

 

 

 

 

 

 

.oome came

a. a. - a. :3“: a. asset
4 II. t --

:.. it. -- $9.3...“

Imo+emv.m

lm0+w@¢.¢

roo+ond.u

2m . 89.22 a
Be. 08.2% a
smasoaga Hausa sew.amtmmmflu
sneeze "3:... Ea. catama— -

 

909-9)"

“:‘POBM'fi-flm

130

O Buffer O Adnrinistration Speed

0 pH of buffer 0 Platelet Concentration

0 Loading Time 0 Agonist Concentration

0 Loading Temp 9 Stir Speed

0 B.S.A. vs no B.S.A. 0 Aggregate vs Non-Aggregate
O Fura-2/AM Concentration O Slit Width

0 Fura-2/AM Source 0 Species

O Lysing Agent 0 Sulfinpyrazone

In each instance the variations in standard procedure attempted did not result
in a si ' cant improvement in the fluorescence or calcium response of the bovine
platelet sample. Specifically, these alterations included: 1) buffer - Ca“ *-Mg+ ,-free
Tyrode’s buffer vs Hank’s buffered saline solution; 2) pH of buffer - 7.4 vs 7.55; 3)
loading time - 5 to 60 minutes; 4) loading temperature - 37°C vs 30°C; 5) addition
of bovine serum albumen (B.S.A.) vs no-B.S.A. in the buffer solution; 6) fura-2/AM
concentration - 2 to 10pM; 7) fura-ZIAM source - Sigma vs Molecular Probes; 8)
lysing agent - Triton-X vs digitonin; 9) administration speed (agonist) - slow
administration with Hamilton syringe vs rapid bolus addition; 10) platelet
concentration - 100,000/pl vs 300,000/p1; ll) lamp slit width - 1.2 vs 1.5; 12)
comparison with platelets from another mammalian species (i.e. canine); and 13) use
of sulfinpyrazone in plasma or buffer vs standard loading of fura-2/AM in autologous

platelet poor plasma.

131
The results obtained from assessment of the last two variables listed revealed

that: 1) the experimental conditions were acceptable to reproduce previously obtained
results for the canine, and, 2) the bovine species was clearly different in its ability to

hydrolyze or leak the loaded fura-2/ AM .

Although fura-2 has displayed many advantages over other fluorescent
indicators, its use has not been without problems.”"""“"“"“"‘7 One of the more
important factors described is the tendency for a high rate of dye leakage into the
extracellular medium in certain cell types. This leakage may be a result of failure to
cleave the acetoxymethyl ester groups from the compound once inside the cell, thus
resulting in fura-2/AM passively moving out through the cell membrane. In contrast,
removal of fura-2 from a cell may be a direct result of an active organic anion

membrane transport system.

Through modifications of previously described methods for the fura-2 Ca2+
assessment procedure, we have developed a protocol for the use of this indicator in
bovine platelet suspensions. This procedure utilizes a 15 minute loading period of the
dye into platelets suspended in buffer containing 300pM sulfinpyrazone.
Sulfinpyrazone is a phenylbutazone-like compound which has both anti-inflammatory
and anti-thrombotic properties 111M. Its effect on platelets m are minimal.”
More importantly, sulfinpyrazone is a known inhibitor of organic anion transport

systems?” In comparative studies, we measured the intensity of fluorescence over

132

time of various forms of suspension medium. Platelet samples were prepared as
described above, however, the platelet pellet in each sample was resuspended in either
PPP, buffer or buffer containing sulfinpyrazone (300pM). Fura-2/AM was added to
each sample at a concentration of 4pM and the samples incubated at 37°C. At
specified time intervals, 0.5 ml of the platelet sample was removed and the sample
placed in an Eppendorf centrifuge for 20 sec at 12,800 x g. The supernatant was
removed and a 20 pl aliquot was added to 2 ml of Ca*+-Mg++-free Hepes Tyrode’s
buffer in a spectrophotometer cuvette. The intensity of fluorescence of each sample
was then recorded. In addition, Fm and F“ were calculated for each sample as
described above. These studies revealed a marked difference in relative fluorescence
of the supernatant within 5 minutes of addition of dye to platelet samples suspended in

buffer with sulfinpyrazone versus buffer alone or autologous plasma. (Figure 11)

These data suggest that the bovine platelet has a well-developed mechanism for
the transport of certain organic compounds from within the cell interior, and that
sulfinpyrazone may serve as a functional inhibitor of this transport system. In a
similar supernatant study, subsequent to that described above, the concentration of
sulfinpyrazone added to the buffer was varied in order to determine the minimum
concentration necessary to achieve optimum fluorescence values. The concentrations

utilized were 100, 300 and 500pM. Results of this study are shown in Figure 12.

133

Figure 11. Fura-2/AM loading assessment in resting normal bovine supernatant from
platelet samples. Comparison of the effect of plasma (Pls), buffer (But), and buffer

containing 300pM sulfinpyrazone (sz) on intensity of fluorescence over time.

.HH Guzman

”wan-:2:

2Q E

134

 

 

 

l
I

843 see".

oooom

oooov

oooom

cocoa

ooooop

oooome

(sdo)£usuann

135

Figure 12. Comparison of the effect of varying concentrations of sulfinpyrazone on the
uptake (intensity of fluorescence) of fura-2/ AM over time, measured in the supernatant

from resting platelet samples.

136

.NH engage
«22...:

.aaovn.

