MK-383 (tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor

The platelet integrin alphaIIb beta3 (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10(-7) mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.     

Advances in Antiplatelet Therapy in Coronary Artery Disease: Importance of the Platelet GPIIb/IIIa Receptor

It is now widely agreed that platelets are intimately involved in and contribute to the pathogenesis of acute coronary thrombosis. Aspirin, a relatively weak inhibitor of platelet activation, saves lives when administered early after acute myocardial infarction and should be routinely used as lifelong therapy in patients with coronary atherosclerosis. Ticlopidine has a mechanism of action distinct from and additive to that of aspirin; it inhibits activation of platelets mediated by the agonist, adenosine diphosphate (ADP). The reduction in subacute coronary thrombosis attained by the use of combination therapy with aspirin and ticlopidine (for 2–4 weeks) after intracoronary stenting is further evidence of the role of platelets in mediating acute arterial thrombosis. Potent platelet agonists (like thrombin) can override the effect of aspirin and ticlopidine; therefore these agents are of limited efficacy. In contrast, inhibitors of the platelet glycoprotein (GP) IIb/IIIa receptor are potentially more potent inhibitors of adhesive platelet interaction and may therefore be effective in blocking adhesive platelet interactions irrespective of the activating agonist. The GPIIb/IIIa receptor mediates the bridging of platelets (platelet aggregation) via fibrinogen, thus allowing platelet to bind other platelets at the injured vessel wall. Antagonists of this receptor are thus capable of blocking the “effector function” by acting at a step that is downstream to platelet activation. By abrogating the final common pathway of platelet aggregation, antagonists of GPIIb/IIIa also affect the most proximal step in thrombin generation (that most efficiently occurs on the membrane surface provided by platelets). Accordingly, these agents can profoundly inhibit arterial thrombosis. The clinical use of the antibody fragment directed against the GPIIb/IIIa receptor (c7E3 Fab) has truly revolutionized the practice of interventional cardiology and has the potential to effectively treat heparin-resistant intracoronary thrombosis. Synthetic antagonists of fibrinogen binding to the GPIIb/IIIa receptor (the “fibans”) are currently under initial clinical testing.     

MK-383 (Tirofiban) induces a GPIIb/IIIa receptor conformation which differs from the resting and activated receptor

The platelet integrin α_IIb β₃ (GPIIb/IIIa) acts as a receptor for fibrinogen, playing a critical role in platelet aggregation. GPIIb/IIIa antagonists, which block the receptor-ligand interaction, have been accused of causing occasional thrombocytopenia, probably via drug-induced platelet activation or immunogenic neoepitopes. We, therefore, analyzed the effects of the GPIIb/IIIa antagonist MK-383 (tirofiban) on platelet activation and GpIIb/IIIa conformation. At a concentration of 10^-7 mol/l, MK-383 completely inhibited fibrinogen binding to in vitro stimulated platelets. Simultaneously, the GPIIb/IIIa expression density increased, similar to that on activated platelets, but no effect on P-selectin expression or the formation of platelet-leukocyte aggregates could be observed, indicating that MK-383 binding did not induce general platelet activation. The GPIIb/IIIa receptor conformation was further analyzed by fluorescence resonance energy transfer analysis between fluorochrome-labeled antibodies against different GpIIb/IIIa epitopes. As a result, MK-383 induced a receptor conformation that differed from the resting as well as the activated receptor as induced by ADP or TRAP-6. This conformational modulation of GPIIb/IIIa presents an interesting mechanism which may be linked to receptor recruitment without inducing general platelet activation.     

Comparative in vitro efficacy of different platelet glycoprotein IIb/IIIa antagonists on platelet-mediated clot strength induced by tissue factor with use of thromboelastography: differentiation among glycoprotein IIb/IIIa antagonists

