(Circulation. 1999;99:e1-e11.)
© 1999 American Heart Association, Inc.
Circulation Electronic Pages |
Evaluation of Platelet Membrane Glycoproteins in Coronary Artery Disease
Consequences for Diagnosis and Therapy
From 1 Medizinische Klinik, Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universität München, München, Germany.
Correspondence to Meinrad Gawaz, MD, 1 Medizinische Klinik, Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universität München, Lazarettstraße 36, 80636 München, Germany. E-mail gawaz{at}dhm.mhn.de
Key Words: platelets • glycoproteins • coronary disease • thrombosis • platelet aggregation inhibitors
Platelets and Arterial Thrombosis
Platelets play a fundamental role in atherogenesis and
development of ischemic complications.1 2 3 Under
physiological conditions, platelets do not
interact with the vessel wall. Injury of vascular intima disrupts the
antithrombotic properties of endothelium and exposes
the blood to adhesive molecules of the subendothelium.
Platelet adhesion to the damaged vessel wall is the first step in
hemostasis and thrombosis.4 Platelet adhesion is
followed by spreading and activation, resulting in release of
granule components and aggregate formation.5 6 On initial
contact, platelet glycoprotein (GP) Ib/V/IX complex
binds to von Willebrand factor associated with collagen on the
subendothelial surface (Figure 1
)4 5 and thus arrests the
platelet on the vessel surface. The collagen receptor
2ß1 is an important
secondary receptor for platelet adhesion.
2ß1-Collagen
interaction leads to platelet activation and is critical for the
spreading process involving the fibrinogen receptor GP IIb/IIIa to
ensure close contact of the spread platelet with the
surface.5 Other adhesion receptors, including the
fibronectin receptor
5ß1 and the laminin
receptor 6ß1, support
and strengthen secondary adhesion (Figure 1). The fibrinogen
receptor GP IIb/IIIa is particularly important in
platelet-platelet coadhesion, termed aggregation. This requires
conformational changes in GP IIb/IIIa that allow binding of soluble
fibrinogen to the platelet membrane (Figure 2). Thus, fibrinogen bridging allows
formation of platelet aggregates (Figure 2).6 7 8 9
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Platelet adhesion and aggregation induce intracellular signaling,
which mediates several responses, such as formation and secretion of
thromboxane A2 (TXA2),
serotonin, and ADP6 (Figure 1
). These
substances reinforce platelet activation, vasoconstriction, and
slowing of blood flow and therefore increase platelet-platelet
and platelet–vessel wall interaction.3 6
Platelet Membrane Receptors
The most abundant platelet membrane glycoprotein
is the ß3-integrin GP IIb/IIIa (60 000 to
100 000 per platelet and 1% to 2% of the total platelet
protein), the inducible platelet fibrinogen receptor.7
Eighty percent of GP IIb/IIIa is randomly distributed and expressed on
the platelet surface in its resting state, and the remaining 20%
is located within the surface connecting system (SCS) and in
-granule membranes5 10 (Figure 3). GP IIb/IIIa stored in this
internal pool becomes surface expressed as functional receptor on
platelet activation.11 Congenital deficiencies of GP
IIb/IIIa in Glanzmann thrombasthenia lead to defective
platelet aggregation and enhanced bleeding.5 As is the
case with other integrins, GP IIb/IIIa is a heterodimer consisting of
an -subunit (GP IIb) and a ß-subunit (GP IIIa)9
(Figure 4). Although the expression of GP
IIb/IIIa (IIbß3) is
limited to megakaryocytes and platelets, the other
ß3-integrin present on platelets, the
vitronectin receptor
vß3, is more widely
distributed and is also found on endothelial and smooth
muscle cells.12
vß3 shares a common
ß3-subunit with GP IIb/IIIa but is coupled with
a different -subunit.12 Thus, GP IIb/IIIa
antagonists that cross-react with
vß3 (eg, abciximab)
have potential
anti-vß3 activity that
might result in broader pharmacological effects.
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). C indicates
carboxy terminus; N, amino terminus.
