GPIIb/IIIa inhibitors: From bench to bedside and back to bench again

Transcription

1 808 Schattauer 2012 Invited Clinical Focus GPIIb/IIIa inhibitors: From bench to bedside and back to bench again Paul C. Armstrong; Karlheinz Peter Atherothrombosis & Vascular Biology, Baker IDI Heart & Diabetes Institute, Melbourne, Australia Summary From the discovery of the platelet glycoprotein (GP) IIb/IIIa and identification of its central role in haemostasis, the integrin GPIIb/IIIa (αiibβ3, CD41/CD61) was destined to be an anti-thrombotic target. The subsequent successful development of intravenous ligand-mimetic inhibitors occurred during a time of limited understanding of integrin physiology. Although efficient inhibitors of ligand binding, they also mimic ligand function. In the case of GPIIb/IIIa inhibitors, despite strongly inhibiting platelet aggregation, paradoxical fibrinogen binding and platelet activation can occur. The quick progression to development of small-molecule orally available inhibitors meant that this approach inherited many potential flaws, which together with a short half-life resulted in an increase in mortality and a halt to the numerous phar- maceutical development programs. Limited clinical benefits, together with the success of other anti-thrombotic drugs, in particular P 2 Y 12 ADP receptor blockers, have also led to a restrictive use of intravenous GPIIb/ IIIa inhibitors. However, with a greater understanding of this key platelet-specific integrin, GPIIb/IIIa remains a potentially attractive target and future drug developments will be better informed by the lessons learnt from taking the current inhibitors back to the bench. This overview will review the physiology behind the inherent problems of a ligand-based integrin inhibitor design and discuss novel promising approaches for GPIIb/IIIa inhibition. Keywords GP IIb/IIIa, platelet pharmacology, antiplatelet agents, thrombosis Correspondence to: Prof. Karlheinz Peter Atherothrombosis & Vascular Biology Baker IDI Heart & Diabetes Institute PO Box 6492 St Kilda Road Central Melbourne, 8008 VC, Australia Tel.: , Fax: karlheinz.peter@bakeridi.edu.au Received: October 21, 2011 Accepted after major revision: January 20, 2012 Prepublished online: February 28, 2012 doi: /th Thromb Haemost 2012; 107: Discovery of platelet glyocoproteins Glanzmann s thrombasthenia sufferers are characterised by a platelet-based bleeding disorder, first clinically described by Swiss physician Eduard Glanzmann in The underlying pathology of such a disease would remain ill-defined for a further 50 years until the optimisation of platelet washing techniques, protein radiolabelling, protease cleaving of surface receptors and SDS PAGE separation, allowed scientists such as Drs. Alan Nurden, David Phillips, James George and others to finally begin unravelling the complexities of platelet glycoproteins (GP), in particular IIb/IIIa (1). It was such discoveries that ultimately led us to the development of GPIIb/IIIa inhibitors. This vein of research continues in earnest as we continue to seek, and gain, insights into the relationship between glycoprotein structure and function, their suitability as therapeutic targets and, furthermore, the optimisation of therapeutic drug structure and application strategies. GPIIb/IIIa structure and ligand binding The GPIIb/IIIa complex numbers between 60,000 and 80,000 copies per platelet (2, 3). Also known as integrin αiibβ3 or in the CD nomenclature CD41/CD61, it is a heterodimeric complex formed after synthesis of one IIb (α) and one IIIa (β) subunit (4). Each subunit is coded by individual genes, which are closely located, although not commonly regulated, on chromosome 17 (5). During megakaryopoiesis, the α subunit is formed after undergoing intracellular proteolysis resulting in a 105 kda heavy chain and 25 kda light chain. Notable structural features include a betapropeller arrangement at the N-terminus and four divalent binding motifs at the base (6). In contrast, the beta-subunit is a 95 kda polypeptide chain, with an A-domain at the N-terminus containing two-three divalent cation sites, a PSI (plexin/ semaphoring/integrin) domain connecting to a protease resistant domain (7). The subunits utilise these divalent motifs to maintain a cation-dependant arrangement (8). The principal ligand for GPIIb/IIIa is fibrinogen; however, fibronectin, vitronectin and von Willebrand factor (vwf) have also been demonstrated to have the ability to bind (9). From these ligands, two predominant peptide sequences have been found to encode recognition. The first is Lys-Gln-Ala-Gly-Asp-Val (KQAGDV), found only in the carboxyl terminus of the fibrinogen gamma chain (10, 11). The second sequence, Arg-Gly-Asp (RGD) is found in the alpha chain with at least one copy contained in each known ligand. Of the amino acids in the recognised peptide sequences, the positively charged lysine (K) or arginine (R) residues Thrombosis and Haemostasis 107.5/2012