In the present study, the in vitro efficacy of different platelet glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists on platelet-fibrin-mediated clot strength under shear was compared with the antiaggregatory efficacy by using tissue factor (TF) thromboelastography (TEG). The ability of platelets to augment the elastic properties of blood clots under shear conditions was measured by computerized TEG under conditions of maximal platelet activation accelerated by recombinant TF. Under these conditions, platelets significantly enhance clot strength 8-fold (relative to platelet-free fibrin clots). This effect was inhibited to a different extent by various platelet GPIIb/IIIa receptor antagonists; this inhibition appears to be dependent on the transmission of platelet contractile force to fibrin via the GPIIb/IIIa receptors. The GPIIb/IIIa antagonists with high binding affinity for resting and activated platelets and slow platelet dissociation rates (class I) but not those with fast platelet dissociation rates (class II) demonstrated potent and comparable inhibition of platelet aggregation and TF-TEG clot strength. Platelet GPIIb/IIIa antagonists of class I, such as XV459 (free-acid form of roxifiban), DMP802, XV454, and c7E3, demonstrated comparable inhibitory dose responses of TF-TEG clot strength and platelet aggregation, with an IC(50) of 50 to 70 nmol/L. In contrast, platelet GPIIb/IIIa antagonists from class II, with comparable antiaggregatory efficacy, such as DMP728, YZ202 (free-acid form of orbofiban), YZ211 (free-acid form of sibrafiban), YZ751, and other antagonists, have a much lower efficacy in altering the strength of TF-mediated clot formation (IC(50) >1.0 micromol/L). These data suggest differential efficacy among different GPIIb/IIIa antagonists in inhibiting platelet-fibrin clot retraction despite of equivalent antiaggregatory potency.     

Is platelet adhesion assay able to quantify drug-induced platelet dysfunction?

The platelet adhesion assay (PADA) is an innovative method for the detection of both normal, pathologically increased or decreased platelet adhesiveness. Adhesion is the first important phase of platelet activation, followed by shape change and aggregation. Adhesion is triggered by glycoproteins (GP) on the platelet surface, mainly by GPIIb/IIIa, and to a lesser extent also by GPIb/V/IX. Since fibrinogen serves as adhesive protein for GPIIb/IIIa receptors, and since the PADA uses polymer particles that become coated with fibrinogen, the PADA is able to monitor GPIIb/IIIa receptor antagonists and to detect overdosing, potentially leading to bleeding complications. Ex vivo, citrated whole blood from healthy volunteers and patients was spiked with increasing GPIIb/IIIa inhibitor concentrations and PADA was measured. Comparing these results with GPIIb/IIIa receptor occupancy, determined by FACS, a basic consistency of the data was shown. Via intracellular signaling, the adenosine diphosphate (ADP) receptor mechanism is closely involved in the activation of GPIIb/IIIa receptors so that also ADP receptor antagonists of the thienopyridine type, especially clopidogrel, can be quantitatively determined by the PADA. In patients under clopidogrel therapy, the therapeutic effect was monitored and also individual dose adjustments were realized. Furthermore, patients having partial or full clopidogrel resistance were identified. Overdoses can be detected as well.     

Glycoprotein IIb/IIIa and P2Y12 receptor antagonists yield additive inhibition of platelet aggregation, granule secretion, soluble CD40L release and procoagulant responses

Glycoprotein IIb/IIIa (GPIIb/IIIa) antagonists, including abciximab and tirofiban, are administered concurrently with clopidogrel, a P2Y12 antagonist, and aspirin in some patients undergoing percutaneous coronary intervention. We studied the effects of, and interactions between, abciximab, tirofiban, aspirin and the P2Y12 antagonist cangrelor on platelet aggregation, alpha and dense granule secretion and procoagulant responses in vitro. Blood was obtained from healthy volunteers. Platelet aggregation, dense granule secretion, alpha granule secretion (PAI-1 and soluble CD40 ligand levels) and procoagulant responses (annexin-V and microparticle formation) were assessed using collagen and thrombin receptor activating peptide (TRAP) as agonists. All the antagonists used singularly inhibited collagen-induced responses. Combinations of abciximab or tirofiban with aspirin and/or cangrelor gave additive inhibition with the greatest effect seen when abciximab or tirofiban was combined with both aspirin and cangrelor. Cangrelor inhibited TRAP-induced responses and, again, there was additive inhibition of these parameters when abciximab or tirofiban were combined with cangrelor. The GPIIb/IIIa receptor plays an important role in amplification of platelet activation such that there are important interactions between GPIIb/IIIa antagonists and inhibitors of both P2Y12 receptor activation and, to a lesser extent, thromboxane A2 generation. These interactions are likely to have important influences on the safety and efficacy of combination anti-platelet therapies.     

Glycoprotein IIb/IIIa antagonists in acute coronary syndromes: where are we now?

