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40 Part I / Basic Principles
a variety of platelet functions including those involved in coagulation, inflammation, fibri-
nolysis, and vascular cell proliferation.
The function of GPIIb/IIIa in platelet aggregation is often initiated by inside-out
signaling events, which cause expression of the receptor function of GPIIb/IIIa for solu-
ble fibrinogen. On unstimulated platelets, GPIIb/IIIa has low affinity for soluble fibrino-
gen, i.e., it is in an “inactive” conformation. However, after stimulation of platelets by
a variety of agonists, agonists generated at sites of vascular injury such as occurs during
percutaneous interventions or upon rupture of an atherosclerotic plaque (i.e., thrombin,
ADP, fibrillar collagens), GPIIb/IIIa undergoes a rapid conformational change such that
it can now bind soluble fibrinogen. Upon binding to GPIIb/IIIa, the bivalent fibrinogen
as well as other adhesive proteins including fibronectin, vitronectin, and von Willebrand
factor (vWf) can further mediate platelet aggregation by crosslinking the surfaces of acti-
vated platelets via GPIIb/IIIa. The binding of vWf may have a unique role in mediating
aggregation since it is considered to be the key ligand mediating platelet aggregation under
conditions of high shear such as would occur in coronary arteries.
A second signaling reaction of GPIIb/IIIa is also involved in platelet aggregation.
When fibrinogen interacts with GPIIb/IIIa on the platelet surface, or otherwise becomes
immobilized on artificial surfaces, it undergoes a conformational change that allows it
to bind to “inactive” GPIIb/IIIa on unstimulated platelets. This binding results in “out-
side-in” signals being transduced via GPIIb/IIIa, which, in turn, can lead to further plate-
let activation including the activation of other GPIIb/IIIa molecules on the platelet surface,
the induction of platelet secretion, and the consolidation and stabilization of platelet aggre-
gates. These events also occur when fibrinogen is immobilized on the vessel wall. The
signaling pathway initiated by ligand binding to GPIIb/IIIa appears to involve the phos-
phorylation of two tyrosines on the cytoplasmic domain of GPIIIa (4,5). Proteins have
been identified that bind to tyrosine-phosphorylated GPIIIa and are currently being eval-
uated for their potential roles in GPIIb/IIIa signal transduction. These outside-in signal-
ing events including GPIIIa tyrosine phosphorylation are also involved in processes such
as clot retraction, in which GPIIb/IIIa serves as the transmembrane link between extra-
cellular fibrin and intracellular cytoskeletal proteins.
Recent studies have shown that GPIIb/IIIa, in addition to its role in platelet aggrega-
tion and thrombosis, also interacts with CD40L, thereby having a role in inflammation
(6). CD40L is an inflammatory mediator that has recently been identified on platelets. This
protein is shed during platelet stimulation, generating a soluble cleavage product termed
sCD40L. GPIIb/IIIa is involved in this process in two ways. First, cleavage can be blocked
by GPIIb/IIIa antagonists, indicating a role for GPIIb/IIIa in CD40L shedding and in the
generation of this proinflammatory product. Second, sCD40L also binds directly to GPIIb/
IIIa, promoting platelet thrombosis.
This chapter summarizes recent studies pertaining to the structure and function of GPIIb/
IIIa. The themes to be developed are twofold. First, GPIIb/IIIa is not a static receptor,
but one whose adhesive functions respond to, and initiate signal transduction processes
within the platelet and whose distribution changes during platelet stimulation. Second,
whereas the central role of GPIIb/IIIa in aggregation indicates that it is an attractive target
for effective therapeutic regulation of platelet aggregation and thrombosis, the role of
GPIIb/IIIa in the generation of inflammatory mediators suggests that GPIIb/IIIa antago-
nists may also function in the inhibition of inflammation.
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 41
GPIIb/IIIa IN THROMBOGENESIS
Perhaps the most well-studied function of GPIIb/IIIa is that of platelet aggregation.
These studies have resulted in the development of the central tenant of platelet biology:
platelet stimulation induces the activation of the receptor function of GPIIb/IIIa for fibrin-
ogen and vWF, the binding of which mediates platelet aggregation. However, recent data
now show that through secondary activities, GPIIb/IIIa is also required to stabilize plate-
let aggregates and arterial thrombi. Without these secondary events involving both direct
and indirect roles of GPIIb/IIIa, stable thrombosis does not occur. These observations
highlight new functions for GPIIb/IIIa in platelet biology and new considerations in the
use of GPIIb/IIIa antagonists for the treatment of acute coronary thrombosis.