2 Armstrong, Peter: Basics of GPIIb/IIIa inhibitors 809 have been found to interact with the negatively charged aspartic acid 224 residue of the GPIIb beta-propeller. Likewise, the negatively charged aspartic acid (D) amino acid coordinates to the positively charged metal ion-dependent adhesion site (MIDAS) Mg 2+ ion in βi domain of IIIa (6, 12). The integrin nature of GPIIb/IIIa, through its assumption of conformational states, is also fundamental to facilitating the interaction with potential ligands. GPIIb/IIIa exists in a resting conformational state, where the integrin is bent and the headpiece in a closed form, meaning the RGD binding domain is concealed and thus it has only a low affinity for many physiological ligands. Upon appropriate stimulation, an inside-out signal, a conformational change occurs with the integrin transforming from a bent to an extended form with an opening of the headpiece, exposing the extracellular RGD ligand binding domain (13) resulting in the integrin having a much higher affinity for its ligands. This is followed by the unclasping of the tail sections of both subunits, structurally repositioning the transmembrane domains (see Fig. 1A). The intracellular pathways governing inside-out signalling are both numerous and complex. However, strong evidence exists demonstrating an important role for talin-1 and kindlin-3 translocation mediated by CalDAG-GEF1 and Rap1 activation (reviewed in more detail by Ye et al. [14], Petrich [15] and Stefanini et al. [16]). In addition, it is believed that the conformational change of GPIIb/IIIa may induce further signalling promoting actin polymerisation and cytoskeletal reorganisation in a process termed outside-in signalling (17). The exact mechanisms governing this process remain ill-defined, however a variety of molecules have been implicated including Src tyrosine kinases (18, 19), protein kinase C (20), phospholipases (21, 22) and phosphoinositide 3-kinase (23). More recently, Gong et al. have implicated the G-protein subunit G 13 as being a key mediator (24). Crucially, it is the above mentioned details concerning the conformational changes and ligand binding affinity that hold the key to our insights into utilising GPIIb/IIIa as a therapeutic target (25 29). Approved GPIIb/IIIa inhibitors There are currently three approved therapeutic inhibitors targeting GPIIb/IIIa. The first to be used extensively in patients was the recombinant monoclonal antibody (mab) abciximab (Reopro). Developed from the murine-raised 7E3 monoclonal antibody (mab) by Dr. Barry Coller (30), the variable (Fab) antigen-specific region was utilised and the Fc region replaced and humanised using regions of human immunoglobulin to form a chimeric antibody (31), successfully reducing immunogenicity. Abciximab was approved by the US Food and Drug Administration (FDA) in A second inhibitor, eptifibitide (Integrillin), was approved in 1996 and is based on the disintegrin, barbourin, obtained from snake venom. Developed by Drs. David Phillips and Robert Scarborough with COR therapeutics/portola pharmaceuticals; a Lys-Gly-Asp (KGD) sequence, analogous of the recognition sequences, is contained within a disulphide ring and acts as a competitive inhibitor for endogenous fibrinogen (32). In contrast, the third approved inhibitor tirofiban, is a small molecule non-peptide inhibitor developed by Merck. Using the RGD motif as a functional basis, an effective compound structure was established, which maintained the biochemical interactions important to the motif whilst removing the alpha-amino acids peptide bonds; a strategy which increased pharmacological survival time in vivo (33, 34). The first clinical data supporting the intravenous use of GPIIb/ IIIa inhibitors was established in early trials where abciximab (EPIC and EPILOG trials) and eptifibatide (IMPACT II and ESPRIT) were both found to achieve significant improvements in mortality in patients undergoing percutaneous coronary intervention (PCI) (35 38). Subsequent studies such as PRISM-PLUS (39) supported the use of tirofiban. These early studies also identified the importance of a bolus administration followed by an infusion. The relative risk reductions (RRR) achieved at 30-days with 35% and above were impressive and led to a widespread use of GPIIb/ IIIa inhibitors in cardiac catheter laboratories worldwide. However, the early clinical trials on GPIIb/IIIa inhibitors were conducted before the introduction of the routine use of P 2 Y 12 ADP receptor blockers and also before the introduction of newer anticoagulants such as bivalirudin. In more recent trials, pretreatment with the P 2 Y 12 blocker clopidogrel seems to offset the beneficial effects of GPIIb/IIIa inhibitors in patients undergoing elective, lowrisk PCI or in diabetic patients (40, 41). A very recent meta-analysis including trials on