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70 Part I / Basic Principles
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Chapter 4 / Development and Pharmacology of GPIIb/IIIa Antagonists 73
73
From: Contemporary Cardiology: Platelet Glycoprotein IIb/IIIa
Inhibitors in Cardiovascular Disease, 2nd Edition
Edited by: A. M. Lincoff © Humana Press Inc., Totowa, NJ
4 Glycoprotein IIb/IIIa Antagonists
Development of Abciximab and Pharmacology
of Abciximab, Tirofiban, and Eptifibatide
CONTENTS
INTRODUCTION
DEVELOPMENT OF ABCIXIMAB AS A GPIIb/IIIa ANTAGONIST
PHARMACOKINETICS OF APPROVED GPIIb/IIIa ANTAGONISTS
ACKNOWLEDGMENTS
REFERENCES
Barry S. Coller, MD
INTRODUCTION
The rationale for developing platelet glycoprotein (GP) IIb/IIIa antagonists as anti-
thrombotic agents for use in ischemic cardiovascular disease emerged from integrating
data obtained in the early 1980s by many different investigators working in diverse fields.
The data were, however, often controversial or fragmentary, or required extrapolation to
human disease from in vitro systems or animals models. Thus, for those who are consid-
ering embarking on a similar adventure in drug development, it is important to emphasize
that until efficacy and safety was established for these agents in well-controlled, large
clinical trials, many individuals remained skeptical about the potential benefits and safety
of this approach.
DEVELOPMENT OF ABCIXIMAB AS A GPIIb/IIIa ANTAGONIST
Pathophysiology of Coronary Artery Occlusion
Despite strong evidence from postmortem examinations for a major role of thrombo-
sis in the development of ischemic cardiovascular disease dating back to before 1950 (1),
controversies regarding whether the thrombus occurred before or after myocardial infarc-
tion (2) and the role of coronary spasm (3) led to confusion as to the role of thrombosis
in the etiology of ischemic damage. Moreover, a plaque hemorrhage model was favored
by some investigators in which obstruction was primarily caused by a mechanical flap
produced by hemorrhage-induced dissection of the coronary artery (1). It was only with the
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74 Part I / Basic Principles
advent of cardiac catheterization of patients with myocardial infarction in the early 1980s
that there was general acceptance in the cardiologic community that thrombosis initiated
most myocardial infarctions (4). The success of thrombolytic therapy in decreasing mor-
tality from myocardial infarction provided strong support for the importance of throm-
bosis in the pathogenesis of myocardial infarction (5,6).
Even after it was established that thrombosis is a major contributor to myocardial
infarction, however, the relative contributions of platelets and fibrin to this process were
unclear. The efficacy of thrombolytic therapy appropriately focused attention on the con-
tribution of fibrin. These data logically led to attempts to develop strategies to decrease
fibrin deposition by developing more effective inhibitors of thrombin generation and/or
thrombin’s action (7). Detailed pathologic studies identified, however, that a white plate-
let “head” of variable size is usually identifiable at the site of atherosclerotic plaque rup-
ture or erosion, indicating that platelets commonly initiate the thrombotic process (1,8,9).
In this formulation, the commonly identified large red fibrin “tail” reflects subsequent
activation of the coagulation system (facilitated by thrombin generation on the surface
of platelets) and entrapment of erythrocytes in the rapidly forming fibrin meshwork. Bio-
chemical evidence of platelet activation during ischemic vascular events (10) provided
additional evidence of a role for platelets, although it was not possible to exclude the pos-
sibility that platelet activation was an epiphenomenon. Animal models of thrombosis
secondary to vascular injury provided compelling evidence that platelets can make a major
contribution to thrombus formation (11–14), but all the animal models differ significantly
from naturally occurring human cardiovascular disease in the mechanism of injury. More-
over, none of the animal models at that time involved arteries containing the complex
types of atherosclerotic lesions on which virtually all human disease occurs.