Vascular inflammation, coronary constriction, and thrombus formation are central to all acute coronary syndromes (ACSs). Adhesion and aggregation of activated platelets, initially described during thrombosis, now appear pivotal to all three processes. Several platelet adhesion receptors participate but the integrin glycoprotein (GP) IIb/IIIa occupies a critical role. GPIIb/IIIa antagonists used as an adjunct to percutaneous coronary intervention show clear benefit. However in the setting of ACS results have been disappointing. Indeed, trials of oral GPIIb/IIIa antagonists in patients with ACS were associated with increased mortality. Difficulties with drug dosing and variable pharmacodynamics may contribute to suboptimal receptor occupancy, incomplete inhibition of platelet aggregation, paradoxical partial agonist activity, and proinflammatory effects. Moreover, variable responses of patients to GPIIb/IIIa antagonists may reflect population heterogeneity.     

Platelet GPIIb/IIIa antagonist, XV459, in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia (HIT) is a serious, immune-related complication of heparin therapy. One of the most severe manifestations of HIT is the development of thromboembolic events, which is based on platelet activation and aggregation caused by HIT-associated antibodies. Therapeutic options for patients with HIT are limited despite advancement toward the development of alternative (nonheparin) anticoagulants, such as direct thrombin inhibitors and indirect anti-factor Xa agents. Platelet GPIIb/IIIa receptor antagonists have been shown to be the final common pathway for platelet aggregation regardless of the use of activator or anticoagulant. In this study, the ability of a novel platelet GPIIb/IIIa antagonist, a free acid form of roxifiban (XV459), to block platelet activation/aggregation in response to highly characterized heparin-PF4 antibody-positive plasma/heparin was examined using light transmittance aggregometry, serotonin release, and (125)I-fibrinogen binding assays to human platelets. XV459 at 20 nM maximally inhibited (P < 0.001) the platelet-activation/aggregation responses as mediated by the HIT antibody-positive plasma (in the presence of therapeutic heparin concentrations). Compared with controls, both HIT antibodies/heparin and TEAC (a mixture of thrombin [0.1 IU/ml], epinephrine [1 microg/ml], arachidonate [0.1 mM], and collagen [10 microml]) resulted in significantly higher levels of fibrinogen binding to human platelets (5-7-fold increase; P < 0.001). Concentration-dependent profiles of XV459 on the mean percent inhibition of (125)I-fibrinogen binding in the presence of HIT antibodies and TEAC were achieved ( approximately 50% inhibition at 10 nM XV459). The platelet GPIIb/IIIa receptor antagonist (XV459) might be of potential benefit in the management of thrombotic thrombocytopenia produced by heparin and/or related glycosaminoglycans.     

Comparative antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist (XV454) and abciximab (c7E3) in flow models of thrombosis

Glycoprotein (GP) IIb/IIIa is pivotal in homotypic platelet aggregation and may also be involved in the heterotypic adhesion of leukocytes and tumor cells to platelets. This study was primarily undertaken to compare the antiplatelet efficacy of a novel, nonpeptide GPIIb/IIIa antagonist, XV454, to that of abciximab in 2 flow models of platelet thrombus formation: (1) direct shear-induced platelet aggregation imposed by a cone-and-plate rheometer and (2) platelet adhesion onto von Willebrand factor (vWF)/collagen I followed by aggregation in a perfusion system. XV454 inhibited platelet aggregation in a concentration-dependent manner in both experimental models. Maximal inhibition of aggregation was achieved by XV454 at approximately 70% receptor occupancy, which is lower than the >/=85% previously reported for abciximab. At similar levels of receptor blockade (approximately 45%), XV454 appeared to be relatively more effective than abciximab in suppressing platelet aggregation. Neither XV454 nor abciximab inhibited platelet adhesion to collagen. Pretreatment of surface-adherent platelets with either XV454 or abciximab inhibited the attachment of monocytic THP-1 cells under flow. In contrast, the rapidly reversible GPIIb/IIIa inhibitor orbofiban failed to suppress these heterotypic interactions. These findings demonstrate that XV454 is a potent GPIIb/IIIa antagonist with a long receptor-bound lifetime like abciximab and may be beneficial for the treatment/prevention of thrombotic complications.     