GPIIb/IIIa in Platelet Aggregation
On unstimulated discoid platelets such as those that normally exist in the circulation,
GPIIb/IIIa is distributed among the plasma membrane (approx 80,000 copies per plate-
let), the α-granule membrane (approx 40,000 copies per platelet), and the dense body mem-
brane (trace amounts) (7,8). Only limited receptor functions have been attributed to GPIIb/
IIIa on the surface of unstimulated platelets. The primary one is to bind immobilized
fibrinogen—a reaction that may assist in the recruitment of platelets to damaged vessel
walls or to platelet aggregates (9). However, following platelet stimulation, which also may
involve α-granule secretion, there is an up to 50% increase in the GPIIb/IIIa expressed
on the surface membranes of platelets. The amount of GPIIb/IIIa recruited to the platelet
surface during platelet stimulation is a function of the strength of the platelet agonist.
Potent agonists like thrombin cause most of the α-granule GPIIb/IIIa to be recruited with
prebound fibrinogen to the platelet surface, whereas weak agonists like ADP only margi-
nally increase the platelet surface GPIIb/IIIa (10,11). The GPIIb/IIIa on the surface of
activated platelets becomes a receptor for the soluble forms of several adhesive proteins,
most prominantly fibrinogen and vWf. The affinity of fibrinogen for GPIIb/IIIa on stim-
ulated platelets (K
d
approx 500 nmol/L) and the high concentration of fibrinogen in plasma
(approx 11 µmol/L) predicts that GPIIb/IIIa will rapidly become saturated with fibrino-
gen upon platelet stimulation. The binding of fibrinogen (a bivalent ligand) and vWf (a
polyvalent ligand) to the GPIIb/IIIa on stimulated platelets is responsible for the initial
platelet aggregation.
GPIIb/IIIa also shuttles between the plasma membrane and the α-granule membrane,
which is independent of platelet stimulation (12). This shuttling allows for the packaging
of fibrinogen in α-granules during megakaryocytopoiesis (13) and for the uptake of GPIIb/
IIIa ligands in mature platelets (14). The function of GPIIb/IIIa shuttling in circulating
platelets could potentially allow for the uptake of GPIIb/IIIa antagonists during anti-
thrombotic therapy (15). Indeed, it has been demonstrated that abciximab accumulates
in α-granules within 3 h following the initiation of therapy, and as fast as 5 min in vitro,
suggesting a possible mechanism for localization of this antagonist during thrombosis (16).
It is not yet known whether eptifibatide or aggrastat also accumulate in α-granules. The
α-granule pool of GPIIb/IIIa is functionally important to platelet aggregation. Indeed,
thrombin stimulation of platelets in which the plasma membrane GPIIb/IIIa molecules
have been rendered inactive can still induce sufficient functional GPIIb/IIIa to the plate-
let surface to support platelet aggregation (17).
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42 Part I / Basic Principles
Clinical studies have shown that thrombin-induced, but not ADP-induced, platelet aggre-
gation is rapidly restored following discontinuation of infusion of high-affinity GPIIb/IIIa
antagonists like the Fab fragment, abciximab, or L-738,167, a synthetic GPIIb/IIIa antag-
onist (18,19). In these trials, the unbound pool of these antagonists, but not the platelet-
bound pool, is rapidly cleared. Since thrombin, but not ADP, induces maximal surface
expression of the α-granule pool of GPIIb/IIIa, thrombin-induced aggregation is more rap-
idly restored. P-selectin is another α-granule protein that becomes expressed on the sur-
face of activated platelets (20). FACS analysis has shown that thrombosis associated with
percutaneous intervention or unstable angina increases the amount of platelet-associated
P-selectin, implying that these indications increase the amount of activated platelets within
the circulation, and also providing an assay to measure such platelets (21).