Perhaps the most compelling evidence supporting an essential role for platelets in myo-
cardial infarction came from the Second International Study of Infarct Survival (ISIS-2),
a double-blind, randomized, placebo-controlled study involving more than 17,000 patients
with clinically diagnosed myocardial infarction (6,15). This study demonstrated an 18.9%
decrease in 35-d vascular mortality from taking aspirin alone (absolute reduction 2.5%),
a reduction very similar to the 21.2% achieved with intravenous streptokinase alone (6).
Combining aspirin with streptokinase resulted in a 39.4% reduction in mortality, with this
additive effect suggesting that the agents operated through different mechanisms of action.
Data on the combined use of heparin and aspirin in ISIS-2 were especially important in
conceptualizing the roles for antiplatelet and anticoagulant agents. Patients were not ran-
domized with regard to heparin therapy, but the intention of each treating physician to either
use or not use heparin was recorded. Aspirin improved the outcome of patients regardless
of whether intravaneous, subcutaneous, or no heparin use was planned, providing further
evidence that aspirin operates through a mechanism different from that of heparin (6,15).
Even the ISIS-2 data supporting an important role for platelets in myocardial infarction
could be challenged, however, because it could be argued that aspirin’s benefit may have
been owing to its effects on nonplatelet proteins or cells, because many such effects have
been described (16).
The safety data in ISIS-2 also were extremely important in that they showed that aspirin
alone did not increase the risk of hemorrhagic stroke and actually decreased the risk of
any stroke by approx 50% (6,15). Aspirin’s stroke benefit may result from several mecha-
nisms linked to its antithrombotic action. These include: (1) decreasing the size of mural
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Chapter 4 / Development and Pharmacology of GPIIb/IIIa Antagonists 75
thrombi, (2) decreasing the size of the myocardial infarctions so that fewer are transmural
(the feature most predictive of developing a mural thrombus), (3) decreasing the extent
of secondary thrombus formation on emboli that reach the cerebral circulation, and 4)
decreasing microvascular thrombus formation that may result from even temporary inter-
ruption of the cerebral circulation. The stroke data from ISIS-2, when combined with those
of the US Physician’s Health Study of primary prophylaxis of myocardial infarction with
aspirin (17), which identified an increased risk of hemorrhagic stroke with aspirin treat-
ment, led to the interesting paradox that aspirin decreases the risk of stroke during and
immediately after myocardial infarction, a period of high stroke risk, but increases the risk
of hemorrhagic stroke in the normal population, in which the risk of stroke is very low.
Because the protection from death from myocardial infarction afforded by aspirin, a rela-
tively weak antiplatelet agent, extended to only a minority of patients in ISIS-2 and because
aspirin’s hemorrhagic toxicity during the first 30 d of treatment was minimal, it seemed
logical to conclude that there was a potential role for more powerful antiplatelet agents
in the treatment of ischemic cardiovascular events caused by thrombosis.
Theories of the pathogenesis of acute reocclusion after percutaneous coronary inter-
ventions (PCIs), including balloon angioplasty, atherectomy, and similar procedures,
went through phases similar to those relating to the pathogenesis of myocardial infarction.
Thus, in the 1980s, there was considerable controversy as to the relative roles of thrombo-
sis versus mechanical dissection resulting in development of an obstructing intimal flap.
Estimates that I obtained at that time from expert interventionalists for a key role for throm-
bosis ranged from 20 to 80%, with complementary estimates of 80 to 20% for mechanical
dissection. Data from at least one relatively small retrospective study indicated that aspirin
and dipyridamole decreased the risk of abrupt occlusion (18), but the uncertainty about
pathogenesis made it unclear as to whether more potent antiplatelet agents would further
decrease the risk of abrupt occlusion after PCI.