 

INCH—h

 

ooom

oooow

oocmv

oooom

ooomN

oooon

ooomn

oooov

coomv

(sdo)Ausuauu!

137
The results of the above studies using fura-2lAM loading of bovine platelets

have resulted in a modification of the previously well-documented procedure reported
by Daniel et a1 and Tsien et 8.13“" In this modified procedure, platelet loading of
fura-2/AM is performed in buffer containing 300pM sulfinpyrazone and the loading
time has been shortened to 15 minutes maximum at 37°C. Preliminary results in
control bovine platelets stimulated with 1.0 U/ ml thrombin show greater than tlrree-
fold increase in fluorescence of the sample at 340 and 380 nm as well as indication of

a calcium response following addition of agonist and again at calculation of

F‘“.(Figure 13)

Fura-2 has become widely accepted as an indicator of change in cytosolic
calcium in response to stimulation by various agonists in a variety of cell types and
different mammalian species.” 150.151 Use of the modifications described for the fura-

2 use in the bovine platelet may become applicable with other cell lines.

138

Figure 13. A comparison between plasma (A,B) and buffer containing 300pM
sulfinpyrazone (C,D) of the intensity of fluorescence in counts per second (cps),
following the addition of 1.0 U/ ml thrombin in control bovine platelet samples loaded
with 4pM fura-2. [A=340nm; B=380nm; C=340nm; D=380nm. Addition of

agonist=60 sec; Fmax=120 sec; Fminbufiq=l90 sec; Fminpm=240 sec]

mm

.2 seem

 

 

 

 

 

 

 

Honey camp
an . 94v ‘ can can -JMHtt -.... no.90oent
t--wwrwrrwrlqdr :-.:.
#Jttrrrttna+ooo.e
rmo+dph.m
teo+om¢.q
littL toot; . «

 

€25 "8 8.2.... :5. atoms: a
£25 28 3.3... 2m. «meme. a
Sm. cent: mm a
2m. centtmm -

 

 

909-10)"

Heaven-~03

CHAPTER VII: SUMMARY AND CONCLUSIONS

The studies presented in this dissertation were undertaken in order to
characterize a defect in platelet function seen in purebred Simmental cattle. Previous
reports of this bleeding diathesis considered principally the clinicopathologic findings
and disclosed that there was a defective platelet aggregation response to stimulation by
the agonist ADP. The laboratory procedures described for this research project,
therefore, were selected to establish a cellular mechanism for the platelet dysfunction

in what we have termed Simmental Hereditary Thrombopathy.

To further evaluate the aggregation and secretion responses of affected bovine
platelets, an extended range of agonists was selected. These agonists included ADP,
A23187, collagen, PAF, PMA, and thrombin. The SHT platelet aggregation response
to the first three agonists listed was minimal or inapparent; however, in each case,
shape change was noted, either within aggregation tracings, or, on transmission
electron microscopy. Even when the affected platelets were incapable of aggregating,
the occurrence of shape change indicated that SHT platelets have a functional receptor
for each of the agonists which were utilized. Affected platelets changed shape and
aggregated in response to PAP and thrombin; however, the maximum % aggregation
was decreased compared to normal bovine platelet samples. In the case of the agonist
PMA, the SHT aggregation response closely correlated with the response in control
PMA-induced aggregation. Concurrent studies of platelet dense granule ATP

secretion using ADP and PAF as agonists exhibited similar values for maximum ATP

140

141

secretion and time to maximum ATP secretion when comparing the control platelets
to SHT platelets. Transmission electron microscopy, assessment of platelet calcium
through I.C.P. , platelet aggregation response to variations in citrate concentration,
and assessment of plasma von Willebrand Factor antigen revealed no apparent
differences between the affected bovine samples and control bovine samples. In
addition, it was not possible to differentiate between the platelet proteins of normal

and affected platelets using l-D reduced SDS polyacrylamide gel electrophoresis.

Studies aimed at examination of the response of bovine platelets to varying
concentrations of external Ca++ revealed that thrombin-stimulated aggregation in
control platelets was enhanced by increasing the concentration of external Ca”. The
aggregation potential of affected platelets, however, was relatively unresponsive to

similar increases in the external Ca”.

Results of a quantitative analysis of clot retraction demonstrated a marked
difference in the final degree of retraction of the clot at 24 hours in affected blood
samples. The SHT remaining clots were soft, smaller and friable. In addition, the
amount of sediment remaining in the serum of SHT samples was increased compared
to controls. Clot retraction is a phenomenon dependent upon the number of platelets
in the sample, the amount of fibrinogen, and the ability of platelets to mobilize
external calcium. In affected animals used in this study, the fibrinogen level and
platelet number are normal. Further evaluation of Ca“ 1 was thus indicated. The

results of the external calcium concentration study, the lack of an aggregation

142
response to ADP and A23187, and the unusual response of affected platelets in the

clot retraction study suggested that affected platelets may have a defect in the

mechanism controlling the influx of external calcium.

