Platelet Protocols Research and Clinical Laboratory
Procedures
Visit the link below to download the full version of this book:
https://medipdf.com/product/platelet-protocols-research-and-clinical-laboratory-
procedures/
Click Download Now
This Page Intentionally Left Blank
PLATELET PROTOCOLS
RESEARCH AND CLINICAL
LABORATORY PROCEDURES
Melanie McCabe White, B.A.
and
Lisa K. Jennings, Ph.D.
Department of Medicine
University of Tennessee, Memphis
Selected Illustrations by Michael P. Condry
Academic Press
San Diego New York Boston
London Sydney Tokyo Toronto
This book is printed on acid-free paper. @
C o p y r i g h t 9 1999 by ACADEMIC PRESS
All Rights Reserved
No part of this publication may be reproduced or transmitted in any form or by any
means, electronic or mechanical, including photocop3~ recording, or any information
storage and retrieval system, without permission in writing from the Publisher.
Academic Press
a division of Harcourt Brace & Company
525 B Street, Suite 1900, San Diego, California 92101-4495
http://www.academicpress.com
Academic Press Limited
24-28 Oval Road, London NW1 7DX, UK
http://www.hbuk.co.uk / ap /
Library of Congress Catalog Card N u m b e r : 98-83126
PRINTED IN THE UNITED STATES OF AMERICA
99 00 01 02 03 04 EB 9 8 7 6 5 4 3 2 1
CONTENTS
Preface . . . . . . . .............................. ix
Acknowledgments .............................. xi
ONE BASICINTRODUCTION TO PLATELETS
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Platelets in Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Membrane Surface Proteins . . . . . . . . . . . . . . . . . . . . . . . . . 6
GPIb-IX-V . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
GPIIb-IIIa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
GPIV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
CD9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
Events Associated with Platelet Aggregation . . . . . . . . . . . . . . . 9
GPIIb-IIIa Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Platelet Cytoskeleton . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Storage Granules and the Release Reaction . . . . . . . . . . . . . . 15
Platelet Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Adenosine Diphosphate (ADP) . . . . . . . . . . . . . . . . . . . . . 17
Epinephrine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
Thrombin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Antibody Activation of Platelets . . . . . . . . . . . . . . . . . . . . 20
Coagulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
TWO LABORATORY EVALUATIONOF PLATELET FUNCTION
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Variables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Anticoagulants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Glass vs. Plastic Tubes . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Aggregometer Stir Speed . . . . . . . . . . . . . . . . . . . . . . . . 35
Platelet Count . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Fibrinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Hemolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
Red Blood Cell Contamination . . . . . . . . . . . . . . . . . . . . . 38
Lipemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Venipuncture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
V
vi Contents
Drawing and Processing Blood for Platelet Aggregation . . . . . . . . 39
Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Platelet-Rich Plasma Correction Formula . . . . . . . . . . . . . . . 40
Platelet-Rich Plasma Dilution Table . . . . . . . . . . . . . . . . . . 42
Platelet Aggregation and Secretion . . . . . . . . . . . . . . . . . . . . 43
Platelet Aggregation Method . . . . . . . . . . . . . . . . . . . . . . 44
Platelet Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Lumiaggregation: Method . . . . . . . . . . . . . . . . . . . . . . . 51
Other Methods Used for Measuring Granule Secretion . . . . . . . 52
Platelet Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Clot Retraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Whole Blood Clot Retraction . . . . . . . . . . . . . . . . . . . . . . 58
PRP Clot Retraction . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Platelet Adhesion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Flow Cytometric Analysis of Platelet Surface Proteins . . . . . . . . . 61
ReceptorOccupancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Receptor Occupancy Measurement . . . . . . . . . . . . . . . . . . 64
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
THREE FUNCTIONAL ABNORMALITIES OF PLATELETS
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Storage Pool Disorders (SPDs) and Release Defects . . . . . . . . . . . 70
Storage Pool Disorders . . . . . . . . . . . . . . . . . . . . . . . . . 71
Release Defects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
FOUR THERAPEUTIC APPROACHES TO INHIBITION
OF PLATELET FUNCTION
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