elective PCI concluded that the use of GPIIb/ IIIa blockers in combination with ADP receptor blockers did not improve mortality but are associated with a significant reduction of non-fatal myocardial infarction (49). Numerous clinical trials investigating GPIIb/IIIa treatment in patients with unstable angina, non-st-elevation as well as ST-elevation myocardial infarction, who are pretreated with P 2 Y 12 blocker, demonstrated no additional benefits of GPIIb/IIIa inhibitors and thus resulted in recommendations that limit GPIIb/IIIa blockers to high-risk cases only, such as high thrombus burden for example (42, 43). However, if patients did not receive timely pretreatment with an ADP receptor blocker or, as demonstrated in the recent 3T/2R study (44), reveal a low response to treatment with aspirin and/or clopidogrel then GPIIb/IIIa inhibitors are recommended for patients undergoing PCI. Two concerns, which also arose during these studies, were the occurrence of thrombocytopenia and the increased risk of major and minor bleeding complications. Thrombocytopenia is more frequently reported with abciximab than with eptifibatide and tirofiban (45, 46). Nevertheless, severe clinical problems arising from thrombocytopenia have not been seen to significantly influence outcome in the numerous clinical trials conducted with GPIIb/IIIa inhibitors. However, bleeding complications are more and more recognised as major determinant of clinical outcome in PCI (47 49). This is particularly true for the currently clinically used GPIIb/IIIa inhibitors. For example, although GPIIb/IIIa inhibition was hoped to be pathophysiologically beneficial when given in combination with fibrinolytic drugs, as these have been shown to have a platelet-activating side effect, increased bleeding Schattauer 2012 Thrombosis and Haemostasis 107.5/2012

3 810 Armstrong, Peter: Basics of GPIIb/IIIa inhibitors complications have resulted in the recommendation not to combine fibrinolysis and GPIIb/IIIa inhibition (42, 49 51). Overall in comparison to the wide spread use of GPIIb/IIIa blockers directly following their approval, the use of these drugs nowadays has dropped substantially. Based on contradictory study outcomes and changing use, as well as availability of other new antithrombotic drugs in particular P 2 Y 12 receptor blockers, the clinical role of GPIIb/IIIa inhibitors is poorly defined. In addition, it will keep changing and remain to be a matter of controversial discussion amongst cardiologists. Most cardiologists though agree that the specific but rather small group of patients without pretreatment with P 2 Y 12 blockers as well as high-risk patients in regards to lesion pathology (e.g. large thrombus burden) will benefit from GPIIb/IIIa inhibitors. Figure 1: Cartoon models of the conformational states of GPIIb/IIIa under physiological conditions or in presence of inhibitors. A) GPIIb/IIIa exists in a bent conformation in the resting state (stage 1) whereupon insideout signalling induces a change to an extended conformation with increased ligand affinity (stage 2). Engagement of a ligand binding, such as fibrinogen, causes the IIb and IIIa subunits to unclasp and induce outside-in signalling (stage 3). B) Ligand (RGD)-mimetic therapeutics are the basis for the currently approved GPIIb/IIIa inhibitors. These are able to bind in a low affinity resting state but induce integrin extension causing a conformational change (also termed: priming). When these agents leave their interaction site and the drug plasma levels drop, fibrinogen or other ligands are able to bind. C) Future therapeutics include allosteric agents which hold the integrin in the bent resting conformation, GPIIb-specific structures, which do not intrinsically induce nor inhibit extension to stage 2, or activation-specific singlechain antibodies (scfv), which block ligand binding but only once stage 2 has been achieved. Figure based on Zhu et al. (12). Thrombosis and Haemostasis 107.5/2012 Schattauer 2012

4 Armstrong, Peter: Basics of GPIIb/IIIa inhibitors 811 Failure of oral inhibitors The approved and initially developed GPIIb/IIIa inhibitors could only be administered intravenously. Therefore, orally available structures were developed by several companies as a potential primary and secondary prevention therapy, potentially providing benefits to millions of patients (thus the term superaspirin was used for the proposed oral GPIIb/IIIa inhibitors). In total five major phase III trials (including over 42,000 patients) testing four different compounds were completed, reviewed in more detail by Cox (52). Two studies EXCITE and SYMPHONY, found no significant improvement over existing therapy (aspirin) and OPUS and BRAVO were terminated early due to excess mortality, including cardiovascular mortality (53 57). This negative clinical outcome essentially ended the prophylactic potential for GPIIb/IIIa inhibitors and