To further evaluate this hypothesis, studies were initiated to examine and
compare the platelet [Ca* *], in control versus affected platelets, following agonist
stimulation. A standard laboratory method chosen for this evaluation, the use of the
fluorescent calcium indicator dye fura-2, gave unexpected results when the procedure
was attempted. A prominent species difference was revealed between normal bovine
and canine platelets, both in the percent of dye which could be loaded into the cell as
well as in the intensity of fluorescence. These differences necessitated the
development of modifications of the established methodology to generate a protocol
which would be useful for [Ca* *1, in the bovine platelet. This protocol involved
shortening the loading time to 15 minutes, loading the acetoxymethyl ester form of
furs-2 in buffer instead of plasma, and incorporating the use of the organic anion
transport system inhibitor, sulfinpyrazone. The supernatant studies designed to
evaluate the use of this product showed a marked inhibition of the increase in
supernatant fluorescence of fura-2/AM over time when compared with plasma or
buffer-only samples. These findings indicated that fura-2/AM was being taken up and
retained within the bovine platelet when sulfinpyrazone was added. These results
further suggested that the problem in fura-2/AM loading of bovine platelets may be

due to a combination of factors including: 1) the effect of bovine plasma on fura-

143

2/AM uptake and/or spontaneous hydrolysis; and, 2) fura-2 is actively transported out

of the bovine platelet through an organic anion transport system.

From the results presented in this dissertation, the following conclusions can

be made.

1.

The bleeding diathesis in Simmental Hereditary Thrombopathy is
properly classified as a primary platelet function defect.

Affected cattle commonly exhibit recurrent episodes of epistaxis and
bleed excessively as a result of minor trauma.

Platelets from affected animals fail to aggregate in response to the
agonists ADP, A23187 and collagen.

Affected platelets exhibit no indication of a structural defect when
examined by transmission electron microscopy or l-D reduced SDS
PAGE.

Affected platelets appear to have altered mobilization or utilization of
external calcium, as determined by evaluation of the platelet responses
to variations in external calcium and results of clot retraction studies.
The standard fura-2/AM loading procedure requires significant
modification for bovine platelet study.

The frequency of expression and the distribution of SHT we have
observed in male and female affected animals, both in our experimental
herd and from observations reported in the literature, strongly implicate

an autosomal recessive mode of inheritance.

144
The characteristics of the thrombopathy described in SHT do not coincide with

any previously described platelet defect, and, cannot be fully explained by the current
knowledge of this dysfunction. Further investigations of SHT should include the
continued comparative analysis of platelet [Ca* *], in control and affected samples
using fura-2/AM, and extended examination of the platelet structural proteins through
the use of 1-D non-reduced SDS PAGE and 2-D reduced-non-reduwd gel
electrophoresis. In addition, the evaluation of platelet plasma membrane high and
low-affinity Ca** binding sites through the use of 4’Ca** binding studies may reveal
if the defect is associated with Ca* * uptake. Evaluation of the genetic mode of
inheritance and incidence of carrier animals through complete breeding studies will
provide valuable information to the researcher, veterinarian and livestock producer.
Finally, the development of a laboratory method to detect carrier animals should be
considered a key goal in future studies. The carrier detection may incorporate an
analysis of the parameters of clot retraction. The continued investigation of the
biochemical mechanisms of the unique platelet dysfunction in Simmental Hereditary

Thrombopathy is certain to disclose valuable information about platelet function.

LIST OF REFERENCES

10.

LIST OF REFERENCES

Searcy GP, Petrie L. Clinical and laboratory findings of a bleeding disorder
in eight Simmental cattle. Can Vet J 31:101-103, 1990.

George IN, Reimann TA. Inherited disorders of the platelet membrane:
Glanzmann’s thrombasthenia and Bemard-Soulier disease. In: Hemgstasis
andlhmmlnsjs. Colman, I-Iirsh, Marder, Salzman (eds). Philadelphia,
PA:J.B. Lippencott Co. 1982, pp. 496-506.

Steficek BA, Bell TG, Thomas JS. A primary platelet function disorder in
purebred Simmental cattle. Thromb and Haemost 69:590, 1993.

Steficek BA, Thomas JS, Baker JC, Bell TG. Hemorrhagic Diathesis
Associated with a Hereditary Platelet Disorder in Simmental Cattle. J. of
Veterinary Diagnostic Investigation 5:202-207, 1993.

Kociba GJ. Partial characterization of a hemostatic defect in Simmental cattle.
Vet Clin Path 9:446-47, 1980.

Leipold H Moore W Bovine thrombopatm In: W
2 DJ ‘1‘..‘§,W.J. Dodds(ed)ILAR

 

News, 21: A14, 1977.

Searcy GP, Sheridan D, Dobson KA. Preliminary studies of a platelet
function disorder in Simmental cattle. Can J Vet Res 54:394-396, 1990.

Searcy GP, Frojmovic MM, McNicol A, Robertson C, Wong T, Gerrard JM.
Platelets from blwding Simmental cattle mobilize calcium, phosphorylate
myosin light chain and bind normal numbers of fibrinogen molecules but have
abnormal cytoskeletal assembly and aggregation in response to ADP. Thromb
and Haemost 71:240-246, 1994.

Marcus AJ, Safier LB. Thromboregulation: multicellular modulation of
platelet reactivity in hemostasis and thrombosis. FASEB J 7:516-522, 1993.

Hawiger J. Formation and regulation of platelet and fibrin hemostatic plug.
Human Path 18:111-122, 1987.

145

ll.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

146

Ruggeri ZM. Mechanisms of shear-induced platelet adhesion and aggregation.
Thromb and Haemost 70:119-123, 1993.

Weiss HJ. Platelet Mechanisms. In: 1 ‘
Wm New York, NY. Alan R. Liss, Inc., 1983, pp. 1-27.