Differential efficacy of different platelet glycoprotein IIb/IIIa antagonists on platelet/fibrin-mediated clot dynamics under different conditions using thrombelastography: the critical need for anticoagulant

BACKGROUND: Intravenous GpIIb/IIIa antagonists demonstrate various significant clinical benefits depending on the agent used. In contrast, oral delivery of GpIIb/IIIa antagonists failed in achieving clinical benefits. This raises the question about the differences among different GpII/IIIa antagonists. METHODS: The effect of various platelet glycoprotein (GP) IIb/IIIa antagonists on the dynamics of platelet/fibrin clot formation and strength was determined using thrombelastography under different conditions. RESULTS: GPIIb/IIIa antagonists with high affinity for resting and activated platelets and with slow rates of dissociation from GPIIb/IIIa (Class I antagonists) demonstrated potent and comparable inhibition of platelet aggregation and platelet-mediated clot strength under different conditions. In contrast to antagonists that dissociate rapidly from GPIIb/IIIa (class II antagonists). Class I antagonists such as the free acid form of roxifiban inhibited platelet-mediated clot strength, with the inhibiting concentration required for 50% effect (IC50) = 70 n mol/l, whereas the IC50 of the class II antagonists such as the free acid forms of orbofiban, sibrafiban, lotrafiban, integrilin or aggrastat ranged from 1 to 15 micromol/l. The IC50s for class II antagonists in inhibiting platelet/fibrin clot formation and strength were substantially greater (10-15 fold) than their clinically achievable concentrations. The limited efficacy for class II antagonists in inhibiting platelet-mediated clot dynamics was enhanced by the combination with heparin. CONCLUSIONS: Thus, these data indicated that there are differences in the efficacy of various GPIIb/IIIa antagonists in inhibiting platelet/fibrin clot formation and strength, which might be corrected by heparin. Data also suggest that inhibition of platelet aggregation may not be the sole determinant for the in-vivo efficacy of various GPIIb/IIIa antagonists.     

In-vitro efficacy of different platelet glycoprotein IIb/IIIa antagonists and thrombolytics on platelet/fibrin-mediated clot dynamics in human whole blood using thrombelastography.

Suppressing platelet activation improves efficacy of thrombolytic therapy for stroke and acute myocardial infarction. Combination treatment with recombinant tissue plasminogen activator (r-tPA) and glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitor that binds with high affinity to platelets may therefore improve the efficacy of thrombolytic therapy. The effect of platelet GPIIb/IIIa antagonists and/or r-tPA on the dynamics of platelet/fibrin clot formation, strength, and lysis was determined using thrombelastography in human blood under thrombin or tissue factor stimulation. The study utilized platelet GPIIb/IIIa antagonists with high affinity and slow off-rate (Class I) from resting and activated platelets in comparison with Class II antagonists (lower affinity and fast off-rate from platelet GPIIb/IIIa receptors). The combination of the active form of roxifiban (XV459; Class I) or the active form of orbofiban (Class II) with a subeffective concentration of r-tPA resulted in a synergistic effect in clot lysis with roxifiban active form XV459 but not with that of orbofiban at therapeutically achievable concentrations that inhibit human platelet aggregation. These data indicate differential enhanced thrombolysis of low levels of r-tPA with high-affinity Class I but not with low-affinity Class II GPIIb/IIIa antagonists in the absence of anticoagulants.     

In vitro measurement of platelet glycoprotein IIb/IIIa receptor blockade by abciximab: interindividual variation and increased platelet secretion