GP IIb/IIIa on nonstimulated platelets cannot bind soluble,
adhesive, RGD-containing glycoproteins such as fibrinogen,
von Willebrand factor, vitronectin, or fibronectin
(low-affinity state) (Figure 5).9 Stimulation of
platelets with agonists7 induces signaling
transduction events followed by conformational change within GP
IIb/IIIa extracellular domains ("inside-out signaling") that allows
binding of fibrinogen (high-affinity state)9 (Figure 5). Distinct amino acid sequences such as RGD or KQAGDV, which
are present in the fibrinogen molecule, bind to specific regions of
the GP IIb/IIIa complex.9 13 RGD- and KQAGDV-containing
peptides compete with fibrinogen for a common binding
site14 (Figure 4). Under certain circumstances,
platelets can bind fibrinogen at near-normal affinity but fail to
aggregate. These observations suggest a role of "postoccupancy
receptor events" in aggregation. Postoccupancy receptor events
involve cytoskeletal anchorage and organization of GP IIb/IIIa within
the plasma membrane.9 15
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Activation-dependent binding of fibrinogen or activation-independent
binding of small fibrinogen mimetic peptides to GP IIb/IIIa results in
a further conformational change of GP IIb/IIIa (ligand-occupied state).
Thus, the RGD sequence in ligands functions both as part of the binding
site and as a trigger for secondary conformational changes leading to
expression of additional ligand-induced binding sites
(LIBS)9 16 (Figure 5). This leads to receptor
clustering and generation of transmembrane cell signaling
("outside-in signaling"), which results in transduction signaling
(eg, tyrosine phosphorylation), and irreversible
binding of fibrinogen (postoccupancy events) (Figure 5).12 16
In addition to the ß3-integrins
IIbß3 and
vß3, 3
ß1-integrin receptors have been identified to
date on platelets, including a receptor for collagen
(2ß1), fibronectin
(5ß1), and laminin
(6ß1)5
(Table 1). Two additional
nonintegrin receptors are also involved in platelet adhesion, the
leucine-rich glycoprotein GP Ib/IX/V (receptor for von
Willebrand factor) and GP IV (GP IIIb) as a receptor for
collagen and thrombospondin (Table 1).5 Congenital
deficiencies of GP Ib in Bernard-Soulier syndrome result in a
functional defect of platelet adhesion and in an increase in
abnormal bleeding.5
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P-selectin (GMP-140, PADGEM, cluster of determinants [CD]62P) is a
membrane glycoprotein located in the -granules of
platelets and in the Weibel-Palade bodies of
endothelial cells.5 Platelet
activation leads to its fusion with the SCS and expression on the
platelet membrane surface.5 P-selectin has been shown
to be the major surface receptor for neutrophils and monocytes on
thrombin-activated platelets.5 Its expression
may facilitate recruitment of leukocytes to sites of
thrombosis.4 Lysosomes contain another
glycoprotein, GP 53,5 that shares structural
similarity to lysosome integral membrane proteins (LIMPs)
(Table 1). Moreover, the platelet
surface expresses immunoglobulin-type adhesion molecules such as
platelet–endothelial cell adhesion molecule
(PECAM)-1 and intercellular adhesion molecule (ICAM)-2 (Table 1).5
Coronary Atherosclerosis
Platelet -granules contain a variety of
mitogenic growth factors, such as platelet-derived
growth factor (PDGF) and transforming growth factor
(TGF),6 which might cause migration and proliferation of
smooth muscle cells and thus intima proliferation at sites of enhanced
platelet activation.1
In patients with diabetes, platelet surface expression of P-selectin and microparticle formation were significantly increased.17 The alteration in platelet function in diabetes might result from primary release of larger platelets with enhanced thromboxane formation and increased surface density of platelet membrane glycoproteins.18 Hypercholesterolemia is associated with increased fibrinogen binding to activated platelets19 and an increased degranulation of P-selectin.20 Thus, coronary risk factors might induce early mechanisms of atherogenesis via enhanced systemic activation and degranulation of circulating platelets.
Unstable Angina
The pioneering studies of Willerson and colleagues3
showed that the conversion from stable to unstable angina is associated
with platelet aggregation at sites of vascular injury followed by
release of mediators that promote vasoconstriction and further
platelet aggregation. We found that surface expression of
P-selectin and of the activated GP IIb/IIIa complex (LIBS-1)
was significantly increased in patients with crescendo unstable angina
compared with those with stable angina.21 The alterations
in platelet membrane glycoproteins were associated with
increased platelet-neutrophil coaggregates and enhanced leukocyte
activation.21 Recently, we22 and
others23 showed that activated platelets
induce the expression and secretion of cytokines in leukocytes.