Brass LF. Regulation of biological responses in platelets. In: W

Wm Meyer and Manehe. (edS).
1989, pp. 59-92.

Gentry PA. The mammalian blood platelet: Its role in haemostasis,
inflammation and tissue repair. J Comp Path 107:243-270, 1992.

White JG, Gerrard JM. Anatomy and structural organization of the platelet.

In: W Colman Hirsh Marder, Salzman (e48)
Philadelphia, PA: I. B. Lippencott Co. 1982, pp. 343-363.

White JG. Interaction of membrane systems in blood platelets. Am J Path
66:295-312, 1972.

Brass LF. Ca“ homeostasis in unstimulated platelets. J Biol Chem
259:12563-12570, 1984.

Zucker-Franklin D, Benson KA, Meyers KM. Absence of a surface-connected
canalicular system in bovine platelets. Blood 65:241-244, 1985.

Phillips DR, Parise LV, Fitzgerald LA, Fox JE. Calcium regulation of
platelet membrane and cytoskeletal proteins. In. W
W Oates, Hawiger, Ross (eds). Bethesda, MD. American
Physiological Society, 1985, pp. 21-33.

Cohen 1. Contractile Platelet Proteins. In: W
Colman, Hirsh, Marder, Salzman (eds). Philadelphia, PA:J.B. Lippencott Co.
1982, pp. 459-471.

Handagarna PJ, Shuman MA, Bainton DE. The origin of platelet tat-granule
' 1‘1 '31 ' -, ‘._ , , U, , . .

 

Wiley-Liss, Inc., 1990,pp. 119-130.

Harrison P, Savidge GF, Cramer EM. The origin and physiological relevance
of alpha granule adhesive proteins. Brit J Haematol 74:125-130, 1990.

White JG. Delivery of platelets from megakaryocytes. In: Molecular
WW Wiley- Liss Inc 1990 PP
25.-39

24.

26.

27.

28.

29.

30.

31.

32.

33.

35.

36.

147

Levin J. Thrombopoiesis. In: W Colman, Hirsh,
Marder, Salzman (eds). Philadelphia, PA:J.B. Lippencott Co. 1982, pp. 225-
236.

Meyers KM. Pathobiology of animal platelets. Adv in Vet Sci and Comp
Med 30:131-165, 1985.

Hawiger J, Kloczewiak M, Timmons S. Receptor-mediated activation of

platelets. In: WWW]. Oates. Hawiger.
Ross (eds). Bethesda, MD: American Physiological Society, 1985, pp. 1-19.

Hantgan RR, Hindriks G, Taylor RG, Sixma JJ, de Groot PG. Glycoprotein
Ib, vonWillebrand factor, and glycoprotein IIb:IIIa are all involved in platelet
adhesion to fibrin in flowing whole blood. Blood 76:345-353, 1990.

Furman MI, Gardner TM, Goldschmidt-Clermont PJ. Mechanisms of
cytoskeletal reorganization during platelet activation. Thromb and Haemost
70:229-232, 1993.

Marcus 41- Platelet aggregation. In: Hemortasiundflmmhcsis. Colman.
Hirsh, Marder, Salzman (eds). Philadelphia, PA:J.B. Lippencott Co. 1982,
pp. 380-389.

Weiss HJ, Turitto VT, Baumgartner HR. Further evidence that glycoprotein
IIb-IIIa mediates platelet spreading on subendothelium. Thromb and Haemost
65:202-205, 1991.

Holmsen H. Platelet secretion. In: W Colman,
Hirsh, Marder, Salzman (eds). Philadelphia, PA:J.B. Lippencott Co. 1982,
pp. 390-403.

Seiss W. Molecular mechanisms of platelet activation. Physiol Rev 69:5 8-
178, 1989.

Kroll MH, Schafer AI. Biochemical mechanisms of platelet activation. Blood
74:1181-1195, 1989.

Sage SO, Reast R, Rink TJ. ADP evokes biphasic Ca“ influx in fura-2-
loaded human platelets. Biochem J 265 :675-680, 1990.

Jefferson JR, Harmon JT, Jamieson GA. Identification of high-affinity (K.
0.35 pmollL) and low-affinity (K, 7.9 pmollL) platelet binding sites for ADP
and competition by ADP analogues. Blood 71:110-116, 1988.

Lips JPM, Sixma JJ, Schiphorst ME. Binding of adenosine diphophate to
human blood platelets and to isolated blood platelet membranes. Biochim et
Biophys Acta 628:451-467, 1980.

37.

38.

39.

40.

41.

42.

43.

45.

46.

47.

48.

49.

148

Colman RW. Platelet activation: Role of an ADP receptor. Seminars in
Hematology 23:119-128, 1986.

Colman RW. Aggregin: a platelet ADP receptor that mediates activation.
FASEB J 4:1425-1435, 1990.

Colman RW, Puri RN, Zhou F, Colman RF. Aggregin: The platelet receptor
mediating activation by ADP. In: We:

WWW AlanR Liss, Ine(ed8).
1988, pp. 263-277.

White JG, Rao GHR, Gerrard JM. Effects of the ionophore A23187 on blood
platelets. Influence on activation and secretion. Am J Pathol 77: 135-150,
1974.

Roth GJ. Platelet arachidonate metabolism and platelet-activating factor. In

Bi ' f Pl 1 Phillips, Shuman (eds). Orlando, FL: Academic
Press, Inc. 1986, pp. 69-115.