Theories about the pathophysiology of unstable angina and acute non-ST segment
elevation myocardial infarction (NSTMI) focused on the role of platelet-mediated throm-
bosis, since there was biochemical evidence of systemic platelet activation and aspirin
was demonstrated to be effective in reducing the risk of death or developing a myocardial
infarction (19,20). Moreover, adding heparin to aspirin provided additional benefit, but
the effect was not dramatic (20). The role of fibrin in these syndromes was uncertain, since
thrombolytic therapy did not improve the outcome of patients with unstable angina and
NSTMI and may have actually made the outcome worse (21–23). Thus, judging by empiric
observations on response to therapy, the pathophysiology of unstable angina and NSTMI
appeared to differ from that of acute myocardial infarction. This difference may reflect
heterogeneity in the population diagnosed with these disorders, a contention supported
by the observation that the group can be divided on the basis of troponin elevation (24),
and the variability in angiographic findings (25).
Glanzmann Thrombasthenia
Although first described in 1918, it was not until the 1960s that Glanzmann thrombas-
thenia was categorized as an autosomal recessive inherited disorder characterized by muco-
cutaneous hemorrhage, marked prolongation of bleeding time, abnormal clot retraction,
and the hallmark failure of platelets to aggregate in response to all the agonists thought to
operate in vivo, including ADP, epinephrine, collagen, and thrombin (26,27). Subsequently,
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76 Part I / Basic Principles
it was observed that the platelets of patients with Glanzmann thrombasthenia are deficient
in two different glycoproteins, identified as GPIIb and GPIIIa based on their migration in
polyacrylamide gels after treatment with sodium dodecyl sulfate (28,29). Soon thereafter,
it was established that these proteins exist as a calcium-dependent complex (30).
The nature and severity of the clinical hemorrhagic syndrome caused by Glanzmann
thrombasthenia were important factors in the decision to consider GPIIb/IIIa as a target
for antithrombotic therapy, since the patients’ symptoms could be considered the likely
equivalent of maximal GPIIb/IIIa blockade by an antagonist. The spectrum of severity
is wide among patients (26,27,31), but easy bruising, menorrhagia, epistaxis, and variable
gingival bleeding (depending largely on dental hygiene and tooth repair) are most common.
Severe gastrointestinal bleeding and bleeding in other organs occurs, but it is usually
episodic and infrequent, unless there is a separate anatomic abnormality. Of particular
note is that, unlike the bleeding in hemophilia A and B (factors VIII and IX deficiencies),
spontaneous central nervous system bleeding is not common in Glanzmann thrombasthe-
nia (26), highlighting the variability of bleeding syndromes that occur with defects in dif-
ferent components of the hemostatic system. Thus, because it was known that complete
absence of GPIIb/IIIa receptors is not incompatible with life, there was reason to hope
that even high-grade GPIIb/IIIa blockade could be sustained for at least a short period
with acceptable toxicity.
The ability to extrapolate these data to the potential use of GPIIb/IIIa antagonists to treat
thrombotic cardiovascular disease was limited, however, by the lack of knowledge about
the impact of using a GPIIb/IIIa antagonist in combination with heparin, aspirin, a throm-
bolytic agent, or perhaps all of them together, since these agents are scrupulously avoided
in thrombasthenia patients. Moreover, the invasive instrumentation that is required for PCI
and that frequently occurs during the management of myocardial infarction would add a
serious hemostatic challenge. Thus, the safety considerations that could be deduced from
analysis of patients with Glanzmann thrombasthenia were complex; GPIIb/IIIa antago-
nists were likely to cause a significant, but perhaps not unacceptable risk of hemorrhage;
the use of aspirin, heparin, and thrombolytic agents would probably increase the risk sig-
nificantly, and there would probably be a need for fastidious attention to the vascular sites
of entry of invasive devices.
Observations on patients with Glanzmann thrombasthenia had little to offer with
regard to the potential efficacy of GPIIb/IIIa antagonists. Because it is such a rare disorder,
few patients who have Glanzmann thrombasthenia are old enough to have thrombotic
ischemic vascular disease. Nonetheless, neither we nor Dr. Uri Seligsohn and his colleagues
in Israel have identified any patients with Glanzmann thrombasthenia who have had an
acute ischemic cardiovascular event (32). Glanzmann thrombasthenia does not, how-
ever, appear to afford protection from developing subclinical atherosclerosis, as judged
by ultrasound assessment of carotid arteries for intima-media thickness and the presence
of atherosclerotic plaques (33).