Activated platelets induce the oxidative burst in
neutrophils by a fibrinogen-mediated event.24 Furthermore,
activated platelets induce surface expression of ICAM-1,
interleukin (IL)-6, monocyte chemoattractant protein-1, and IL-8 in
cultured endothelial cells via CD40L-mediated
mechanisms.25 26 27 Moreover, we found that
activated platelets induce activation of the transcription
factor nuclear factor-
B (NF-B) in monocytes22 and in
endothelial cells26 through an
IL-1–mediated mechanism (Gawaz et al, unpublished data, 1997).
NF-B–regulated gene expression plays a role in systemic
inflammatory response.28 Recently, Liuzzo and coworkers
showed that patients with unstable angina and elevated systemic
C-reactive protein have a poor prognosis,29 whereas
aspirin treatment reduces systemic inflammation and improves
prognosis.30 Thus, platelets might be a promising
pharmacological target for anti-inflammatory treatment in acute
coronary syndromes.
Acute Myocardial Infarction and Reperfusion
Reocclusion of the recanalized infarct-related artery after fibrinolysis remains a serious limitation during the acute hospital phase.31 The therapeutic effect of fibrinolysis in acute myocardial infarction (AMI) seems to be limited by increased platelet activation and TXA2 biosynthesis. Thromboxane metabolite excretion is elevated in postinfarction angina, with increased risk for ischemic events.32
P-selectin expression is enhanced for days after AMI, which implies enhanced platelet activity.33 Activation of GP IIb/IIIa occurs within 72 hours after fibrinolysis despite treatment with aspirin and heparin.34 This coincides with an increased risk of thrombotic reocclusion of infarct-related vessels.31 We35 showed that fibrinogen receptor activity and P-selectin expression on circulating platelets in AMI decreased early (4 to 8 hours) after direct PTCA, coinciding with decreased peripheral platelet count and increased generation of microparticles. Fibrinogen receptor activity and P-selectin expression increased again 24 to 48 hours after PTCA. Enhanced surface expression of platelet adhesion receptors in the early postinfarction period also seems to influence platelet–endothelial cell adhesion, mediated through vitronectin receptor36 and P-selectin.37 Enhanced platelet–endothelial cell adhesion might contribute to impairment of microcirculation and compromise myocardial salvage during reperfusion.
Coronary Angioplasty and Stenting
In pioneering studies, Scharf and colleagues38 showed
that surface expression of platelet activation markers increases
during coronary angioplasty (Table 2). In coronary blood samples
obtained at the site of angioplasty, we found enhanced platelet
LIBS-1 expression.39 Platelet activation is enhanced
for days after coronary interventions.40 41
Nonionic as opposed to ionic contrast media causes profound
platelet degranulation,42 which indicates that the
type of contrast media modulates platelet function.
We43 44 showed that coronary stenting but not
conventional PTCA results in significant platelet activation
despite anticoagulation treatment with phenprocoumon and heparin in
combination with aspirin. In contrast, platelet fibrinogen receptor
activity was significantly reduced in patients treated with combined
antiplatelet therapy consisting of ticlopidine and aspirin, which
is associated with a reduced incidence of stent
thrombosis.44 In addition, we45 found that
combined antiplatelet therapy with ticlopidine plus aspirin is
superior in inhibiting platelet activation after stenting.
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Flow Cytometric Functional Assays
Flow cytometry of platelets in whole blood, as first
described by Shattil and colleagues,46 enables the
evaluation of platelet membrane glycoproteins and is
the current method of choice to study functional aspects of
platelets.47 Unlike other platelet activation
markers (PF4 [platelet factor-4],
ß-thromboglobulin [ßTG], and
TXA2), flow cytometry allows the detection of
specific activation-dependent modification in the platelet membrane
surface (Figure 6).
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GP IIb/IIIa
The availability of monoclonal antibodies (MAbs) enables us to
distinguish between the resting, activated, and ligand-occupied
forms of GP IIb/IIIa (Table 2) (Figures 6 and 7). The high-affinity state of
activated GP IIb/IIIa can be assessed by PAC-1, which
recognizes the ligand binding site in GP IIb/IIIa48
(Figure 7). The ligand-occupied state can be characterized by
MAbs specific for LIBS (LIBS epitopes; LIBS-1 or PMI-1) (Figure 7).49 These epitopes are not expressed by resting
GP IIb/IIIa or by activated GP IIb/IIIa not bound to ligand
(Figure 5). LIBS epitopes decorate both subunits of the
receptor, and 1 epitope (PMI-1) has been localized to the carboxy
terminus of the heavy chain of GP IIb9 (Figure 4).
