Table Of ContentBiochemistry
of Platelets
Edited by
David R. Phillips
Gladstone Foundation Laboratories for Cardiovascular Disease,
Cardiovascular Research Institute,
and Department of Pathology
University of California, San Francisco
San Francisco, California
Marc A. Shuman
Department of Medicine, and Cancer Research Institute
University of California, San Francisco
San Francisco, California
1986
ACADEMIC PRESS, INC.
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LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA
Main entry under title:
Biochemistry of platelets.
Includes bibliographies and index.
1. Blood platelets. I. Phillips, David R.,
Date II. Shuman, Marc A. [DNLM: 1. Blood
Platelets —physiology. WH 300 B615]
QP97.B52 1986 612M17 85-11066
ISBN 0-12-553240-7 (alk. paper)
ISBN 0-12-553241-5 (paperback)
PRINTED IN THE UNITED STATES OF AMERICA
86 87 88 89 9 8 7 6 5 4 3 2 1
Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Dorothy F. Bainton (257), Department of Pathology, University of California
School of Medicine, San Francisco, California 94143
Thomas C. Detwiler (1), Department of Biochemistry, State University of New
York Downstate Medical Center, Brooklyn, New York 11203
Thomas F. Deuel (347), Department of Medicine, and Department of Biological
Chemistry, Washington University School of Medicine, The Jewish Hospi
tal of St. Louis, St. Louis, Missouri 63110
Joan Ε. B. Fox (115), Gladstone Foundation Laboratories for Cardiovascular
Disease, Cardiovascular Research Institute, and Department of Pathology,
University of California, San Francisco, San Francisco, California 94140
James N. George (159), Department of Medicine, University of Texas Health
Science Center at San Antonio, San Antonio, Texas 78284
Mark H. Ginsberg (225), Department of Immunology, Research Institute of
Scripps Clinic, La Jolla, California 92037
Charles S. Greenberg1 (319), Department of Medicine, and Cancer Research
Institute, University of California, San Francisco, San Francisco, California
94143
Evelyn Mei Huang (1), Department of Biochemistry, State University of New
York Downstate Medical Center, Brooklyn, New York 11203
Jung San Huang2 (347), Department of Medicine, and Department of Biological
Chemistry, Washington University School of Medicine, The Jewish Hospi
tal of St. Louis, St. Louis, Missouri 63110
Shuan Shian Huang (347), Department of Medicine, and Department of Biolog-
1 Present address: Department of Medicine, and Department of Pathology, Duke University Medi
cal Center, Post Office Box 3934-M, Durham, North Carolina 27710.
2Present address: Department of Biochemistry, St. Louis University Medical School, St. Louis,
Missouri 63104.
xi
xii CONTRIBUTORS
ical Chemistry, Washington University School of Medicine, The Jewish
Hospital of St. Louis, St. Louis, Missouri 63110
Richard F. Levine (417), Veterans Administration Medical Center, and George
Washington University, Washington, District of Columbia 20422
Shirley P. Levine (377), Department of Medicine, University of Texas Health
Science Center, and Audie L. Murphy Veterans Hospital, San Antonio,
Texas 78284
Kenneth G. Mann3 (295), Hematology Research Section, Mayo Clinic and Foun
dation, Rochester, Minnesota 55901
Gerard A. Marguerie4 (225), Unite 217 INSERM, Institut de Pathologie Cel-
lulaire, Hopital de Bicetre, Le Kremlin-Bicetre, France
Alan T. Nurden (159), Unite 150 INSERM, Hopital Lariboisiere, 75475 Paris,
France
David R. Phillips (159, 443), Gladstone Foundation Laboratories for Car
diovascular Disease, Cardiovascular Research Institute, and Department of
Pathology, University of California, San Francisco, San Francisco, Califor
nia 94140
Edward F. Plow (225), Department of Immunology, Research Institute of
Scripps Clinic, La Jolla, California 92037
Gerald J. Roth5 (69), Division of Hematology and Oncology, Department of
Medicine, University of Connecticut Health Center, Farmington, Connecti
cut 06032
Marc A. Shuman (319, 443), Department of Medicine, and Cancer Research
Institute, University of California, San Francisco, San Francisco, California
94143
Paula E. Stenberg (257), Department of Pathology, University of California
School of Medicine, San Francisco, California 94143
Paula B. Tracy6 (295), Department of Pathology, University of Rochester
School of Medicine and Dentistry, Rochester, New York 14642
3Present address: Department of Biochemistry, University of Vermont College of Medicine,
Burlington, Vermont 05405.