BACKGROUND AND OBJECTIVES: Inhibition of soluble fibrinogen binding to activated platelets represents the target of pharmacologic approach with antagonists of the glycoprotein IIb/IIIa (GPIIb/IIIa) complex. In this study we assessed the effects of abciximab, a recombinant chimeric Fab fraction of the antibody against GPIIb/IIIa, on several markers of platelet activation. DESIGN AND METHODS: The platelet surface expression of GPIIb/IIIa was measured by a flow cytometry technique using a two-color assay. GPIIb/IIIa was detected by FITC-conjugated antibodies in whole blood, either unstimulated or exposed to platelet stimuli. The following antibodies were used: CD41, which recognizes the IIb/IIIa complex both in activated and non-activated conformers, and PAC-1, which is directed toward the activated conformer of GPIIb/IIIa. In addition, the same blood sample was incubated with CD62 antibody to measure P-selectin, as a marker of a-granule degranulation. The effect of abciximab was also assessed by experiments carried out on shear stress-induced platelet aggregation, a test that appears to be a predictor of platelet hemostatic function. RESULTS: Abciximab inhibited CD41 binding to glycoprotein IIb (GPIIb) in a concentration-dependent manner and also inhibited the binding of PAC-1 to active GPIIb/IIIa. In contrast, membrane-associated P-selectin was significantly increased by the drug, which suggests that blockade of GPIIb/IIIa receptors results in an increased platelet degranulation in response to agonists. Shear stress-induced platelet aggregation was inhibited by abciximab, with a more pronounced effect on blood filtration, which represents an index of platelet aggregate formation. INTERPRETATION AND CONCLUSIONS: Our results indicate that GPIIb/IIIa blockade by abciximab is accompanied by an increase of a-granule secretion, suggesting that different mechanisms regulate these aspects of platelet activation. The described flow cytometry technique, that allows the simultaneous in vitro detection of several platelet markers, is a suitable method for assessing the effects of agents which interfere with platelet function.     

Interaction of porcine von Willebrand factor with the platelet glycoproteins Ib and IIb/IIIa complex

Porcine von Willebrand factor (PvWF) induces platelet aggregation which is thought to be responsible for the thrombocytopenia that occurs in haemophilic patients treated with commercial preparations of porcine factor VIII. This study demonstrates that such aggregation can be completely inhibited by a monoclonal antibody against human platelet glycoprotein GPIb and partially inhibited by an antibody directed against platelet GPIIb/IIIa. The interaction of PvWF with GPIb is also demonstrated by the inhibitory effect of purified glycocalycin on aggregation. The binding site of PvWF to GPIb is very close to that of human vWF, since a recombinant peptide blocks the binding of both molecules to GPIb. When platelets are incubated with PvWF, the GPIIb/IIIa receptor is activated and binds fibrinogen. PvWF also binds to GPIIb/IIIa when platelets are stimulated with thrombin, suggesting that the molecule has the same RGD sequence as other adhesive proteins (human vWF, fibrinogen, fibronectin and vitronectin). These findings identify the dual mechanisms responsible for in vivo platelet aggregation induced by PvWF, i.e. binding to GPIb and activation of the GPIIb/IIIa receptor.     

The GPIIb/IIIa antagonist eptifibatide markedly potentiates platelet-leukocyte interaction and tissue factor expression following platelet activation in whole blood in vitro

Tissue factor (TF) is the most important initiator of intravascular coagulation. Activated platelets are able to adhere to leukocytes and this heterotypic cell-cell interaction results in a CD62P-dependent TF expression on monocytes. GPIIb/IIIa antagonists are inhibitors of the common pathway of platelet aggregation and they are widely used in patients with acute coronary syndromes undergoing coronary interventions. As GPIIb/IIIa antagonists do not prevent platelet activation we investigated the effect a GPIIb/IIIa antagonist, eptifibatide, on the formation of platelet-leukocyte conjugates and leukocyte TF expression. Flow cytometry was used to detect conjugates and TF. When platelets in citrated human blood were stimulated for 30 min with collagen there was a increase in the number of both neutrophils and monocytes with the platelet-specific antigen CD42a, indicating the formation of platelet-neutrophil (P/N) and platelet-monocyte (P/M) conjugates. P/M formation was associated with about a 2.5-fold increase in TF expression on monocytes, whereas P/N formation changed TF expression neutrophils only by about 10%. Eptifibatide enhanced dose-dependently (0.0625-1.5 microg/ml) both collagen-induced P/M formation and monocyte TF expression. Maximum enhancement by about 60 and 120%, respectively, was observed at 0.5 microg/ml eptifibatide. In contrast, eptifibatide had only a minor effect on P/N formation and no effect on neutrophil TF expression. The augmented P/M formation and monocyte TF expression in the presence of a GPIIb/IIIa antagonist may be relevant to the poor antithrombotic efficiency of oral GPIIb/IIIa antagonists as shown in recent large clinical trials.     