The advantage of PAC-1 and LIBS antibodies is the detection of
activated platelets, stimulated by low doses of agonists
such as ADP, when secretion has not occurred.49 In
contrast to PAC-1, LIBS-1 binding to GP IIb/IIIa does not interfere
with the ligand binding pocket. Another approach to characterize
activated platelets is to quantify adhesive proteins bound
to activated GP IIb/IIIa50 51 by binding of
specific MAbs directed against the ligand. A more subtle way to detect
ligand binding is to use conformation-dependent antibodies, termed
anti-RIBS (receptor-induced binding site), which recognize exclusively
receptor-bound fibrinogen that has undergone conformational
change49 (Figures 5 and 7). Because GP
IIb/IIIa is a promiscuous receptor (Table 1), detecting bound
fibrinogen could underestimate the quantity of bound ligand if a
significant percentage of receptor is already occupied by other
ligands, such as von Willebrand factor. With the use of
fluorochrome-conjugated RGD-containing polypeptides such as FITC
echistatin, the accessibility of the fibrinogen binding site within the
GP IIb/IIIa complex can be evaluated (Figure 7).
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GP Ib/IX/V
In contrast to activation-dependent MAbs directed to GP IIb/IIIa,
binding of GP Ib–specific antibodies to thrombin-activated
platelets is decreased owing to internalization of receptor
complexes within the SCS (Figure 7).47 Thus,
enhanced in vivo activity of thrombin may be monitored by reduction in
surface expression of GP Ib.
Granula Markers
Binding of anti-thrombospondin and anti–P-selectin antibodies
specifically indicates release reaction of -granules; binding of
anti-GP 53 indicates primarily lysosome secretion (Figure 7) (Table 2).5
Platelet-Leukocyte Aggregates
Flow cytometric evaluation of a single platelet population
might underestimate platelet activation in vivo. Analysis
of circulating platelet-leukocyte aggregates (Figures 8 and 9)
detects activated platelets adhering to circulating
leukocytes.22 52 53 Determination of
platelet-leukocyte aggregates allows the study of inflammatory
aspects of platelet activation. Platelet adhesion to leukocytes
triggers activation mechanisms within the leukocytes22 23 24
that result in enhanced expression of adhesion receptors (eg, MAC-1) or
shedding of L-selectin on the activated leukocyte
surface.21
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Platelet Microvesicles
Platelets have been shown to shed microvesicles during
clotting of whole blood and after activation by thrombin, collagen, and
the complement protein C5b-9 (Figure 2).5
Microvesicles are particularly rich in anionic phospholipids, GP Ib, GP
IIb/IIIa, and receptors for coagulation factors Va, VIII(a), and
IXa.5 54 Thus, microvesicles provide a catalytic surface
for the transformation of prothrombin to thrombin. Flow cytometry is a
sensitive method to detect platelet microparticles and enables the
evaluation of platelet-coagulation
interaction.46 47
Genetic Analysis of Platelet Membrane Receptors
Little is known about genetic risk factors of platelet
membrane glycoproteins in arterial thrombotic
disease. Several platelet membrane glycoproteins are
known to be polymorphic, having 
2 allelic forms present in
the human gene pool.55 Restriction fragment length
analysis enables the study of polymorphisms of platelet
glycoproteins (Figure 10).
In addition, with allele-specific antibodies, polymorphism of
GP IIb/IIIa can be rapidly determined by flow cytometry (Figure 11).
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Weiss and colleagues56 demonstrated a high frequency of the PlA2 polymorphism of GP IIIa in patients who became symptomatic before the age of 60. However, the potential link between PlA2 and occurrence of acute coronary syndromes is controversial.57 Recently, Walter and coworkers58 found a strong association between subacute stent occlusion and the PlA2 allele after coronary stenting that was independent of classic risk factors such as reduced left ventricular function or residual dissection.
Platelet Analysis and Risk Stratification
Increased platelet reactivity correlates with an increase in mortality and ischemic heart disease. Spontaneous platelet aggregation in vitro predicts coronary events and mortality in survivors of AMI.59 Platelet hyperaggregability in the morning is associated with increased frequency of AMI and sudden cardiac death.60 Mean platelet volume is an independent risk factor for the development of recurrent acute coronary ischemic events in survivors of AMI.61
Tschoepe and colleagues41 found that an increased fraction
of platelets surface expressing P-selectin or thrombospondin is
predictive for occurrence of ischemic events after PTCA.