Platelet Physiology
The development of the platelet aggregometer in the 1960s (34,35) opened the modern
era of platelet function analysis, with rapid characterization of the aggregation response
to ADP, epinephrine, collagen, and thrombin, among others (36). It also allowed for appre-
ciation of the complex interrelation of platelet aggregation and the release reaction (37).
It further permitted the characterization of antiplatelet agents, most notably aspirin, which
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Chapter 4 / Development and Pharmacology of GPIIb/IIIa Antagonists 77
gives a characteristic pattern of inhibition involving the second wave of aggregation (38,
39), the wave that requires thromboxane A
2
production and is associated with the release
reaction (40,41).
Platelet aggregation induced by ADP, epinephrine, collagen, and thrombin (the ago-
nists that fail to aggregate platelets from patients with Glanzmann thrombasthenia) was
established in the late 1970s to result from the binding of the bivalent adhesive glycopro-
tein fibrinogen to the platelet surface (42,43), followed by the development of fibrinogen
molecule bridges between receptors on two different platelets. Since the GPIIb/IIIa recep-
tor was known to be abnormal in Glanzmann thrombasthenia, it became the logical
choice to be the fibrinogen receptor, but even here there was controversy as to whether
Glanzmann thrombasthenia patients lacked the receptor or lacked the ability to activate
the receptor (44,45). Although several lines of evidence supported the conclusion that the
GPIIb/IIIa receptor was, in fact, the receptor that bound fibrinogen (45,46), it was this
lingering controversy that led us in the early 1980s to try to develop monoclonal antibodies,
which were then coming into general use, to help resolve this issue. We wanted to focus
on antibodies that would affect platelet function, and thus our screening assay built on
our earlier studies (45) demonstrating that platelets could agglutinate fibrinogen-coated
beads. In fact, the antibodies we produced that blocked platelet-mediated fibrinogen bead
agglutination (10E5 and 7E3) bound to the GPIIb/IIIa complex, providing very strong
evidence for GPIIb/IIIa being responsible for the binding of fibrinogen (47,48).
At approximately the same time, additional molecular biologic data from investigators
studying adhesion phenomena in many different tissues provided evidence that GPIIb/IIIa
was a member of a large family of receptors with similar structures (49). Hynes (49) termed
these receptors integrins to emphasize that they have binding domains on both the exterior
of the cell and the cytoplasmic face; the former interact with adhesive ligands, whereas the
latter interact with cytoskeletal proteins and proteins involved in both receiving and send-
ing signals. GPIIb/IIIa is a prototypic integrin, being a heterodimer of an α-subunit (GPIIb,
αIIb) and a β-subunit (GPIIIa, β3) (50,51). Monoclonal antibody studies demonstrated
that GPIIb/IIIa is essentially specific for platelets and megakaryocytes and is expressed
at very high density on platelets [approx 80,000 receptors per platelet (52)], making it one
of the densest adhesion/aggregation receptors in any biologic system. There is also an
internal pool of GPIIb/IIIa receptors associated with α-granule membranes and perhaps
other structures (53,54). At least some GPIIb/IIIa receptors probably cycle between the
platelet surface and the internal pools (54).
The β3-subunit of GPIIb/IIIa can also pair with another α-subunit, termed αV, to form
the αVβ3 receptor, commonly called the vitronectin receptor (55). This is present on plate-
lets at extremely low levels [approx 50–100 molecules per platelet (56)] and is also present
on many other cell types, including endothelial cells, osteoclasts, smooth muscle cells,
and activated lymphocytes (57–59).