GPIIL-IIIa Antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Other Anti-Platelet Therapies . . . . . . . . . . . . . . . . . . . . . . . 84
Aspirin (ASA) and Nonsteroidal Antiinflammatory
Drugs (NSAIDs) . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
Ticlopidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
Drugs that May Affect Platelet Function . . . . . . . . . . . . . . . .. 86
Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Dipyridamole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Fibrinolytics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Dextran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Anesthetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Thrombin Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
Clinical Trials and Pharmacodynamic Measurements
of Platelet Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
Contents vi i
APPENDIX
Recipes for Citrate-Based Anticoagulants . . . . . . . . . . . . . . . . . 94
Inhibitors Used in Platelet Techniques . . . . . . . . . . . . . . . . . . 95
Cytoskeletal Preparation from PRP . . . . . . . . . . . . . . . . . . . . 95
Cytoskeleton Preparations from Washed Platelets . . . . . . . . . . . . 96
Preparing Gel-Filtered Platelets . . . . . . . . . . . . . . . . . . . . . . 97
Preparing Washed Platelets . . . . . . . . . . . . . . . . . . . . . . . . . 99
SDS Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Gel Recipes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Gel Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Separating Gel Solutions . . . . . . . . . . . . . . . . . . . . . . . . . 104
Electrophoresis Sample Buffers . . . . . . . . . . . . . . . . . . . . . 107
Western Blots (of Acrylamide Gels) . . . . . . . . . . . . . . . . . . . . 109
Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . 109
Western Blotting Using Chemiluminescence Substrates . . . . . . . 111
Staining of Platelets for Flow Cytometric Analysis . . . . . . . . . . . 112
Whole Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
PRP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
Sources of Aggregometers and Aggregation Reagents . . . . . . . . . 114
INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
This Page Intentionally Left Blank
PREFACE
Platelets are required for hemostasis and are key participants in
pathologic thrombosis. Individual platelets circulate in an un-
activated state. In response to vascular injury; platelets adhere
to the subendothelium, where adhesion and platelet activation
lead to formation of the thrombus. When hemostasis is initi-
ated by a pathologic event such as plaque rupture, thrombus
formation can lead to vasoclusion, ischemia, or infarction. A
growing body of knowledge in hemostasis and thrombosis
over the last twenty years has demanded a current handbook
on the evaluation of platelet function in both the basic research
and clinical laboratories.
In this book we have endeavored to provide a systematic
review of key points important in evaluation of platelet func-
tion and to outline the methods necessary for proper platelet
function testing. The first chapter is a basic introduction to
platelets. The major platelet membrane proteins, the events
associated with platelet aggregation, and the fundamental pla-
telet agonists are described. The second chapter outlines labo-
ratory evaluation of platelet function, including the variables
that can affect the platelet aggregation response, the proper
methods for collecting and processing blood for platelet aggre-
gation, methods for measuring platelet aggregation and secre-
tion, as well as other platelet function testing. Chapter three is
an introduction to the functional abnormalities of platelets.
This is not intended to serve as a comprehensive fund of
knowledge, but it does provide key information with regard to
abnormalities that can affect the platelet response. The final
chapter introduces anti-platelet drugs that have an effect on
x Preface
platelet function, including the newly developed ticlopidine
and clopidogrel, as well as the GPIIb-IIIa antagonists. A dis-
cussion of the pharmacodynamic effects of these drugs is in-
cluded along with the criteria for evaluating their effect on
platelet function.
This book will provide information for evaluation of platelet
function that is timely and logically organized. The text will be
useful to basic science investigators, laboratory staff, and clini-
cians who evaluate or manage patients in the area of thrombo-
sis and hemostasis. We also hope to satisfy the great new
demand for a text that concisely outlines the current protocols
for platelet function evaluation which has arisen with the rapid
development of the new anti-platelet therapies.
Lisa Kyle Jennings
Melanie McCabe White
ACKNOWLEDGMENTS
A sincere thank you to Dr. David R. Phillips for giving me the
opportunity to learn and value scientific inquiry.
L.K.J.
I would like to thank Dr. Mervyn A. Sahud for introducing
me to the wonders of platelets in 1972 and for his continued
mentorship through the ensuing thirteen years.