GPIIb/IIIa in Thrombus Stability
In a simple aggregation reaction, such as that occurring with a stirred suspension of plate-
lets activated by agonists like as collagen, ADP, or thrombin in a cuvette during labora-
tory analysis, it is likely that fibrinogen, which is bivalent, bridges the surfaces of activated
platelets and is sufficient to stabilize platelet-platelet contacts (Fig. 1). Aggregation reac-
tions under flow such as that occurring during thrombosis and hemostasis within the vas-
culature are much more complicated, primarily because the effects of shear require the
involvement of additional factors both to induce thrombus growth and to stabilize plate-
let-platelet interactions. There are multiple pathways in which GPIIb/IIIa is involved in
thrombus stability, only four of which will be described.
First, the initial reversible binding of fibrinogen to GPIIb/IIIa becomes irreversible
(22). Although the mechanism of converting a reversible fibrinogen interaction to one
that is irreversible is not known, the lack of this conversion might explain the destabili-
zation of the arterial thrombi observed in the fibrinogen-deficient mice (23).
Second, GPIIb/IIIa outside-in signaling is partly induced by the tyrosine phosphory-
lation of the cytoplasmic domain of GP IIIa (see below). One consequence of outside-
in signaling during platelet aggregation is the release of autocrine factors from platelet
storage organelles, which are required to induce further platelet activation to stabilize
aggregates. Such factors include ADP and serotonin, two dense body constituents, and
Gas6, a protein released from α-granules. Although the roles of ADP and serotonin in
aggregate stability are well known, the function of Gas6 is new and was discovered by
observations showing that Gas6-deficient mice produce only loosely packed thrombi
(24). It is also of interest that the long form of the leptin receptor has been detected in
platelets, and high concentrations of leptin have been noted to potentiate platelet aggre-
gation (25,26). Moreover, it has been proposed that the conversion from the reversible
to the irreversible binding of fibrinogen to GPIIb/IIIa is facilitated by the factor XIIIa-
catalyzed coupling of secreted serotonin to fibrinogen on the platelet surface, thus per-
mitting the high-affinity binding of derivatized protein to serotonin receptors (27).
Third, sCD40L generated from stimulated platelets plays a cofactor role in the produc-
tion of stable arterial thrombi. GPIIb/IIIa is not only involved in the processing of CD40L
to sCD40L, it also serves as the sCD40L receptor (see below).
Fourth, the generation of thrombin on the surface of platelet aggregates not only pro-
vides an additional platelet stimulus, but also catalyzes the formation of fibrin, both of
which cause increased aggregate stability. The generation of the procoagulant surface is
another consequence of outside-in GPIIb/IIIa signaling.
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 43
More generally, aggregation and stabilization cofactors released from activated/aggre-
gated platelets will undoubtefully become a major center of scientific interest, as both
CD40L- and Gas6-deficient mice exhibit a normal bleeding time, even though they have
abnormal thrombosis. In addition, since serotonin is now believed to enhance the binding
of prothrombotic ligands on activated platelets (27), it is highly likely that other mole-
cules will emerge as key players of the aggregation and stability processes.
GPIIb/IIIa STRUCTURE
GPIIb/IIIa is a member of the integrin family of adhesion receptors. All integrins con-
sist of two subunits, termed α and β (Fig. 2). In mammals, the integrin family is quite large,
Fig. 1. Schematic illustrating the role of GPIIb/IIIa in the multiple steps leading to thrombus forma-
tion under high shear rate. On resting discoid platelets, the GPIIb/IIIa is present in an “inactive”
conformation and is unable to bind soluble fibrinogen. 1, Upon vessel wall injury, von Willebrand fac-
tor (vWF) binds to the exposed collagen and undergoes a conformational change that allows the plate-
let to adhere via an interaction with the GPIbα. Other receptors such as α
2
β
1
and GPVI participate
in platelet adhesion. GPIIb/IIIa becomes activated and mediates final spreading onto the thrombo-
genic surface. 2, The platelet undergoes a shape change, and the GPIIb/IIIa expressed on the luminal
side is activated such that it can now bind its soluble ligands, fibrinogen and vWf. 3, Those ligands
will now be able to sustain the recruitment of unactivated platelets (platelet aggregation) via GPIbα and
GPIIb/IIIa. 2', Activated platelets also release their granules contents and are procoagulant. Released
ADP and newly synthesized thrombin will be able to accentuate platelet recruitment further by acti-
vating other GPIIb/IIIa. 4, α-Granules containing intracellular GPIIb/IIIa are present in both resting
and stimulated platelets. The contents of the α-granules are released as a consequence of outside-in
signaling, resulting in the secretion of factors involved in inflammation, vascular remodeling, plate-
let recruitment, and coagulation. CD40L is expressed during this process. 5, The surface-expressed
CD40L is rapidly cleaved, and the soluble form of CD40L (sCD40L) further enhances thrombosis
via its binding to GPIIb/IIIa and also activates endothelium, inducing the expression of proinflamma-
tory and procoagulant proteins. The cleavage mechanism of CD40L is GPIIb/IIIa-dependent and can
be blocked by GPIIb/IIIa antagonists.