led to the cancellation of all development programmes in this drug class with large financial loss to many of the pharmaceutical companies (52). Ligand-mimetic GPIIb/IIIa inhibition: The wrong strategy for the right target? Eighteen years on from the initial regulatory approval and the fast following commercial success, beginning with the failure of the oral GPIIb/IIIa inhibitor program and continuing with ongoing negative outcomes of large clinical trials, the enthusiasm that was once held for GPIIb/IIIa inhibitors has diminished (58). The unexpected finding of increased mortality caused by oral GPIIb/IIIa inhibitors and the limited benefits seen with intravenous GPIIb/IIIa inhibitors has not only questioned the specific GPIIb/IIIa inhibitors but also GPIIb/IIIa as a therapeutic target in general. However, there is increasing evidence that it is not the target as such but the targeting strategy that caused problems and based on a better understanding of the integrin physiology promising alternative targeting strategies are currently under development (58 60). Integrin function was initially considered purely as mechanotransducer connecting the cell with ligands in the extracellular matrix or on the surface of other cells. Thus, the concept of using reagents that imitate ligands to inhibit this function was logical. Of the three approved agents, the R(K)GD-based reagents tirofiban and eptifibatide directly imitate fibrinogen binding, whereas the mab abciximab, binds close to the fibrinogen binding pocket (61), all three can be considered to function as a ligand mimetic. However, integrins also function as mechano-sensors and detect binding of ligands by conformational change that is then transferred as an outside-in signal into the cell (62). Du et al. demonstrated in 1991 that RGD peptides can cause a conformational change of GPIIb/IIIa (63). This response implied that integrins may be used by cells to sense their surroundings through ligandspecific responses; however, at this time the full implications were not known. Ligand-mimetic GPIIb/IIIa inhibitor induced conformational change and its link to paradoxical fibrinogen binding and potential platelet activation was first demonstrated by Peter et al. (29). Electron microscopy and crystallisation studies of GPIIb/ IIIa occupied by ligand-mimetic inhibitors by Xiao et al. later provided proof of concept as well as a detailed structural model of how ligand-mimetic inhibitors induce a conformational change of GPIIb/IIIa and thereby induce outside-in signalling (7). This platelet activation is now known to induce the expression of CD40L, P-selectin, CD63, and phosphatidylserine as well as the release of dense granule content and other stored pro-inflammatory mediators (64 67). The release of such mediators may contribute to concurrent paracrine platelet activation or inflammatory stimuli. One consequence of the induced conformational change of GPIIb/IIIa is the exposure of what have been termed ligand-induced binding sites (LIBS, see Fig. 1). A small number of patients have preformed circulating antibodies against these LIBS. When treated with intravenous ligand-mimetic GPIIb/IIIa inhibitors this can result in a rapid immunological reaction, causing thrombocytopenia (68 70). It is the paradoxical fibrinogen binding to GPIIb/IIIa that is thought to be especially pertinent to the failure of the oral inhibitor class. As reversible small molecule ligand-mimetic inhibitors with relatively short half-lives (31) it would continually be binding and leaving the receptor, inducing GPIIb/IIIa to adopt a high-affinity conformation. During periods of high plasma drug level, sufficient drug is present to successfully compete with any physiological ligand. However, during the troughs in the plasma drug levels, the GPIIb/IIIa would remain in a high affinity confirmation with the binding site exposed and more accessible to ligands (71 73). The repeated peaks and troughs of drug plasma levels and the paradoxical induction of fibrinogen binding are hypothesised, but not proven, to be contributing factors to the increased mortality associated with this drug class. Findings with other integrins indicate that ligand-mimetic inhibition might indeed have particular side effects at low concentrations (46). As an important example for this Reynolds et al. recently demonstrated that RGD-mimetic inhibitors cause paradoxical effects at low concentrations resulting in the induction instead of inhibition of tumour growth (74). Another characteristic of the currently used GPIIb/IIIa inhibitors, although the relevance is difficult to determine, is the lack of specificity for the target integrin GPIIb/IIIa. Abciximab binds to the integrins αvβ3 (75) as well as αmβ2 (Mac-1, CD11b/CD18) (76), whereas tirofiban and eptifibatide possess various levels of cross-reactivities to integrin receptors that share RGD containing ligands, such