Lapetina EG. Platelet-activating factor stimulates the phosphatidylinositol
cycle. J Biol Chem 257:7314-7317, 1982.

Murphy CT, Elmore M, Kellie S, Westwick J. The relationship between
cytosolic Ca**, sn-1,2-diacylg1ycerol and inositol 1,4,5-trisphosphate elevation
in platelet-activating-factor-stimulated rabbit platelets. Biochem J 278:255-
261 , 1991 .

Coughlin SR. Thrombin receptor structure and function. Thromb and
Haemost 70:184-187, 1993.

Brass LF, Hoxie JA. Signaling through G proteins and G protein-coupled
receptors during platelet activation. Thromb and Haemost 70:217-223, 1993.

Crouch MF, Lapetina EG. A role for G, in control of the thrombin receptor-
phospholipase C coupling in human platelets. J Biol Chem 263:3363-3371 ,
1988.

Lapetina EG. The signal transduction induced by thrombin in human platelets.
FEBS 268:400-404, 1990.

Hashimoto Y, Ogihara A, Nakanishi S, Matsuda Y, Kurokawa K, Nonomura
Y. Two thrombin-activated Ca* * channels in human platelets. J Biol Chem
267: 17078-17081, 1992.

Font J, Azula FJ, Marino A, Nieva N, Trueba M, Macarulla JM. Intracellular
Ca* * mobilization and, not calcium influx promotes phobol ester-stimulated

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

61.

149

thromboxane A, synthesis in human platelets. Prostaglandins 43:383-395,
1992.

Shattil SJ, Bennett J S. Platelets and their membranes in hemostasis:
Physiology and pathOphysiology. Annals of Int Med 94: 108-118, 1980.

Clementi E, Scheer H, Zacchetti D, Fasolato C, Poznan T, Meldolesi J.
Receptor-activated Ca‘M influx. J Biol Chem 267:2164-2172, 1992.

Clemetson KJ. Biochemistry of platelet membrane glycoproteins. In: Platelet
u' 1211’ {avg-10 oa' tub-)1 icit‘ 11 ll 0 014 it. t'u as -l'

Pamelggy. New York, Alan R. Liss, Inc. 1988, pp. 33-75.

Ginsberg MH, Xiaoping D, O’Toole TE, Loftus JC, Plow EF. Platelet
Integrins. Thromb and Haemost 70:87-98, 1993.

Shattil SJ . Regulation of platelet anchorage and signaling by integrin a153,.
Thromb and Haemost 70:224-228, 1993.

Lewis JC, Hantgan RR, Stevenson SC, Thomburg T, Kieffer N, Guichard J,
Breton-Gorius J. Fibrinogen and glycoprotein IIb/IIIa localization during
platelet adhesion. Amer J of Path 136:239-252, 1990.

Puszkin EG, Mauss EA, Zucker MB. Assembly and gpIIIa content of
cytoskeletal core in platelets agglutinated with bovine von Willebrand factor.
Blood 76: 1572-1579, 1990.

Sims PJ, Ginsberg MH, Plow EF, Shattil SJ. Effect of platelet activation on
the conformation of the plasma membrane glycoprotein IIb-IIIa complex. J
Biol Chem 266:7345-7352, 1991.

Weasel-drake JD. Plasma membrane gpIIb/IIIa. Evidence for a cycling
receptor pool. Amer J of Path 136:61-70, 1990.

Nurden AT, George JN, Phillips DR. Platelet membrane glycoproteins:
Their structure, function, and modification 1n disease. In: W
Platelets, Phillips, Shuman (eds). Orlando, FL: Academic Press, Inc. 1986,
pp. 160-224.

Peterson DM, Stathopoulos NA, Giorgio TD, Hellums JD, Moake JL. Shear-
induced platelet aggregation requires von Willebrand factor and platelet
membrane glycoproteins Ib and IIb-IIIa. Blood 69:625-628, 1987.

Roth GJ . Developing relationships: Arterial platelet adhesion, glycoprotein
Ib, and leucine-rich glycoproteins. Blood 77:5-19, 1991.

62.

63.

67.

68.

69.

70.

71.

73.

74.

75.

150

Rink TJ, Sage SO. Calcium signaling in human platelets. Annu Rev Physiol
52:431-439, 1990.

Detweiler TC, Charo IF, Feinman RD. Evidence that calcium regulates
platelet function. Thromb and Haemostas 40:207-211, 1978.

Campbell AJ. Calcium and cell movement. In: W
W19; John Wiley & Sons Ltd, 1983, pp. 206-256.

Gerrard JM, Peterson DA, White JG. Calcium mobilization. In: Blateletfln
WM Gordon (ed) Elsevier/North-Holland Biomedical
Press, 1981, pp. 407-436.

Brass LF. Ca“ transport across the platelet plasma membrane. J of Biol
Chem 260:2231-2236, 1985.

Rink TJ, Smith SW, Tsien RY. Cytoplasmic free Ca“ in human platelets:
Ca“ thresholds and Ca-independent activation for shape-change and secretion.
FEBS Letters 148:21-26, 1982.

Heemskerk JWM, Vis P, Feijge MAH, Hoyland J, Mason WT, Sage SO.
Roles of phospholipase C and Ca**-ATPase in calcium responses of single,
fibrinogen-bound platelets. J Biol Chem 268:356-363, 1993.