Investigators studying the α5β1 integrin receptor in the early 1980s made the remark-
able discovery that a very small region of the large fibronectin molecule was crucial for the
binding (60). Ultimately they localized this to a three-amino acid motif, arginine-glycine-
aspartic acid [single-letter code, RGD (60)]. They went on to show that small peptides
containing this sequence could actually block the binding of ligands to the α5β1 receptor
(61). At approximately the same time, aided by the availability of monoclonal antibodies,
it was discovered that the GPIIb/IIIa receptor could bind not only fibrinogen, but also von
Willebrand factor (vWf), fibronectin, vitronectin, and other adhesive glycoproteins (62).
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78 Part I / Basic Principles
With only one exception, as the genes for these proteins were cloned and their amino
sequences determined, it became clear that the regions responsible for binding to plate-
lets are the same regions that contain RGD sequences (63). Moreover, the binding of all
of these ligands to platelet GPIIb/IIIa could be inhibited by RGD-containing peptides
(64,65). The one exception, ironically, is fibrinogen itself, since its binding to GPIIb/IIIa
does not appear to be mediated by the RGD-containing domains, but rather by the C-ter-
minal dodecapeptide of the γ-chain (66,67). This peptide contains a glycine-aspartic acid
(GD) sequence, as well as a crucial lysine residue (which, like arginine, is positively charged)
two amino acids upstream (68). γ-Chain peptides can also inhibit the binding of fibrino-
gen to platelet GPIIb/IIIa (69). The crystal structure and electron microscopic appearance
of fibrinogen demonstrate that the γ-chain dodecapeptides are at the very ends of this
highly extended, dimeric molecule (70,71). Thus, even platelets that are almost 500 Å
apart can be bridged by a single fibrinogen molecule. It is uncertain whether the RGD and
γ-chain peptides bind to the same site on GPIIb/IIIa (72).
The αVβ3 receptor binds many of the same ligands as GPIIb/IIIa, and its ligand binding
can also be blocked by RGD peptides (59,73). It appears to have preference for vitronectin
(56), however, and, unlike GPIIb/IIIa, it binds to osteopontin when activated (74,75).
Moreover, it appears to bind fibrinogen via the latter’s C-terminal RGD sequence in the
Aα chain (76), and perhaps amino acids 190–202 and 346–358 in the γ-chain (77), but
not the γ-chain C-terminal dodecapeptide. A crystal structure of the extracellular segment
of the αVβ3 receptor has been reported, which includes points of contact between the αV
and β3 (GPIIIa) chains and regions thought to be involved in ligand binding (78). In addi-
tion, the crystal structure of αVβ3 in complex with an RGD peptide has been determined,
and it was elegantly demonstrated that the binding site is a point of contact between αV and
β3, with arginine binding to αV and aspartic acid binding to β3 near the metal ion-depen-
dent adhesion site (MIDAS) domain (79). Based on nuclear magnetic resonance (NMR)
data, Beglova et al. (80) have proposed that activation of αVβ3 involves a change in
struc-ture from a bent form to an upright form by a “switchblade” mechanism, but other
models have been suggested (79).
The multiplicity of ligands that can bind to GPIIb/IIIa adds complexity to its biology.
Shear conditions may be important in determining the preferred ligand since vWf appears
to be the most important ligand under high shear conditions, whereas fibrinogen is favored
at low shear (81). One unique feature of the GPIIb/IIIa-fibrinogen interaction is that GPIIb/
IIIa activation is required for platelets to bind fibrinogen in the fluid phase (82), but plate-
let activation is not required for GPIIb/IIIa to mediate adhesion to immobilized fibrino-
gen (45,83).
The recognition that RGD-containing peptides could inhibit the GPIIb/IIIa receptor
opened up exciting new opportunities to synthesize low-molecular-weight peptides, pepti-
domimetics, and nonpeptides that would have favorable pharmacologic features. This led
to the production of literally thousands of compounds, including orally active compounds,
that have high affinity for GPIIb/IIIa and high specificity for GPIIb/IIIa, compared to αVβ3
and other
integrin receptors (see below) (84–86).