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44 Part I / Basic Principles
Fig. 2. Schematic diagram of GPIIb/IIIa and its interacting proteins. GPIIb/IIIa structure: The
schematic for the extracellular domain is adapted from the crystal structure of α
v
β
3
(65), which is
anticipated to be similar to that of GPIIb/IIIa (30). The ligand binding domain of the complex exists
at the contact between the distal, amino terminal domains of each subunit at the top of the figure.
Each subunit has a single transmembrane domain with short cytoplasmic tails. GPIIb/IIIa ligands: The
known physiologic ligands of GPIIb/IIIa (i.e., fibrinogen, vWf, and CD40L) are indicated. GPIIb/IIIa
antagonists function by competing for the binding of these proteins. Intramembraneous GPIIb/IIIa
binding proteins: Four transmembrane proteins have been shown to form intramembraneous com-
plexes with GPIIb/IIIa: CD9, CD151, CD98, and integrin-associated protein (IAP). Cytoplasmic GPIIb/
IIIa binding proteins: Proteins known to bind to the cytoplasmic domains of GPIIb/IIIa include CIB
(to GPIIb), β3-endonexin (to GPIIIa), and several cytoskeletal proteins (i.e., talin, myosin, α-acti-
nin). Proteins involved in GPIIb/IIIa signaling: Signaling proteins involved in the activation of the
receptor function of GPIIb/IIIa (Inside-out) and in mediating platelet activation in response to GPIIb/
IIIa ligands (Outside-in) are indicated. GPIIIa phosphorylation sites: The two cytoplasmic tyro-
sines phosphorylated during outside-in GPIIb/IIIa signaling are indicated.
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 45
consisting of at least 16 α subunits (one of which is GPIIb, or αIIb in the integrin nomen-
clature) and 8 β-subunits (e.g., GPIIIa, also termed β3). Although the tissue distribution
of GPIIb/IIIa is quite limited, being restricted to platelets and other cells of the megakary-
ocytic lineage, the β subunit of this integrin, i.e., β3, is widely distributed and complexed
with an α-subunit, αv, generating the α
v
β
3
integrin. The limited tissue distribution of
GPIIb/IIIa permits specific targeting of platelets by GPIIb/IIIa antagonists.
The GPIIb subunit of GPIIb/IIIa consists of two disulfide-linked subunits, GPIIbα
(M
r
= 125 kDa) and GPIIbβ (M
r
= 22 kDa), which are formed when the precursor GPIIb
polypeptide undergoes cleavage into a heavy and light chain. In contrast, GPIIIa (M
r
=
105 kDa) is a single polypeptide that contains intrachain disulfide linkages. In both cases
the proteins have large extracellular domains, single transmembrane domains, and rela-
tively short cytoplasmic domains (47 amino acids for GPIIIa and 20 for GPIIb). The
extracellular structure of GPIIb/IIIa can be inferred from the crystal structure of α
V
β
3
,
the integrin predicted to have the highest similarity to GPIIb/IIIa (identical β-subunits,
highly homologous α-subunits). This structure consists of 12 domains assembled into
an ovoid head, which contains the binding site for fibrinogen (28), and two legs, which
anchor the integrin to the membrane. X-ray crystallography has shown that the binding
pocket for the RGD (argining-glycine-aspartic acid) peptide sequence, used by many of
the adhesive proteins that bind GPIIb/IIIa, and mimicked by many GPIIb/IIIa antago-
nists, appears to reside in the head at the juncture of the MIDAS domain of GPIIIa and the
β-propeller domain of GPIIb. Although the direct identification of the RGD binding site
on GPIIb/IIIa predicts where fibrinogen, vWF, CD40L, and other proteins bind GPIIb/IIIa,
it is recognized that the binding of such ligands will be more complex and possibly involve
additional domains within the head of GPIIb/IIIa. Nuclear magnetic resonance (NMR)
analysis of GPIIb/IIIa cytoplasmic tails has also been performed and reveals that they are
both relatively flexible (29).