Brass LF, Shattil SJ. Changes in surface-bound and exchangeable calcium
during platelet activation. J of Biol Chem 257: 14000-14005, 1982.

Meyer T, Wensel T, Stryer L. Kinetics of calcium channel opening by
inositol 1,4,5-trisphosphate. Biochemistry 29:32-37, 1990.

Alonso MT, Sanchez A, Garcia-Sancho J. Monitoring of the activation of
receptor-operated calcium channels in human platelets. Biochem Biophys REs
Comm 162:24-29, 1989.

Milani D, Malgaroli A, Guidolin D, Fasolato c, Skaper so, Meldolesi J,
Pozzan T. Ca“ channels and intracellular Ca“ stores in neuronal and
neuroendocrine cells. Cell Calcium 11:191-199, 1990.

Brown AM. A cellular logic for G protein-coupled ion channel pathways.
FASEB J 5:2175-2179, 1991.

Brown AM. Ionic channels and their regulation by G protein subunits. Annu
Rev Physiol 52:197-213, 1990.

Feinstein MB, Walenga R. The role of calcium m platelet activation. In:

mmmmmefletgmgfims AlanR Liss Inc (edS).1981
pp. 279-293.

76.

78.

79.

80.

81.

82.

83.

86.

87.

88.

151

Finch EA, Turner TJ, Goldin SM. Calcium as a coagonist of inositol 1,4,5-
trisphosphate-induced calcium release. Science 252:443-446, 1991.

Irvine RF. Inositol phophates and Ca* * entry: toward a proliferation or a
simplification? FASEB J 6:3085-3091, 1992.

Brass LF, Joseph SK. A role for inositol triphosplate in intracellular Ca**
mobilization and granule secretion in platelets. J our of Biol Chem
260:15172-15179, 1985.

Rink TJ. Cytosolic calcium in platelet activation. Experientia 44:97-100,
1988.

Gerrard JM, Peterson D. A. and White J. G. Calcium mobilization. In:

Womanizer-hm Gordon (ed) Elsevier/North-Holland
Biomedical Press, 1981, pp. 407-436.

Alonso MT, Allvarez J, Montero M, Sanchez A, Garcia-Sancho J. Agonist-
induced Ca* * influx into human platelets is secondary to the emptying of
intracellular Ca** stores. Biochem J 280:783-789, 1991.

Bosia A, Losche W, Pannocchia A, Treves S, Ghigo D, Till U, charmona
G. Regulation of arachidonic acid-dependent Ca** influx in human platelets.
Thromb and Haemost 59:86-92, 1988.

Ware JA, Johnson PC, Smith M, Salzman EW. Effect of common agonists on
cytoplasmic ionized calcium concentration in platelets. J Clin Invest 77:87 8-
886, 1986.

Pollock WK, Rink TJ. Thrombin and ionomycin can raise platelet cytosolic
Ca** stores: studies using fura-2. Biochem Biophys Res Comm 139:308-
314, 1986.

Sage SO, Rink TJ. Kinetic differences between thrombin-induced and ADP-
induwd calcium influx and release from internal stores in fura-2-loaded human
platelets. Biochem Biophys Res Comm 136:1124-1129, 1986.

Rink TJ. Receptor-mediated calcium entry. FEBS 268:381-385, 1990.

Hallman TJ, Rink TJ. Agonists stimulate divalent cation channels in the
plasma membrane of human platelets. FEBS 186:175-179, 1985.

Lecompte T, Potevin F, Champeix P, Morel MC, Favier R, Hurtaud MF,
Schiegel N, Samama M, Kaplan C. Aequorin-detected calcium changes in
stimulated thrombasthenic platelets. Aggregation-dependent calcium movement
in response to ADP. Thromb Res 58:561-570, 1990.

89.

91.

93.

94.

95.

96.

98.

99.

100.

101.

152

Powling MJ, Hardisty RM. Glycoprotein IIb-IIIa complex and Ca“ influx
into stimulated platelets. Blood 66:731-734, 1985.

Yamaguchi A, Yamamoto N, Kitagawa H, Tanoue K, Yammki H. Ca**
influx mediated through the gpIIb/IIIa complex during platelet activation.
FEBS 225:228-232, 1987.

Shattil SJ, Brass LP. Extracellular and surface-bound calcium in human

Platelet function. In: Interacdoacfplateletssdthshuesselxall. Oates,
Hawiger, Ross (eds). Bethesda, MD: American Physiological Society, 1985,

pp. 35-46.

Robblee LS, Shepro D. The efl‘ect of external calcium and lanthanum on
platelet calcium content and on the release reaction. Biochim et Biophys Acta
436:448-459, 1976.

Ryu SH, Lee SY, Lee K, Rhee SG. Catalytic properties of inositol
trisphosphate kinase: activation by Ca** and calmodulin. FASEB J 1:388-
393, 1987.

Connolly TM, Bansal VS, Bross TE, Irvine RF, Majerus RW. The
metabolism of tris- and tetraphosphates of inositol by 5-phosphomonoesterase
and 3-kinase enzymes. J Biol Chem 262:2146-2149, 1987.

Freissmuth M, Casey PJ, Gilman AG. G proteins control diverse pathways of
transmembrane signaling. FASEB J 3:2125-2131, 1989.

Nishizuka Y. Turnover of inositol phospholipids and signal transduction.
Science 225:1365-1370, 1984.