We43 62 showed that GP IIb/IIIa levels before stenting
were an independent risk factor for stent thrombosis (Figure 12).
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Monitoring of Antiplatelet Therapy
Because fibrinogen binding to activated platelets is the crucial step in platelet aggregation, several pharmacological agents have been developed to antagonize the binding of fibrinogen to GP IIb/IIIa.14 63 Abciximab (c7E3; ReoPro), a GP IIb/IIIa blocker, reduces ischemic events among patients undergoing PTCA.63 Administration of synthetic low-molecular-weight GP IIb/IIIa antagonists such as Integrelin or tirofiban, which might have advantages with respect to rapid reversibility of the antiplatelet effect and reduced immunogenicity,14 was less effective than administration of abciximab in reducing adverse cardiac events after PTCA.63 Thus, although the above-mentioned compounds are all very powerful GP IIb/IIIa blockers, the clinical efficacy might be greatly dependent on their pharmacological characteristics. Because GP IIb/IIIa blockers exhibit an extremely steep dose-response relationship and are characterized by variable individual response, it is necessary to monitor GP IIb/IIIa therapy to establish the optimum degree of platelet inhibition and thereby improve clinical efficacy and reduce the risk of bleeding.
In our laboratory, we use flow cytometric techniques and a
fluorescein-conjugated disintegrin, echistatin, to evaluate
receptor blockade after administration of GP IIb/IIIa
antagonists (Figure 7). Both abciximab and
Integrelin similarly block echistatin binding to circulating
platelets during drug administration (Figures 13 and 14). However, after termination of GP
IIb/IIIa antagonist infusion, echistatin binding was
rapidly restored within hours in Integrelin-treated patients, whereas
normalization of GP IIb/IIIa accessibility in patients receiving
abciximab required days (Figures 13 and 14).
Thus, evaluation of echistatin binding may be helpful in monitoring
long-acting (eg, abciximab) and short-acting (eg, Integrelin) GP
IIb/IIIa antagonists. In addition, binding of GP IIb/IIIa
inhibitors during or after administration can be easily
monitored by a competitive assay with
fluorescein-conjugated forms of the inhibitor
(Figure 14).
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In contrast to conventional platelet assays, flow cytometry enables
the study of the pharmacodynamic aspects of GP IIb/IIIa
antagonists. Binding of small peptide ligands to GP
IIb/IIIa produces a conformational change within the receptor molecule
resulting in expression of new epitopes, termed LIBS (Figure 5).
Du and coworkers64 found that RGD peptides induce
proaggregatory conformation of the GP IIb/IIIa complex.
Profound thrombocytopenia is one major side effect of GP IIb/IIIa
antagonists and occurs independently of the nature of the
antagonists (antibody or
synthetic).63 65 The
pathophysiological mechanisms are unclear but might
be related to expression of LIBS and thus "intrinsic" GP
IIb/IIIa–receptor binding activity ("outside-in
signaling").66 One might speculate that a GP IIb/IIIa
blocker–induced conformational change of GP IIb/IIIa and the
subsequent induction of postoccupancy events might alter the adhesive
properties of circulating platelets or that endogenous
LIBS-like antibodies bind to the LIBS-positive GP IIb/IIIa ligand
complex, thus contributing to immunotype thrombocytopenia. This might
particularly be involved during long-term anti-GP IIb/IIIa therapy. As
shown in Figure 15, there are
significant differences between GP IIb/IIIa antagonists in
inducing LIBS expression. Preclinical evaluation of ligand-induced
conformation-dependent epitopes (LIBS-1 and PMI-1) might be helpful in
disclosing differences in "intrinsic" activities of various GP
IIb/IIIa inhibitors and in development and design of new
antagonists (Figure 15).
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-chain (KQAGDV) peptide H12, abciximab, Integrelin, tirofiban, or
lamifiban for 30 minutes in the presence of MAb FITC-LIBS-1 or
FITC-PMI-1, respectively. Binding of LIBS-1 (top) and PMI-1 (bottom)
was quantified by platelet flow cytometry.
Acknowledgments
This study was supported by grants from the Deutsche Forschungsgemeinschaft (Ga 381/1-1 and 1-2; Ga 381/2-1; and Ne 540/1-2). We thank Drs Andreas Ruf, Karlheinz Peter, Melchior Seyfarth, and Silja Rüdiger for critical reading of the manuscript. The authors are grateful to Dr Mark Ginsberg for helpful discussions and for generously supporting us with material.
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