as fibronectin (77). The three approved GPIIb/IIIa inhibitors differ in their pharmacology, particularly in their binding affinity towards GPIIb/IIIa with abciximab demonstrating the strongest affinity and longest half-life (78). The question arises whether these drugs also differ in their characteristic to mimic ligand binding to GPIIb/IIIa and thus their ability to cause paradoxical fibrinogen binding and potentially platelet activation. Using LIBS exposure as a measure of conformational change of GPIIb/IIIa, abciximab induced less binding of an anti-libs antibody to GPIIb/IIIa than eptifibatide and tirofiban (79). Whether this results in less paradoxical side effects with the use of abciximab compared to eptifibatide or tirofiban has not been investigated yet. In the few trials, in which GPIIb/IIIa block- Schattauer 2012 Thrombosis and Haemostasis 107.5/2012

5 812 Armstrong, Peter: Basics of GPIIb/IIIa inhibitors ers were compared to each other, abciximab seems to provide greater benefits compared to small molecule GPIIb/IIIa inhibitors (80 82). However, several factors besides differences in induced conformational change may account for this, such as differences in receptor affinity and differences in the percentage of GPIIb/IIIa receptors blocked by the three competing GPIIb/IIIa inhibitors. Future novel strategies targeting GPIIb/IIIa Whilst the currently approved inhibitors have not been as successful as originally hoped or intended, GPIIb/IIIa remains to be a promising target for future developments of anti-platelet drugs. Recent work by Drs. Robert Blue and Barry Coller has identified a novel type of inhibitor, RUC-1, which effectively inhibits fibrinogen binding to GPIIb/IIIa and thus agonist-induced platelet aggregation (83, 84). However, in contrast to the ligand-mimetic based antagonists discussed above, this novel compound only binds to the GPIIb subunit but not to GPIIIa ( Fig. 1C). This represents a novel and attractive strategy of inhibiting GPIIb/IIIa, without the intrinsic property of inducing a conformational change of GPIIb/IIIa (12). By avoiding such a change it is anticipated that associated thrombocytopenia, induction of fibrinogen binding and paradoxical platelet activation would be prevented. Whether or not the current molecule structure is to prove clinically successful, it offers an informative basis for the rational design of future anti-gpiib/iiia drugs but also anti-integrin drugs in general. Another potential alternative approach aiming to avoid a conformational change of the non-activated GPIIb/IIIa, is to block the activated form of the integrin receptor GPIIb/IIIa only ( Fig. 1C). Using phage display technology, Schwarz et al. (71, 85) generated human single-chain antibodies that only bind to the activated form of GPIIb/IIIa. These single-chain antibodies, which are small recombinant antibody fragments consisting of the variable regions of the antibody heavy and light chain fused together to a single molecule, have attracted major interest from the pharmaceutical industry based on their expected low immunogenicity especially when human antibody libraries are used (71, 85, 86). These anti- GPIIb/IIIa single-chain antibodies were systematically tested in vitro and in vivo and it was shown that they do not cause a conformational change of GPIIb/IIIa and thus do not induce LIBS epitope expression, which makes the development of thrombocytopenia less likely. Furthermore, they do not induce paradoxical fibrinogen binding to GPIIb/IIIa or paradoxical platelet activation (85). Their selective binding and blocking of activated platelets can explain the unique and promising in vivo finding of strong antithrombotic properties without bleeding time prolongation (85). Whereas platelet adhesion was still intact, platelet aggregation and therefore thrombus formation was effectively inhibited (85). Furthermore, this GPIIb/IIIa inhibition approach is highly flexible as additional effector molecules can be fused to these activation-specific single chain antibodies allowing not only blocking of GPIIb/ IIIa but also targeting and thus enrichment for example of fibrinolytics or anticoagulants at the activated platelet and thus clot (87). In addition such a genetic fusion for example of a molecular switch, allowed the development of a unique temperature steerable GPIIb/IIIa inhibitor that can be turned on by hypothermia and off by rewarming to body temperature. This would allow the use of a GPIIb/IIIa blocker as effective anti-thrombotic protection during hypothermic extracorporeal circulation with immediate recovery of platelet function on rewarming at the end of surgery (88). Allosteric inhibition has been shown to be possible for the integrins LFA-1 (αlβ2) (89) and Mac-1 (αmβ2) using small molecules (90). This potential approach would involve holding GPIIb/ IIIa in the bent conformation ( Fig. 1C). One proposed approach is to