Platelet Adhesion and Aggregation
Our current view is that occlusive thrombus formation in coronary arteries probably
begins with deposition of platelets on a ruptured or eroded atherosclerotic plaque. The
adhesion of platelets to the plaque is primarily mediated by constitutively active receptors
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Chapter 4 / Development and Pharmacology of GPIIb/IIIa Antagonists 79
on the platelet surface interacting with subendothelial proteins, including but not limited
to GPIb/IX (vWf); GPIIb/IIIa (αIIbβ3; immobilized fibrinogen); GPIa/IIa (α2β1) (colla-
gen); GPIc*/IIa (α5β1) (fibronectin); GPIc/IIa (α6β1) (laminin); GPVI (collagen); and
perhaps αVβ3 (fibrinogen, vitronectin, vWf, osteopontin) and GPIV (thrombospondin,
collagen) (37). The adhesive proteins may be exposed by the initial injury (e.g., collagen
and vWf), deposited onto subendothelial proteins either from plasma or from platelet
releasates (e.g., binding of vWf to collagen) (87), or deposited from plasma or from plate-
let releasates onto newly formed fibrin (e.g., vWf) (88). Data demonstrating tissue factor
in the lipid-rich core of athersclerotic plaques, however, raise the possibility that the gen-
eration of at least small amounts of thrombin and local fibrin formation may be early events
in at least some circumstances (89). The adhesion process is likely to be much more com-
plex after the release or generation of platelet agonists at the site of vascular injury since
these agents may be able to cause nearby circulating platelets to expose P-selectin and
activate GPIIb/IIIa to a high affinity ligand binding state. Inflammatory cytokines may
also affect the process, with platelets rolling or skipping along the surface before settling
into stable interactions (87,90). Under the relatively high shear rates found in coronary
arteries, the GPIb/IX-vWf interaction appears to play a crucial role (87).
The first layer of adherent platelets probably has little effect on blood flow. It is the
recruitment of additional layers of platelets, mediated primarily or exclusively by the
GPIIb/IIIa receptor, that poses the greatest risk of platelet thrombus formation and result-
ing vasoocclusion. Platelet deposition probably also initiates thrombin generation and
fibrin deposition, since platelet aggregates have been demonstrated to bind the initiator of
the extrinsic pathway of coagulation, tissue factor, which circulates in whole blood (91–
93). Moreover, the activated platelets in the thrombus, as well as microparticles released
from platelets, furnish highly effective catalytic surfaces on which thrombin can be gener-
ated, leading to the initiation of fibrin deposition and further platelet activation and adhe-
sion (94,95). Platelet-rich thrombi resist thrombolysis because platelets facilitate clot
retraction and fibrin crosslinking and release inhibitors of fibrinolysis (see Mechanisms
of Action section below).
Platelets can also contribute to both local and systemic inflammation. Thus, activated
platelets can recruit both neutrophils and monocytes via interactions between P-selectin
on the surface of activated platelets and P-selectin glycoprotein ligand-1 (PSGL-1) on the
surface of the leukocytes (reviewed in ref. 96). Other platelet receptors, including GPIIb/
IIIa, αVβ3, and GPIb/IX may also contribute to strengthening the interactions. Platelets
can release cytokines and cytokine precursors, as well as platelet-activating factor, that
can activate leukocytes. In addition, activated platelets express CD40 ligand and interleu-
kin-1 (IL-1), potent activators of a number of different inflammatory responses. Finally
platelet-neutrophil and platelet-monocyte aggregates circulate in normal blood, and the
latter increase when platelets are activated, as occurs after PCI (97–99). Leukocytes that
circulate after PCI and those with attached platelets show evidence of activation, as judged
by increased expression of αMβ2 (100,101).
Thus, the rationale for targeting the GPIIb/IIIa receptor focused on its pivotal role in
mediating the platelet-platelet interactions crucial for vasoocclusive platelet thrombus
formation (and the downstream effects of platelet thrombi on coagulation, fibrinolysis,
and inflammation), regardless of the agonist responsible for platelet activation. As such,
it represents the final common pathway for platelet aggregation. This model predicts that
GPIIb/IIIa receptor blockade would have little effect on platelet adhesion, which is largely
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