LIGAND BINDING PROPERTIES OF GPIIb/IIIa
Although fibrinogen, vWf, and sCD40L are ligands for GPIIb/IIIa, these molecules
utilize different sequences to bind to the GPIIb/IIIa, and their binding is regulated dif-
ferently. Fibrinogen consists of six polypeptide chains, two α, two β, and two γ, arranged
in a symmetric, bivalent orientation. Because peptides containing the Arg-Gly-Asp (RGD)
sequence were the first identified antagonists of fibrinogen binding and because the
fibrinogen α-chain has two RGD sequences, it was initially assumed that one or both of
Fig. 2. (Continued) GPIIb/IIIa signaling: Inside-out signaling: Signaling events originating from
different agents such as collagen, thrombin, ADP, or vWF bound to collagen and transmitted by the
indicated “Inside-out” signaling proteins lead to the activation of the receptor function of GPIIb/IIIa for
soluble ligands (i.e., fibrinogen, vWf, CD40L). Although the mechanism for GPIIb/IIIa activation
is not known, possible candidate GPIIb/IIIa binding proteins for this function include CIB, β
3
-endo-
nexin, and talin. Outside-in signaling: Upon platelet aggregation, a process mediated by the binding
of fibrinogen, vWf, and CD40L to GPIIb/IIIa, the cytoplasmic domain of GPIIIa becomes tyrosine
phosphorylated on residues 747 and 759, allowing the association of GPIIb/IIIa with the cytoskeletal
protein myosin. Aggregation also initiates signaling events transmitted by the indicated outside-in
signaling proteins, which serve to induce platelet secretion reactions, further GPIIb/IIIa activation
to produce stable aggregates, and clot retraction. Outside-in signaling is mediated, in part, by the
tyrosine phosphorylation of GPIIIa. For abbreviations, see Glossary.
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46 Part I / Basic Principles
these sequences were used to bind GPIIb/IIIa (reviewed in ref. 30). Surprisingly, how-
ever, mutations of either RGD sequence in recombinant fibrinogen has no effect on fibrin-
ogen-GPIIb/IIIa interactions as measured either by platelet aggregation (31) or platelet
adhesion to immobilized fibrinogen (32). An additional sequence in fibrinogen that binds
GPIIb/IIIa is the carboxy-terminal hexapeptide on the γ- chain, Lys-Gln-Ala-Gly-Asp-
Val (KQAGDV), an activity first identified by Hawiger and coworkers (33).
This sequence is contained within both outer nodules of fibrinogen, the structural
entity of fibrinogen that binds GPIIb/IIIa (28). Several lines of evidence indicate that this
is the GPIIIb/IIIa binding domain of fibrinogen: antibodies to this sequence block fibrin-
ogen binding (34); fibrinogen lacking this sequence does not support platelet aggre-
gation; and a bleeding diathesis is induced when mouse fibrinogen is mutated so that it
lacks this sequence (35). Novel technologies such as Laser Tweezers have now revealed
the interaction strength between fibrinogen and its receptor GPIIb/IIIa (36). Fibrin also binds
to GPIIb/IIIa but appears to utilize a different sequence than fibrinogen in that fibrin har-
boring a deletion truncation of the γ-chain QAGDV sequence is still capable of support-
ing clot retraction (37). Since clot retraction depends on the fibrin adhesive property of
GPIIb/IIIa, this finding suggests that conversion of fibrinogen to fibrin exposes a differ-
ent GPIIb/IIIa binding site.
Although the binding of soluble fibrinogen to GPIIb/IIIa requires prior activation of
the integrin, fibrinogen immobilized on surfaces or bound to the GPIIb/IIIa on the surface
of aggregated platelets is fully capable of binding to GPIIb/IIIa on unstimulated platelets.
It thus appears that fibrinogen bound to the GPIIb/IIIa on the surface of an aggregated
platelet undergoes a conformational change into an “activated” form that is capable of
mediating further cellular interactions. One is to bind the GPIIb/IIIa on unstimulated
platelets (38), which, through a process known as “outside-in” signaling, induces platelet
binding and subsequent stimulation. Another is to bind Mac-1 on neutrophils to initiate
the adhesion of these cells (39). Thus these activities of bound fibrinogen are important
as they allow for the recruitment of unstimulated platelets and neutrophils, respectively,
into a growing thrombus. Both reactions, the interaction of bound fibrinogen with the GPIIb/
IIIa on unstimulated platelets and the activation of GPIIb/IIIa by vWf, serve to promote
thrombus growth by mechanisms mediated primarily through the binding of adhesive pro-
teins to GPIIb/IIIa.