Comparison of the effects of cellulose triacetate and polysulfone membrane on GPIIb/IIIa and platelet activation

BACKGROUND: During hemodialysis session, several adverse reactions can occur on platelets, which are attributable to bioincompatibility of the dialysis membrane. Glycoprotein IIb/IIIa (GPIIb/IIIa) is the receptor for fibrinogen, which mediates platelet aggregation and adhesion. Accordingly, we compared the influence of a cellulose triacetate (CTA) and polysulfone (PS) membrane on GPIIb/IIIa and platelet activation. METHODS: Blood samples from 5 patients on hemodialysis were taken at 0 time, 15 min, 30 min, 60 min and 240 min, during a single hemodialysis session, by a crossover design using CTA or PS. Platelet count and plasma concentration of GPIIb/IIIa, beta-thromboglobulin (beta-TG) and platelet factor 4 (PF-4) were measured. GPIIb/IIIa was measured by flow cytometry. beta-TG and PF-4 were measured by ELISA. RESULTS: There was no significant change in the total amount of GPIIb/IIIa during dialysis session between the CTA and PS. However, the level of bound GPIIb/IIIa was significantly (p < 0.0002) increased from 1,426 +/- 435 to 40,446 +/- 2,777 mol/PLT with PS. In contrast, there was no significant change with CTA (3,258 +/- 1,469 to 4,301 +/- 1,422 mol/PLT). The platelet counts and beta-TG and PF-4 behavior during the dialysis session did not show significant change between the PS and CTA. CONCLUSION: The characterization of changes in platelet membrane receptor (GPIIb/IIIa) may be a useful marker for studying the biocompatibility of dialysis membranes. On platelet aggregation, CTA might be more biocompatible membrane than PS.     

New antiplatelet agents

Aspirin is an established therapy for the management of acute myocardial infarction (AMI) and unstable angina. Secondary prevention with chronic aspirin therapy is also indicated for patients with stable angina.
Aspirin inhibits cyclo-oxygenase-I, a key enzyme in the biosynthetic pathway leading to the production of thromboxane A₂. It therefore inhibits only one of the many activation pathways leading to platelet aggregation. Other antiplatelet agents that have also been evaluated in clinical trials include ticlopidine and clopidogrel, which inhibit adenosine diphosphate-mediated platelet aggregation, but these agents are known to be effective against only one of the 90 known agonists that stimulate platelet aggregation.
The final common pathway for platelet aggregation involves the glycoprotein IIb/IIIa receptor combining with fibrinogen. Several inhibitors of the glycoprotein IIb/IIIa receptor have been developed and have an important role as adjunctive therapy in angioplasty. Recent trials have been performed in patients with unstable angina, and trials of adjunctive therapy are currently underway in patients receiving thrombolysis for AMI, and for secondary prevention.
These drugs have various different features, including specificity for blockade of the glycoprotein IIb/IIIa receptor, half life, duration of the haemostatic effect and potential for antigenicity. Recently concluded and ongoing trials of both intravenous and oral agents are expected to provide further support for the introduction of these agents into clinical management of patients with acute coronary syndromes.     

Perspectives on the Long Term Management of Heparin-Induced Thrombocytopenia

The glycoprotein (GP) IIb/IIIa receptor antagonists used widely in the medical treatment of acute coronary syndromes and during percutaneous coronary interventions, prevent fibrinogen cross-linking and platelet aggregation, critical initiating steps in arterial thrombosis. Their anticoagulant properties, particularly when administered conjunctively with heparin preparations, are less well-characterized. In a series of in vitro studies, increasing concentrations of abciximab, tirofiban, and eptifibatide either alone or in combination with unfractionated heparin (UFH) or fractionated heparin (enoxaparin) were added to washed platelets suspended in Tyrode's buffer. Following platelet activation and prothrombinase assembly, thrombin generation was determined by enzyme-linked immunosorbent assay (ELISA). There was a concentration-dependent reduction in platelet-dependent thrombin generation with each of the GPIIb/IIIa receptor antagonists. The combination of tirofiban and UFH yielded percent, absolute and relative reductions (compared with tirofiban alone) of 48.0%, 16.9%, and 35.2%, respectively. The corresponding values for eptifibatide and abciximab were 38.0%, 13.5%, 35.5%, and 55.1%, 3.8%, 8.4%, respectively. Thrombin generation was decreased by an additional 2 to 3% (absolute reduction) with high concentrations of enoxaparin in combination with either eptifibatide or abciximab. Platelet GPIIb/IIIa receptor antagonists, beyond their ability to prevent fibrinogen-mediated aggregation, inhibit platelet-dependent prothrombinase activity and thrombin generation in a concentration-dependent manner. Heparin facilitates the existing anticoagulant properties, supporting combination therapy in clinical practice. The potential added benefit of fractionated heparin over UFH will require further investigation.     