4Present address: Departement de Recherche Fondamentale, Laboratoire d'Hematologic, Unite
217 INSERM, Centre d'Etudes Nucleaires, 38041 Grenoble, France.
5Present address: Hematology Section, Seattle Veterans Administration Medical Center, Seattle,
Washington 98108.
6Present address: Department of Medicine and Department of Biochemistry, University of Ver
mont College of Medicine, Burlington, Vermont 05405
Preface
The molecular basis for the various biological processes in which platelets
participate has been elucidated to a greater extent in the last five years than at any
time previously. These studies have led to a remarkable increase in our under
standing of how platelets function. Thus, we have moved from phe-
nomenological observations to the molecular basis for complex biochemical
reactions involving platelets. There are numerous examples illustrating this evo
lution. For example, the initial observation made several years ago that
fibrinogen is required for platelet aggregation has been extended to identifica
tion, purification, and characterization of the fibrinogen receptor and its recon-
stitution in liposomes. Similarly, it had been known for several years that
platelets enhance activation of clotting, but the mechanism for this process had
not been elucidated. Recently, specific receptors for coagulation factors have
been identified on platelets; binding of clotting factors to these receptors is now
known to be required for normal hemostasis. At the subcellular level, initial
observations that Ca2 + flux is associated with platelet activation have now been
extended by the identification of mechanisms for translocation of Ca2 + into the
cytoplasm and by the demonstration that Ca2+ functions in conceit with phos
pholipid metabolites to exert its action. A final example that illustrates the
tremendous acceleration of our understanding of basic biochemical processes
underlying platelet function is platelet-derived growth factor (PDGF), which was
discovered more than a decade ago. Recent studies have identified the cellular
receptor for PDGF. In addition, the structure of PDGF has been shown to be
related to a tumor-transforming protein.
Because the various areas of platelet research are diverse and rapidly expand
ing, there is a real need for a source book to pull together the current, important
observations in this field. The purpose of this book is to present a comprehensive
and up-to-date review of the significant advances in our understanding of platelet
function. The book is intended to serve as a reference for investigators involved
in platelet research as well as a source of information for those working in other
areas of biological investigation.
xiii
1
Stimulus-Response Coupling
Mechanisms
EVELYN MEI HUANG and THOMAS C. DETWILER
Department of Biochemistry
State University of New York Downstate Medical Center
Brooklyn, New York
I. Introduction 1
A. Morphological Responses of Platelets to Agonists 2
B. Metabolic and Structural Changes in Response to Agonists 6
C. Platelet Agonists 7
II. Experimental Strategies for Definition of Mechanisms of Stimulus-
Response Coupling in Platelets 10
A. Establishing Cause-Effect Relationships 10
B. Feedback Mechanisms 11
III. Possible Coupling Mechanisms 12
A. Calcium 12
B. Protein Phosphorylation 19
C. Phosphoinositide Metabolism 25
D. Prostanoids 34
E. Cyclic Nucleotides 35
F. Other Mechanisms 42
IV. Integration of Coupling Mechanisms 48
References 50
I. Introduction
The essence of platelet function is their response to stimuli. On activation,
platelets undergo an impressive array of rather dramatic changes. They change
shape, aggregate, and secrete the contents of at least two types of secretory
granules. The morphological changes are accompanied by such diverse meta
bolic changes as calcium fluxes, protein phosphorylation, arachidonate oxygena
tion, increased energy metabolism, and many other less-well-defined processes.
There is a wide variety of platelet agonists, including such diverse agents as
BIOCHEMISTRY OF PLATELETS 1 Copyright © 1986 by Academic Press, Inc.
All rights of reproduction in any form reserved.
2 EVELYN MEI HUANG AND THOMAS C. DETWILER
thrombin (a protease), collagen (a structural protein), and platelet-activating
factor (a phospholipid), as well as many more that resist simple classification.