Williams J A, Ashby B, Daniel JL. Ligands to the platelet fibrinogen receptor
glycoprotein IIb-IIIa do not affect agonist-induced second messengers Ca** or
cyclic AMP. Biochem J 270: 149-155, 1990.

Sage SO, Heemskerk JWM. Thromboxane receptor stimulation inhibits
adenylate cyclase and reduces cyclic AMP-mediated inhibition of ADP-evoked
responses in fura-2-loaded human platelets. FEBS 298: 199-202, 1992.

Vezza R, Roberti R, Nenci GG, Gresele P. Prostaglandin E, potentiatcs
platelet aggregation by priming protein kinase C. Blood 82:2704-2713, 1993.

George JN, Shattil SJ. The clinical importance of acquired abnormalities of
platelet function. New Eng J Med 324:27-39, 1991.

Searcy G. Platelet disorders. In: W Thomson EG,
(ed)., 1988, pp. 291-297.

102.

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

153

Weiss HJ. Antiplatelet drugs: Pharmacologic aspects. In: PlateletL

Eathcrzhrtsielogunianfinlatelemgrmeranx. NewYorhNY: AlanR Liss
Inc., 1983, pp. 45-73.

Nurden AT, Pico M, Heilmann E, Jallu V, Hourdille P. Inherited disorders

of Platelets and megakaryocytee In: WWW
W 333-346,1990. Wiley-Liss, Inc.

White JG. Structural defects m inherited and giant platelet disorders. In:
W 133-234 1990

White JG. Inherited abnormalities of the platelet membrane and secretory
granules. Human Path 18:123-139, 1987.

Bell TG, Meyers KM, Prieur DJ, Fauci AS, Wolfe SM, Padgett GA.
Decreased nucleotide and serotonin storage associated with defective function
in Chediak-Higashi syndrome cattle and human platelets. Blood 48: 175-184,
1976.

Bell TG. Dissertation: The function of storage pool deficient platelets in
primary hemostasis of Chediak-Higashi syndrome animals. Washington State
University, 1976.

Menard M, Meyers KM, Prieur DJ. Primary and secondary lysosomes in
megakaryocytes and platelets from cattle with the Chediak-Higashi syndrome.
Thromb and Haemost 64:156-160, 1990.

Meyers K, Seachord C. Identification of dense granule specific membrane
proteins in bovine platelets that are absent in the Chediak-Higashi Syndrome.
Thromb and Haemost 64:319-325, 1990.

Thomas J S. Dissertation: Basset hound hereditary thrombopathy:
Measurement of platelet cytosolic calcium, pH and 1,2-diacylglycerol
production. Michigan State University, 1991.

McConnell MF. Dissertation: Functional and biochemical abnormalities in
Basset hound hereditary thrombopathy. Michigan State University, 1989.

Patterson WR, Estry DW, Schwartz KA, Borchert RD, Bell TG. Absent
platelet aggregation with normal fibrinogen binding in Basset hound hereditary
thrombopathy. Thromb and Haemost 62:1011-1015, 1989.

Patterson WR, Kunicki TJ, Bell TG. Two—dimensional electrophoretic studies
of platelets from dogs affected with Basset hound hereditary thrombopathy:
A thrombastheia-like aggregation defect. Thromb Res 42: 195-203, 1986.

114.

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

154

Cattaneo M, Lecchi A, Randi AM, Mcgregor JL, Mannucci PM.
Identification of a new congenital defect of platelet function chracterized by
severe impairment of platelet responses to adenosine diphosphate. Blood
80:2787-2796, 1992.

Parker RI, Bray GL, McKeown LP, White JG. Failure to mobilize
intracellular calcium in response to thrombin in a patient with familial
thrombocytopathy characterized by macrothrombocytopenia and abnormal
platelet membrane complexes. J Lab Clin Med 122:441-449, 1993.

Bondy GS, Gentry PA. Characterization of the normal bovine platelet
aggregation response. Comp Biochem Physiol 92C:67-72, 1989.

Gentry PA, Trombay RRM, Ross ML. Failure of aspirin to impair bovine
platelet function. Am J Vet Res 50:919-922, 1989.

Taylor RG, Christiansen J A, Goll DE. Immunolocalization of the calpains and
calpastatin in human and bovine platelets. Biomed Biochim Acta 50:491-498,
1991. '

Clemmons RM, Bliss EL, Dorsey-Lee MR, Seachord CL, Meyers KM.
Platelet function, size and yield in whole blood and in platelet-rich plasma
prepared using differing centrifugation force and time in domestic and food-
producing animals. Thromb and Haemost 50:838-843, 1983.

Marzee U, Johnston GG, Bernstein EF. Platelet function in calves: A study
of adhesion, aggregation, release, clotting factor activity and life span. Trans
Amer Soc Artif Int Organs 21:581-585, 1975.

Meyers KM, Katz JB, Clemmons RM, Smith JB, Holmsen H. An evaluation
of the arachidonate pathway of platelets from companion and food-producing
animals, mink, and man. Thromb Res 20:13-24, 1990.

Morimoto T, Ogihara S, Takisawa H. Anchorage of secretion-competent
dense granules on the plasma membrane of bovine platelets in the absence of
secretory stimulation. J of Cell Biol 111:79-86, 1990.

White JG. The secretory pathway of bovine platelets. Blood 69:878-885,
1987 .

Liggett HD, Leid RW, Huston L. Aggregation of bovine platelets by acctyl
glyceryl ether phosphorylcholine (platelet activating factor). Vet Immun and
Immunopath 7:81-87, 1984.