target residues on the β3 subunit, as this region has been identified as being a fulcrum to the conformational hinge and as such offers a potential allosteric inhibition site (91). Each of these approaches is so far experimental in nature only and is yet to reach clinical testing. It is also an open question whether after the massive financial losses of the pharmaceutical industry in the development of oral ligand-mimetic GPIIb/IIIa blockers and the fading financial benefits of intravenous GPIIb/ IIIa blockers there will be enough financial support available that is necessary for the clinical testing of new GPIIb/IIIa blockers. Nevertheless, it is hoped that this review might provide the basis for a better understanding of the problems of the current clinically used GPIIb/IIIa blocking strategy and might help to promote the development of newer and better approaches to inhibit thrombus formation by targeting GPIIb/IIIa. Conclusion GPIIb/IIIa plays a critical role in the regulation of platelet aggregation and clinical thrombosis and was, and still is, therefore a rational target in the search for improved anti-thrombotic therapeutics. However significant limitations associated with the currently approved inhibitors, coupled with the failure of the oral GPIIb/IIIa inhibitors, has led to reduced expectations and questioning of the suitability of GPIIb/IIIa as a good target for anti-platelet/anti-thrombotic therapy. The currently clinically used intravenous GPIIb/IIIa inhibitors have helped to establish the benefits of anti-platelet therapy in PCI. However, the emergence of P 2 Y 12 ADP receptor blockers as routine pretreatment of patients undergoing elective PCI or PCI whilst suffering from acute coronary syndrome has offset many of the benefits initially seen with intravenous GPIIb/IIIa inhibitors. Nevertheless, in patients without timely or effective P 2 Y 12 blocker treatment and in high risk patients (e.g. high thrombus burden) and despite a potentially problematic inhibition strategy the use of the currently clinically available GPIIb/IIIa inhibitors provide some proven benefits. In addition, it is in part due to these limitations and lack of success that has directed research to further investigate and gain greater understanding of the mechanisms of GPIIb/IIIa inhibition. The progress in this research area, as outlined above, infers that we may now be able to consider the current inhibitors as first-generation and with this increased knowledge as well as additional bench work we can im- Thrombosis and Haemostasis 107.5/2012 Schattauer 2012

6 Armstrong, Peter: Basics of GPIIb/IIIa inhibitors 813 prove future drug design appropriately and identify more suitable strategies for maximising the protection offered by GPIIb/IIIa inhibition. The second generation of GPIIb/IIIa inhibitors remain to be clinically tested. Since bleeding complications have been the reason for major clinical limitations of the currently clinically used GPIIb/IIIa, the activation-specific anti-gpiib/iiia inhibitors seem to be particularly attractive for further drug development. Conflicts of interest None declared. References 1. Nurden AT, et al. Platelet membrane glycoproteins: historical perspectives. J Thromb Haemost 2006; 4: Wagner C, et al. Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets. Blood 1996; 88: Quinn M, et al. Quantifying GPIIb/IIIa receptor binding using 2 monoclonal antibodies: discriminating abciximab and small molecular weight antagonists. Circulation 1999; 99: Duperray A, et al. Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-gpiib and GPIIIa is a prerequisite for expression of the complex on the cell surface. Blood 1989; 74: Thornton MA, et al. The Human Platelet αiib Gene Is Not Closely Linked to Its Integrin Partner β3. Blood 1999; 94: Xiong JP, et al. Crystal structure of the extracellular segment of integrin alpha Vbeta3 in complex with an Arg-Gly-Asp ligand. Science 2002; 296: Xiao T, et al. Structural basis for allostery in integrins and binding to fibrinogenmimetic therapeutics. Nature 2004; 432: Calvete JJ. Clues for understanding the structure and function of a prototypic human integrin: the platelet glycoprotein IIb/IIIa complex. Thromb Haemost 1994; 72: Phillips DR, et al. The platelet membrane glycoprotein IIb-IIIa complex. Blood 1988; 71: Weisel JW, et al. Examination of the platelet membrane glycoprotein IIb-IIIa complex and its interaction with fibrinogen and other ligands by electron microscopy. J Biol Chem 1992; 267: Farrell DH, et al. Role of fibrinogen alpha and gamma chain sites in platelet aggregation. Proc Natl Acad Sci USA 1992; 89: Zhu J, et al. Closed headpiece of integrin alphaiibbeta3 and its complex with an alphaiibbeta3-specific antagonist that does not induce opening. Blood 2010; 116: Ma YQ, et al. Platelet integrin αiibβ3: activation mechanisms. J Thromb Haemost 2007; 5: Ye F, et al. Molecular mechanism of