Most of these stimuli are presumed to act on specific membrane receptors, which
must in some way be modified or activated, but it is unclear as to what extent the
receptors are unique for each different agonist. All of the agonists are syn
ergistic, and some show specific down-regulation, whereby platelets become
refractory after reaction with low concentrations of agonists. The mechanisms of
synergism and of the apparent down-regulation are unknown.
The interactions of agonists with their surface receptors are presumably cou
pled to the various platelet responses through a series of intracellular second
messengers. In this chapter, we describe the morphological and metabolic re
sponses to agonists, and we review the evidence for the involvement of certain
processes in the coupling of agonist-receptor interactions to platelet responses.
The literature is far too extensive to permit a comprehensive review of each
aspect of this diverse subject, and many topics are covered in detail in other
chapters in this book. Our intention is therefore to survey the field, to discuss
problems and research strategies, and to attempt to give some insight into this
complex subject.
A. Morphological Responses of Platelets
to Agonists
The morphological responses of platelets to agonists can be observed best by
electron microscopic studies, which are outside the scope of this review. De
scriptions of the major changes, in the approximate order of occurence, follow.
A stimulated platelet undergoes a rapid change from a disc to a sphere with long
pseudopods. The platelets then aggregate and expel the contents of their a-
granules and dense granules. The aggregate, which is at first 4'loose" with
considerable extracellular space, subsequently retracts and consolidates into a
compact aggregate with essentially no extracellular space and little apparent
demarcation between individual platelets. At some point in this process, the
contents of lysosome-like granules are also released. This process of formation
of a tight aggregate at the site of vascular injury represents the primary hemo
static mechanism. The mutual support of platelet activation by coagulation en
zymes and of blood coagulation by the surface of platelets greatly complicates an
understanding of the exact physiological involvement of platelets in hemostasis,
and it is difficult to relate the primary hemostatic role of platelets to parameters
that can be measured readily in controlled, in vitro experiments. Work on stim
ulus-response coupling to date has almost exclusively concerned early events:
shape change, aggregation, secretion, and some metabolic and structural changes
described below.
1. STIMULUS-RESPONSE COUPLING MECHANISMS 3
7. In Vitro Measures of Morphological Responses
a. Shape Change
Shape change is an important platelet response in studies of stimulus-response
coupling because it is the most sensitive, it requires the least agonist, and it is the
most resistant to inhibition. Shape change is usually inferred from changes in
light scattering during the course of measurements of aggregation (see Fig. 1). If
shape change alone is studied, interference due to aggregation can be minimized
by measurement of right-angle scattered light rather than the more conventional
measurement of change in a straight-ahead light transmittance (Michal and Born,
1971).
b. Aggregation
The measurement of platelet aggregation has played a major role in develop
ment of our current understanding of platelet function. This is due in part to the
assumed primary relationship of in vitro aggregation to in vivo platelet function,
but it is also because aggregation can be measured so easily. In 1962, Born
(1962) and O'Brien (1962) described a simple photometric measurement of
platelet aggregation; when an agonist was added to a suspension of stirred
platelets, the light transmittance of the suspension increased as the platelets
aggregated. The value of this simple technique resulted in the consumption of
many miles of chart paper, thousands of publications, and major advances in our
understanding of how platelets respond to agonists. While there have been the
oretical studies of platelet aggregation (e.g., Frojmovic, 1973; Frojmovic and
Panjwani, 1975; Latimer et al., 1977), they have not led to a sufficiently clear-
cut (or simple) explanation of this phenomenon to have greatly affected the use
of aggregometry. It is still usually used as an empirical observation, and many
quantitative interpretations are, unfortunately, unjustified.
Several important aspects of the photometric measurement of aggregation are
illustrated in Fig. 1. With the proper concentration of many agonists, the increase
in light transmittance takes place in two distinct phases (MacMillan, 1966)
(biphasic aggregation). This phenomenon has been of considerable importance in
studies of stimulus-response coupling, because the second-phase aggregation
apparently represents the result of platelet regulatory processes that were trig
gered by incomplete activation by the initial agonist. Second-phase aggregation
is always accompanied by secretion of the contents of dense granules (and
probably α-granules) and by synthesis of prostanoids. Second-phase aggregation
(and secretion and prostanoid synthesis) is inhibited by aspirin, one of the few in
vitro functional effects of a drug that causes an impairment of primary hemo-
stasis in vivo. With a higher concentration of agonist, a full aggregation response
4 EVELYN ME1 HUANG AND THOMAS C. DETWILER
ι—LuUu—ι
Fig. 1. Photometric measurement of platelet shape change, aggregation, and dense granule
secretion. Suspensions of human platelets in citrated plasma were activated with different concentra
tions of ADP. Light scattering was recorded in the upper traces, and luminescence from the reaction
of released ATP with added luciferin and luciferase was recorded in the lower traces. Details of the
procedure have been described by Charo et al. (1977). Addition of ADP is indicated by the arrow.