Adhesive proteins other than fibrinogen that bind to GPIIb/IIIa, e.g. vWf, fibronectin,
and vitronectin, most likely do so by the RGD sequences contained within their primary
amino acid structures. Of these proteins, the binding of vWf is unique and has features
that make it ideally suited to mediate platelet adhesion and aggregation under conditions
of high shear, as can be found in stenosed coronary arteries. vWf is a large, disulfide-linked
multimer of approx 220 kDa subunits that has two motifs that bind to receptors on plate-
lets; in addition to the RGD (residues 1744–1746), which binds to GPIIb/IIIa , it also has
an A1 domain, a disulfide-linked loop (residues 449–728) that binds the GPIb/IX/V com-
plex (40). Both motifs in soluble vWf are refractory to unstimulated platelets; however,
the conformational change vWf undergoes when it is immobilized on subendothelial
structures, is exposed to conditions of high shear, or even when exposed to the antibiotic
ristocetin or the snake venom protein botrocetin activates it to bind to the GPIb/IX/V com-
plex on the platelet surface, an interaction mediated by the A1 domain. GPIb/IX/V is a
signaling receptor, and vWf binding induces platelet activation including the activation
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 47
of the receptor function of GPIIb/IIIa, which allows it to bind the RGD sequence on vWf
and also to bind fibrinogen (41,42). The ability of vWf to support platelet aggregation
under conditions of high shear (43) may be related to these dual-binding motifs, repeated
on each subunit of vWf, which together would be expected to bind with much higher
affinity than either would singly and are thus more capable of binding platelets under con-
ditions of high shear than is fibrinogen. The finding that aggregation initiated by platelet
adhesion to vWf is GPIIb/IIIa-dependent and is more important at high shear (44) sug-
gests that agonist-induced aggregation (e.g., thrombin- or ADP-induced) may be more
important at low shear. This may, in part, explain why GPIIb/IIIa antagonists are more
effective at preventing arterial thrombosis than are agonist inhibitors like ticlopidine and
clopidogrel (45), since high shear conditions such as occurs in coronary arteries would
increase the importance of GPIIb/IIIa binding to vWf with subsequent GPIIb/IIIa acti-
vation—an interaction blocked by GPIIb/IIIa antagonists but not by the inhibitors that
affect ADP-induced platelet stimulation. It may also explain why direct thrombin inhibitors
caused bleeding as detected by cerebral hemorrage at doses required to inhibit arterial
thrombosis, whereas GPIIb/IIIa antagonists apparently do not (46).
The sequence used by sCD40L to bind to GPIIb/IIIa is Lys-Gly-Asp (KGD). KGD is
a known GPIIb/IIIa recognition motif that was discovered by the specific binding of bar-
bourin, a snake venom disintegrin, to GPIIb/IIIa (47). The disintegrins are a family of pro-
teins found in snake venoms and other hemataophagous organisms that normally express
the RGD motif, which causes them to be potent antagonists of the adhesive protein bind-
ing activity of GPIIb/IIIa and other integrins (e.g., α
v
β
3
, α
5
β
1
) (48,49). Barbourin is a unique
disintegrin, as it expresses a Lys-Gly-Asp (KGD) sequence that causes this disintegrin
to bind specifically to GPIIb/IIIa and inhibits its ligand binding activity (47). Conserva-
tive mutations of this sequence, for example, aspartic acid to glutamic acid (converting the
KGD to KGE), cause a loss of binding. This finding was utilized in the synthesis of the
cyclic heptapeptide eptifibatide (50,51). The role of the KGD sequence in sCD40L bind-
ing to GPIIb/IIIa was established by observations showing that mutation of the KGD in
sCD40L to KGE caused a loss of GPIIb/IIIa binding, and a loss of the platelet effects of this
protein (6). sCD40L is the first of the many endogenous GPIIb/IIIa ligands that is known
to use the KGD sequence for integrin recognition.