inside-out integrin regulation. J Thromb Haemost 2011; 9 (Suppl 1): Petrich BG. Talin-dependent integrin signalling in vivo. Thromb Haemost 2009; 101: Stefanini L, Bergmeier W. CalDAG-GEFI and platelet activation. Platelets 2010; 21: Coller BS, Shattil SJ. The GPIIb/IIIa (integrin alphaiibbeta3) odyssey: a technology-driven saga of a receptor with twists, turns, and even a bend. Blood 2008; 112: Arias-Salgado EG, et al. Src kinase activation by direct interaction with the integrin β cytoplasmic domain. Proc Natl Acad Sci 2003; 100: Flevaris P, et al. A molecular switch that controls cell spreading and retraction. J Cell Biol 2007; 179: Soriani A, et al. A role for PKC theta in outside-in alpha(iib)beta(3) signaling. J Thromb Haemost 2006; 4: Elvers M, et al. Impaired alpha(iib)beta(3) integrin activation and shear-dependent thrombus formation in mice lacking phospholipase D1. Sci Signal 2010; 3: ra Prevost N, et al. Group IVA cytosolic phospholipase A(2) (cpla(2)alpha) and integrin alpha IIb beta 3 reinforce each other's functions during alpha IIb beta 3 signaling in platelets. Blood 2009; 113: Schoenwaelder SM, et al. Identification of a unique co-operative phosphoinositide 3-kinase signaling mechanism regulating integrin alpha(iib)beta(3) adhesive function in platelets. J Biol Chem 2007; 282: Gong H, et al. G protein subunit Galpha13 binds to integrin alphaiibbeta3 and mediates integrin outside-in signaling. Science 2010; 327: Zhu J, et al. Tests of the extension and deadbolt models of integrin activation. J Biol Chem 2007; 282: Gao C, et al. Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcgammaRIIa and the integrin beta3 cytoplasmic domain. J Clin Invest 2009; 119: Jennings LK, et al. Differential expression of a ligand induced binding site (LIBS) by GPIIb-IIIa ligand recognition peptides and parenteral antagonists. Thromb Haemost 2000; 84: Zhu J, et al. Requirement of alpha and beta subunit transmembrane helix separation for integrin outside-in signaling. Blood 2007; 110: Peter K, et al. Induction of fibrinogen binding and platelet aggregation as a potential intrinsic property of various glycoprotein IIb/IIIa (alphaiibbeta3) inhibitors. Blood 1998; 92: Knight DM, et al. The immunogenicity of the 7E3 murine monoclonal Fab antibody fragment variable region is dramatically reduced in humans by substitution of human for murine constant regions. Mol Immun 1995; 32: Topol EJ, et al. Platelet GPIIb-IIIa blockers. Lancet 1999; 353: Phillips DR, Scarborough RM. Clinical pharmacology of eptifibatide. Am J Cardiol 1997; 80: 11B-20B. 33. Egbertson MS, et al. Non-peptide fibrinogen receptor antagonists. optimization of a tyrosine template as a mimic for Arg-Gly-Asp. J Med Chem 1994; 37: Lefkovits J, et al. Platelet glycoprotein IIb/IIIa receptors in cardiovascular medicine. N Engl J Med 1995; 332: The EPIC Investigators. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty. N Engl J Med 1994; 330: The EPILOG Investigators. Platelet glycoprotein IIb/IIIa receptor blockade and low-dose heparin during percutaneous coronary revascularization. N Engl J Med 1997; 336: The IMPACT-II Investigators. Randomised placebo-controlled trial of effect of eptifibatide on complications of percutaneous coronary intervention: IMPACT- II. Lancet 1997; 349: The ESPRIT Investigators. Novel dosing regimen of eptifibatide in planned coronary stent implantation (ESPRIT): a randomised, placebo-controlled trial. Lancet 2000; 356: Theroux P, et al. Glycoprotein IIb/IIIa receptor blockade improves outcomes in diabetic patients presenting with unstable angina/non-st-elevation myocardial infarction: results from the Platelet Receptor Inhibition in Ischemic Syndrome Management in Patients Limited by Unstable Signs and Symptoms (PRISM- PLUS) study. Circulation 2000; 102: Mehilli J, et al. Randomized clinical trial of abciximab in diabetic patients undergoing elective percutaneous coronary interventions after treatment with a high loading dose of clopidogrel. Circulation 2004; 110: Schuhlen H, et al. Abciximab and angiographic restenosis after coronary stent placement. Analysis of the angiographic substudy of ISAR-REACT--a doubleblind, placebo-controlled, randomized trial evaluating abciximab in patients undergoing elective percutaneous coronary interventions after pretreatment with a high loading dose of clopidogrel. Am Heart J 2006; 151: Steg PG, et al. Bleeding in acute coronary syndromes and percutaneous coronary interventions: position paper by the Working Group on Thrombosis of the European Society of Cardiology. Eur Heart J 2011; 32: Mehilli J, et al. Abciximab in patients with acute ST-segment-elevation myocardial infarction undergoing primary percutaneous coronary intervention after clopidogrel loading: a randomized double-blind trial. Circulation 2009; 119: Valgimigli M, et al. Intensifying platelet inhibition with tirofiban in poor responders to aspirin, clopidogrel, or both agents undergoing elective coronary intervention: results from the double-blind, prospective, randomized Tailoring Treatment with Tirofiban in Patients Showing Resistance to Aspirin and/or Resistance to Clopidogrel study. Circulation 2009; 119: Berkowitz SD, et al. Occurrence and clinical significance of thrombocytopenia in a population undergoing high-risk percutaneous coronary revascularization. Evaluation of c7e3 for the Prevention of Ischemic Complications (EPIC) Study Group. J Am Coll Cardiol 1998; 32: Schattauer 2012 Thrombosis and Haemostasis 107.5/2012