For trace 1, 1 μΛί ADP caused shape change followed by primary, reversible aggregation. A slightly
higher concentration of ADP (2 μΜ) in trace 2 induced biphasic aggregation with secretion accom
panying the second phase. For trace 3, 10 μΜ ADP caused full aggregation in a single phase and
secretion identical to that in trace 2. Secondary aggregation is defined as that aggregation accom
panied by secretion. Cyclooxygenase inhibitors would have blocked secretion at either ADP con
centration (since secretion is aggregation-mediated with ADP, as shown in Fig. 2); inhibitors would
have blocked the second-phase aggregation in trace 2, but they would have had little if any effect on
the other traces.
can be observed even if prostanoid synthesis and secretion are inhibited by
aspirin, and it is difficult to determine by aggregometry alone whether secretion
has occurred. The terms primary and secondary (instead of first phase and
second phase) are thus used to refer more generally to aggregation alone and
aggregation with secretion. The important point for this discussion is that the
1. STIMULUS-RESPONSE COUPLING MECHANISMS 5
difference between primary and secondary aggregation is more qualitative than
quantitative. Even with biphasic aggregation, essentially all platelets have aggre
gated during the initial phase. The second phase apparently represents consolida
tion into larger and more dense aggregates (Born and Hume, 1967; Gear and
Lambrecht, 1981), rather than recruitment of more platelets.
c. Secretion
i. Dense Granule Secretion. Human platelet dense granules contain high
concentrations of calcium, 5-hydroxytryptamine (5-HT), ADP, ATP, and
pyrophosphate. These appear to be released simultaneously, presumably as a
package. While this is hard to establish unequivocally, there is no evidence of
selective release of dense granule constituents or of appreciably different time
courses of release of the different substances. Measurement of released 5-HT is
most often used to detect granule secretion. Since platelets actively take up 5-HT
and package it in dense granules, the granule pool of 5-HT is readily labeled with
radioactive 5-HT. Secretion can thus be quantified as the fraction of total radio
activity in the supernatant solution; controls of no agonist and of a maximal
agonist (e.g., thrombin or A23187) should be included. Inhibitors of reuptake
(e.g., imipramine) are frequently included, but caution is necessary, since these
inhibitors can also inhibit some of the regulatory steps being investigated. An
additional complication is that centrifugation may enhance secretion after partial
activation of the platelets (Holmsen and Setkowsky-Dangelmaier, 1977). Fixa
tion with formaldehyde effectively stops further secretion (Costa and Murphy,
1975), avoiding centrifugation-induced secretion and permitting analysis of the
time course of secretion.
There are several methods that permit the on-line, continuous monitoring of
dense granule secretion, with obvious advantages. Released calcium has been
monitored with a metallochromic indicator (Detwiler and Feinman, 1973a) or
with a calcium-sensitive electrode (Kornstein et al., 1977; Akkerman et al.,
1979). The indicator requires the use of a dual wavelength spectrophotometer
that is not usually available, while the electrode is less sensitive than desired.
The measurement of released ATP (Detwiler and Feinman, 1973b) by its lumi
nescent reaction with firefly luciferin and luciferase is considerably easier for
routine use. The necessary equipment is commercially available or easily con
structed (Charo et al., 1978). Advantages of this method include great sen
sitivity, a continuous trace instead of discrete measurements, immediate results
(without measuring radioactivity and the subsequent calculations required for
measurements of released 5-HT), and the avoidance of preloading with a radio
active tracer. The disadvantage is that additional reagents must be added to the
medium and the requirements for luciferase activity must be met (e.g., Mg2 +
must be present), limiting experimental flexibility.