M.M.W.
This Page Intentionally Left Blank
~.J
.<
-I-
L.;
BASIC
INTRODUCTION
TO PLATELETS
Introduction
Platelets in Hemostasis
Membrane Surface Proteins
GPIb-IX-V
GPIIb-IIIa
GPIV
CD9
Events Associated with Platelet Aggregation
GPIIb-IIIa Signaling
Platelet Cytoskeleton
Storage Granules and the Release Reaction
Platelet Agonists
Adenosine Diphosphate (ADP)
Epinephrine
Collagen
Thrombin
Antibody Activation of Platelets
Coagulation
2 Platelet Protocols
INTRODUCTION
Platelets are anucleate cytoplasmic fragments derived from
human bone marrow megakaryocytes. They have a seminal
role in hemostasis and thrombosis. Human platelets in circula-
tion are discoid in morphology and have a relatively smooth
surface. The ultrastructure of the normal platelet reveals the
surface-connected canalicular system (sccs), which is a con-
tinuous membrane system extending from the plasma mem-
brane (pm) and the dense tubular system (dts) (Figure 1.1). The
plasma membrane contains many proteins that serve as recep-
tors for agonists that initiate platelet activation responses or for
adhesive molecules that mediate platelet adhesion and platelet
aggregation. The surface-connected canalicular system pro-
Figure 1.1. Transmission electron micrograph of normal resting
platelets (x15,000). pm, plasma membrane; dts, dense tubular sys-
tem; sccs, surface-connected canalicular system; d, dense granules; a,
alpha-granules.
Chapter 1. Basic Introduction to Platelets
vides a passage for plasma components as well as for vasoac-
tive substances that are released upon platelet activation. Plate-
lets contain several types of storage granules (d and a), the
contents of which are released upon platelet activation. The
dense tubular system has been shown to be the site from which
the calcium is released that triggers many of the calcium-de-
pendent activation enzymes and contractile events in the plate-
let activation response.
PLATELETS IN HEMOSTASIS
Damage to the vascular endothelium usually leads to exposure
of the subendothelial layer, which encourages adherence
of platelets through various receptors but primarily
through GP (glycoprotein) Ib-IX-V. Adherent platelets become
activated and release the contents of storage granules, recruit-
ing nearby platelets in circulation to form an aggregate (Figure
1.2). The formation of the platelet aggregate or thrombus
occurs via activation of GPIIb-IIIa and binding of multivalent
adhesive ligands, fibrinogen, or von Willebrand factor (vWF),
which crosslink the adjacent activated platelets (Figure 1.3). An
aggregate, as shown in Figure 1.3B, is an amorphous mass of
many platelets. The stronger the agonist, the larger the aggre-
gate size. When platelets are unactivated, the GPIIb-IIIa is in a
low affinity state and cannot bind soluble fibrinogen. Ligand
binding to activated GPIIb-IIIa is regulated by platelet activa-
tion. Upon platelet activation, GPIIb-IIIa undergoes a confor-
mational change that permits fibrinogen to bind. Fibrinogen
contains at least three platelet recognition sites: the Arg-Gly-
Asp-Phe (RGDF) and the Arg-Gly-Asp-Ser (RGDS) at Ac~,
and the dodecapeptide sequence His-His-Leu-Gly-Gly-Ala-
Lys-Gln-Ala-Gly-Asp-Val (HHLGGAKQAGDV) at the ~,400-
411 region of fibrinogen. Even though the fibrinogen molecule
has two RGDX sequences, fibrinogen binding to the platelet
appears to be primarily mediated through the dodecapeptide
sequence. Due to their small size, synthetic peptides of these
three sequences bind GPIIb-IIIa regardless of the GPIIb-IIIa
\'<'
U
D!
+
Figure 1.2. Platelet plug formation in response to vascular injury. Upon vessel injury, platelets adhere to
the exposed subendothelium. Platelet activation and platelet aggregation occur leading to thrombus
formation.
Chapter 1. Basic Introduction to Platelets
Figure 1.3. Transmission electron micrograph of resting discoid
platelets (A) and platelet aggregates (B). Note the presence of numer-
ous dense granules in the resting discoid platelets.