The synthetic GPIIb/IIIa antagonists constitute a wide variety of structures but as a
class do have a common structural feature, a positive and negative charge separated by a
distance of 10–20 Å (52), a distance similar to that between the positive charge of arginine
and the negative charge of aspartic acid on RGD (53), to the positive and negative charge on
KGD, and possibly even to that of the positive charge on lysine and the negative charge of
aspartic acid on the KQAGDV sequence as it exists in a helical conformation (54). Although
it is tempting to speculate that these similarities indicate that synthetic GPIIb/IIIa antago-
nists bind to the RGD or KQAGDV binding site(s) and are classical competitive inhibi-
tors of fibrinogen binding, data are not yet available to support this competitive mode of
inhibition.
Ca
2+
in GPIIb/IIIa Function
The primary amino acid sequence of GPIIb/IIIa predicts five divalent cation binding
sites on the GPIIb/IIIa complex: the four EF hands on GP IIb and the one MIDAS domain
on GPIIIa. Five divalent cations binding to GPIIb/IIIa have indeed been observed, and
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48 Part I / Basic Principles
the affinities of these sites for Ca
2+
predict that all are occupied by this divalent cation
when platelets are suspended in plasma (55,56). Divalent cation binding is reversible, and
reductions in the amount of extracellular cations remove Ca
2+
from GPIIb/IIIa, dramati-
cally affecting its structure, its binding of adhesive proteins, and its binding of antagonists.
For exam-ple, suspension of platelets in solutions containing ionized calcium concentra-
tions of 40–50 µmol/L, such as those achieved in citrate-anticoagulated blood, causes
partial removal of Ca
2+
from GPIIb/IIIa, inducing a loss of fibrinogen binding activity (57).
At <1 µmol/L Ca
2+
, as would occur for example in an EDTA-containing buffer, and at 37ºC
(but to a lesser extent at 25ºC), GPIIb dissociates from GPIIIa within the plane of the plasma
membrane (58). Dissociated subunits irreversibly lose their structure and their ability to
bind fibrinogen and accordingly can no longer mediate platelet aggregation (59). The
close proximity of the second EF hand of GPIIb and the MIDAS domain of GPIIIa to the
site responsible for binding RGD ligands may account for observations suggesting that
binding of Ca
2+
, RGD ligands, and some GPIIb/IIIa antagonists (e.g., eptifibatide) are
mutually competitive (60). Thus, platelet assays relying on GPIIb/IIIa function may not be
valid when performed in buffers containing less than the 1.1 mmol/L Ca
2+
normally found
in plasma.
One illustration of this effect was seen in the pharmacodynamic analysis of eptifibatide.
The reduced Ca
2+
caused by citrate anticoagulation actually increased the binding of eptifib-
atide but decreased the binding of fibrinogen. The combined result of these two effects is
that the apparent inhibitory activity of eptifibatide is markedly overestimated in this con-
dition, an event that led to a dose giving 40–50% inhibition of platelet aggregation at
steady state, versus the 80% inhibition that had been targetted in the IMPACT II trial (61).
The same effect of citrate has been reported for abciximab and tirofiban (62) and may also
have contributed to the suboptimal doses of these drugs achieved in clinical development.
Crystallographic analysis has shown that an additional binding site for divalent cations
is induced upon binding of RGD-containing peptides to α
v
β
3
. The function of this addi-
tional cation binding is not known, nor is it known whether this also occurs in GPIIb/IIIa.
GPIIb/IIIa LIBS Epitope Expression
Ligand binding to GPIIb/IIIa (e.g., adhesive proteins, GPIIb/IIIa antagonists, antibod-
ies) induces a conformational change in the receptor, resulting in the formation of neoepi-
topes, collectively termed ligand-induced binding sites (LIBS) epitopes (63,64). The change
in integrin structure upon ligand binding has been directly observed in α
v
β
3
by crystal-
lographic analysis, which showed that the formation of RGD-integrin complexes induces
a profound change in the structure of the integrin (65). The change in structure undoubtedly
accounts for the neoepitopes generated when fibrinogen or GPIIb/IIIa antagonists bind
GPIIb/IIIa. The response of GPIIb/IIIa to ligands has proved variable in that although all
ligands and antibodies induce LIBS expression (66), the LIBS epitopes expressed are
different. For example, whereas tirofiban, eptifibatide, and abciximab all induce the bind-
ing of the LIBS antibody termed LIBS-1 (67), abciximab fails to induce expression of the
epitope for D-3, although the large size of abciximab may have sterically hindered D-3
access (68). LIBS epitope expression is now recognized as a possible cause of thrombo-
cytopenia following exposure to GPIIb/IIIa antagonists (69–71). The incidence of throm-
bocytopenia is low (from 0.1 to 2% or more depending on the GPIIb/IIIa antagonists used)
and varies between the different GPIIb/IIIa antagonists (72). Antibodies directed against
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Chapter 3 / GPIIb/IIIa in Platelet Aggregation 49
LIBS epitopes (revealed by the drug-dependent binding to GPIIb/IIIa), which secondarily
cause platelet clearance by an immune mechanism, have been observed, for example,
in chimpanzee and Rhesus monkeys in response to both L-738, 167, and L-739,758 (73)
in clinical trials in roxifiban (74) and in other studies in response to abciximab (75,76).