Genu'y PA, Bondy GS. The aggregation of bovine platelets is not dependent
on thromboxane B2 production. Thromb and Haemost 58:460, 1987.

126.

127.

128.

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

155

Grouse LH, Rao GHR, Weiss DJ, Perman V, White JG. Surface-activated
bovine platelets do not spread, they unfold. Amer J Path 136:399-408, 1990.

Steficek BA, Thomas JS, Bell TG, McConnell MP. A primary platelet
disorder of consanguineous Simmental cattle. Thromb Res 72: 145-
153, 1993.

Mattson J C, Borgerding PJ, Craft DL. Fixation of platelets for scanning and
transmission electron microscopy. Stain Technology 52:151-158, 1977.

Miale, J .B. Appendix-methods and quality control. In:
hematglggx. 5th ed. St. Louis,MO:C.V. Mosby Co., 1977, pp. 1061-1062.

Faires LM. Inductively coupled plasma: Principles and horizons. American
Laboratory November, 1982. pp. 18-22.

Laemmli UK. Cleavage of structural proteins during the assembly of the head
of bacteriophage T4. Nature 227:680-685, 1970.

Borle AB. An overview of techniques for the measurement of calcium
distribution, calcium fluxes, and cytosolic free calicum in mammalian cells.
Env Health Perspec 84:45-56, 1990.

Cobbold PH, Rink TJ . Fluorescence and bioluminescence measurement of
cyt0plasmic free calcium. Biochem J 248:313-328, 1987.

Daniel JL,Se1ak MA, Purdon AD, Salganicoff L. Methods for the study of
the role of calcium 1n platelet function. Methods for Studying Platelets and

Megakaryocytes . In WW” AlanR Liss
Inc., 1987, pp. 185-215.

Johnson PC, Ware JA, Cliveden PB, Smith M, Dvorak AM, Salzman EW.
Measurement of ionized calicum in blood platelets with the photoprotein
aequorin. J of Biol Chem 260:2069-2076, 1985 .

Johnson PC, Ware J A, Salzman EW. Concurrent measurement of platelet
ionized calcium concentration and aggregation: Studies with the
lumiaggregometer. Thromb Res 40:435-443, 1985.

Malmgren R, Grunfelt s, Joseph R. On the significance of different aequorin
loading techniques on intracellular aequorin discharge and baseline calcium,
platelet aggregation and aequorin-indicated Ca* *-transients. Thromb and
Haemost 68:352-356, 1992.

Lages B, Weiss HJ. Time dependence of aequorin-indicated calcium levels in
stimulated and unstimulated platelets. Thromb and Haemost 62: 1094-1099,
1989.

139.

140.

141.

142.

143.

144.

145.

146.

147.

148.

149.

150.

156

Yamaguchi A, Suzuki H, Tanoue K, Yamazaki H. Simple method of aequorin
loading into platelets using dimethyl sulfoxide. Thromb Res 44: 165-174,
1986.

Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca“ indicators
with greatly improved fluorescence properties. J Biol Chem 260:3340-3350,
1985 .

Tsien RY, Rink TJ, Poenie M. Measurement of cytosolic free Ca** in
individual small cells using fluorescence microscopy with dual excitation
wavelengths. Cell Calcium 6:145-157, 1985.

Owen CS. Spectra of intracellular fura-2. Cell Calcium 12:385-393, 1991.

Ganz MB, Rasmussen J, Bollag WB, Rasmussen H. Effect of buffer systems
and pI-L on the measurement of [Ca**]; with fura 2. FASEB J 4:1638-1644,
1990.

Lanza F, Beretz A, Kubina M, Cazenave J. Increased aggregation and
secretion responses of human platelets when loaded with the calcium
fluorescent probes quin 2 and fura 2. Thromb and Haemost 58:737-743,
1987.

Ozaki Y, Yatomi Y, Kume S. Evaluation of platelet calcium ion mobilization
by the use of various divalent ions. Cell Calcium 13:19-27, 1992.

Di Virgilio F, Fasolato C, Steinberg TH. Inhibitors of membrane transport
system for organic anions block fura-2 excretion from PC 12 and N2A cells.
Biochem J 256:959-963, 1988.

Sage SO, Rink TJ. The kinetics of changes in intracellular calcium
concentration in fura-2-loaded human platelets. J Biol Chem 262: 16364-
16369, 1987.

Mills, DBC. The mechanism of action of antiplatelet drugs. In: W
W Colman, Hirsh, Marder, Salzman (eds). Philadelphia,
PA:J.B. Lippencott Co. 1982, pp. 1058-1067.

Sage SO, Merritt JE, Hallam TJ, Rink TJ. Receptor-mediated calcium entry
in fura-2-loaded human platelets stimulated with ADP and thrombin. Biochem
J 258:923-926, 1989.

Pollock WK, Rink TJ, Irvine RF. Liberation of [’Hlarachidonic acid and
changes in cytosolic free caclium in fura-2-loaded human platelets stimulated
by ionomycin and collagen. Biochem J 234:869-877, 1986.

157

151. Cavalinni L, Francesconi MA, Ruzzene M, Valente M, Deana R. A
procedure allowing measurement of cytosolic Ca** in rat platelets. Inhibition
of a plasma lipoprotein on fura 2-AM loading. Thromb Res 63:47-57, 1991.