Platelet Glycoprotein IIb/IIIa Integrin Blockade in Coronary Artery Disease: Current State of the Art

Platelets have been shown to play an important role in the pathogenesis of atherosclerosis, acute coronary syndromes, and ischemic complications after percutaneous coronary intervention. Fibrinogen binding via platelet surface glycoprotein (GP) IIb/IIIa receptors constitutes the "final pathway" in platelet aggregation leading to thrombus formation. The GP IIb/IIIa receptor inhibitors, a new class of antiplatelet agents that have emerged in recent years, show great promise in reducing complications of coronary angioplasty and acute coronary syndromes. This review will examine the biology of platelet GP IIb/IIIa receptors, the various classes of GP IIb/IIIa receptor antagonists, the results of the latest clinical trials, and their implications in current clinical practice.     

GPIIb/IIIa Inhibitor-Induced Dethrombosis

GPIIb/IIIa inhibitors have demonstrated clinical benefit in patients with acute coronary syndromes and in those undergoing percutaneous coronary intervention. As opposed to the well documented prevention of platelet aggregation by GPIIb/IIIa inhibitors, we here discuss the less well appreciated reversal of platelet aggregation by, and dethrombotic effects of, GPIIb/IIIa inhibitors. In vivo evidence for GPIIb/IIIa inhibitor-induced dethrombosis includes animal models of arterial thrombosis and human studies (both observational and randomized clinical trials) in the setting of acute myocardial infarction. These human studies demonstrated increased coronary perfusion prior to percutaneous coronary intervention in patients receiving GPIIb/IIIa inhibitors as compared to those patients receiving placebo. Mechanisms of GPIIb/IIIa inhibitor-induced dethrombosis include alterations in thrombus size and structure, reduced capacity for subsequent thrombus growth, inhibition of soluble CD40L release from platelets, and a direct platelet disaggregatory effect that parallels fibrinogens interactions with GPIIb/IIIa. In summary, mechanistic studies, animal models, human observational studies and randomized clinical trials all strongly suggest that GPIIb/IIIa inhibitors have a direct dethrombotic effect in addition to their well-described preventative effects on platelet aggregation.     

Evidence of platelet activation during treatment with a GPIIb/IIIa antagonist in patients presenting with acute coronary syndromes

OBJECTIVES: The study was done to determine the role of partial agonist activity in the lack of effectiveness of the oral GPIIb/IIIa antagonist orbofiban. BACKGROUND: Orbofiban, an oral GPIIb/IIIa antagonist, was found to increase the mortality of patients with acute coronary syndromes (ACS) in the OPUS-TIMI-16 trial, despite the fact that it is a very potent anti-platelet agent and that IV agents have proven very effective. METHODS: Patients (n = 520) with ACS were randomized to orbofiban 30 mg, 40 mg or 50 mg twice daily or 50 mg once daily or placebo. Platelet activity was assessed in 175 patients by examining GPIIb/IIIa receptor conformation, expression of CD63 antigen, and platelet aggregation. RESULTS: Plasma concentrations of orbofiban at the highest dose (74 +/- 6 ng/ml peak, 61 +/- 5 ng/ml trough) exceeded the IC50 for platelet aggregation to adenosine diphosphate (ADP) (29 +/- 6 ng/ml) and thrombin-activating peptide (61 +/- 18 ng/ml). Orbofiban induced a conformational change in GPIIb/IIIa detected as the displacement of the monoclonal antibody mAb2; such conformational changes have been linked to partial agonist activity. Consistent with this, platelet expression of CD63 ex vivo was significantly increased at five time points during the study. In vitro, orbofiban increased platelet aggregation to a submaximal concentration of epinephrine (67 +/- 19% vs. 27 +/- 9%, n = 5) and increased thromboxane formation when the platelet GPIIb/IIIa were clustered using monoclonal antibodies to the receptor. CONCLUSIONS: Orbofiban is both an antagonist and a partial agonist of platelet GPIIb/IIIa. At low concentrations of the drug, this partial agonist activity may enhance platelet aggregation. Along with suboptimal plasma drug levels, these findings may help explain the lack of efficacy seen with orbofiban in patients with ACS.