STIMULUS-INDUCED GPIIb/IIIa ACTIVATION
The activation of the receptor function of GPIIb/IIIa (i.e., inside-out GPIIb/IIIa sig-
naling) is known to occur in response to a wide spectrum of physiologic and pathophysi-
ologic conditions (e.g., vascular trauma to initiate normal hemostasis; atherosclerotic
plaque rupture or percutaneous intervention to initiate thrombosis). It is not surprising,
therefore, that the stimulus pathways resulting in GPIIb/IIIa activation are redundant.
Redundancy is found on at least two levels. First, GPIIb/IIIa activation occurs in response
to multiple agonists. A host of soluble agonists, including thrombin and ADP, several
adhesive proteins in the extracellular matrix including collagen, vWf, and fibrinogen,
and even high shear act on distinct receptors and are each capable of initiating signal
transduction pathways to induce activation of the receptor function of GPIIb/IIIa for
soluble adhesive proteins (reviewed in refs. 77 and 78). This multiplicity of agonists broadens
the physiologic conditions capable of initiating platelet aggregation, which causes throm-
bosis and hemostasis.
Second, it is now appreciated that many of the primary agonists, e.g., thrombin, colla-
gen, and ADP, act on multiple receptors (Table 1). Indeed, it appears that there may be a
synergy between the different receptors with more than one receptor needing to be acti-
vated by a particular agonist for successful platelet activation to occur. In one example,
ADP activates at least three receptors on platelets: the recently cloned P2Y12 (P2Y
ADP
or P2T
AC
), which inhibits adenylylcyclase and induces platelet aggregation (79); P2Y1
(P2Y
ADP
or P2T
PLC
), which is coupled to phospholipase C and mediates mobilization of
Ca
2+
from intracellular stores and platelet shape change; and a ligand-gated ion channel
similar to P2X1 that allows for rapid Ca
2+
influx. The indirectly acting thienopyridine-
based platelet inhibitors clopidogrel and ticlopidine and the directly acting ATP-based
antagonist ARL 66096 all target P2Y12 and inhibit ADP-induced GPIIb/IIIa activation
(79–81). In another example, collagen is now known to bind to at least two receptors, the
integrin α
2
β
1
and a membrane glycoprotein termed GPVI (82–84). The two receptors are
interdependent, as antibodies against either receptor will block collagen-induced platelet
activation (85,86), and individuals lacking either receptor are defective in collagen-induced
GPIIb/IIIa activation and have a bleeding diathesis (87–89). α
2
β
1
appears to be the high-
affinity receptor, which tethers collagen so that it can bind GPVI to initiate a signal trans-
duction reaction culminating in GPIIb/IIIa activation.
A third example is thrombin. It is uniformly recognized that proteolysis of PAR1 by
thrombin initiates signal transduction reactions within platelets and GPIIb/IIIa activation
(90). However, the inability of PAR1 antagonists to block thrombin-induced human plate-
let stimulation completely (91) and the differences between thrombin-induced responses
compared with those induced by TRAP, peptide agonists of PAR1 (92), suggest that the
thrombin responsiveness of platelets may be partly mediated by a receptor in addition to
PAR1. Possible candidates for a second thrombin receptor include PAR4 and the GPIb/
IX/V complex, which contains a thrombin cleavage site (in GPV) and a high-affinity throm-
bin binding site (in GPIbα) and is known to have thrombin signaling capabilities (93–97).
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