7 814 Armstrong, Peter: Basics of GPIIb/IIIa inhibitors 46. Aster RH, et al. Thrombocytopenia associated with the use of GPIIb/IIIa inhibitors: position paper of the ISTH working group on thrombocytopenia and GPIIb/IIIa inhibitors. J Thromb Haemost 2006; 4: Manoukian SV. The relationship between bleeding and adverse outcomes in ACS and PCI: pharmacologic and nonpharmacologic modification of risk. J Invasive Cardiol 2010; 22: Kim YH, et al. Impact of bleeding on subsequent early and late mortality after drug-eluting stent implantation. JACC Cardiovasc Interv 2011; 4: Starnes HB, et al. Optimal use of platelet glycoprotein IIb/IIIa receptor antagonists in patients undergoing percutaneous coronary interventions. Drugs 2011; 71: Brener SJ, et al. The relationship between baseline risk and mortality in ST-elevation acute myocardial infarction treated with pharmacological reperfusion: insights from the Global Utilization of Strategies To open Occluded arteries (GUSTO) V trial. Am Heart J 2005; 150: Sinnaeve PR, et al. Efficacy of tenecteplase in combination with enoxaparin, abciximab, or unfractionated heparin: one-year follow-up results of the Assessment of the Safety of a New Thrombolytic-3 (ASSENT-3) randomized trial in acute myocardial infarction. Am Heart J 2004; 147: Cox D. Oral GPIIb/IIIa antagonists: what went wrong? Curr Pharm Des 2004; 10: Chew DP, et al. Increased mortality with oral platelet glycoprotein IIb/IIIa antagonists: a meta-analysis of phase III multicenter randomized trials. Circulation 2001; 103: Cox D, et al. Evidence of platelet activation during treatment with a GPIIb/IIIa antagonist in patients presenting with acute coronary syndromes. J Am Coll Cardiol 2000; 36: Holmes MB, et al. Increased platelet reactivity in patients given orbofiban after an acute coronary syndrome: an OPUS-TIMI 16 substudy. Orbofiban in Patients with Unstable coronary syndromes. Thrombolysis In Myocardial Infarction. Am J Cardiol 2000; 85: , A The SYMPHONY Investigators. Comparison of sibrafiban with aspirin for prevention of cardiovascular events after acute coronary syndromes: a randomised trial. Sibrafiban versus Aspirin to Yield Maximum Protection from Ischemic Heart Events Post-acute Coronary Syndromes. Lancet 2000; 355: O'Neill WW, et al. Long-term treatment with a platelet glycoprotein-receptor antagonist after percutaneous coronary revascularization. EXCITE Trial Investigators. Evaluation of Oral Xemilofiban in Controlling Thrombotic Events. N Engl J Med 2000; 342: Kristensen S, et al. Contemporary use of glycoprotein IIb/IIIa inhibitors. Thromb Haemost 2012; 107: Lip GY, et al. Management of antithrombotic therapy in atrial fibrillation patients presenting with acute coronary syndrome and/or undergoing percutaneous coronary intervention/ stenting. Thromb Haemost 2010; 103: Winchester DE, et al. Efficacy and Safety of Glycoprotein IIb/IIIa Inhibitors During Elective Coronary Revascularization: A Meta-Analysis of Randomized Trials Performed in the Era of Stents and Thienopyridines. J Am Coll Cardiol 2011; 57: Artoni A, et al. Integrin beta3 regions controlling binding of murine mab 7E3: implications for the mechanism of integrin alphaiibbeta3 activation. Proc Natl Acad Sci USA 2004; 101: Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002; 110: Du XP, et al. Ligands activate integrin alpha IIb beta 3 (platelet GPIIb-IIIa). Cell 1991; 65: Bassler N, et al. A mechanistic model for paradoxical platelet activation by ligandmimetic alphaiib beta3 (GPIIb/IIIa) antagonists. Arterioscler Thromb Vasc Biol 2007; 27: e Peter K, et al. Intrinsic activating properties of GP IIb/IIIa blockers. Thromb Res 2001; 103 (Suppl 1): S Cox D, et al. Integrins as therapeutic targets: lessons and opportunities. Nat Rev Drug Discov 2010; 9: Jones ML, et al. RGD-ligand mimetic antagonists of integrin alphaiibbeta3 paradoxically enhance GPVI-induced human platelet activation. J Thromb Haemost 2010; 8: Attaya S, et al. Acute profound thrombocytopenia with second exposure to eptifibatide associated with a strong antibody reaction. Platelets 2009; 20: Peter K, et al. Platelet activation as a potential mechanism of GP IIb/IIIa inhibitorinduced thrombocytopenia. Am J Cardiol 1999; 84: Bednar B, et al. Fibrinogen receptor antagonist-induced thrombocytopenia in chimpanzee and rhesus monkey associated with preexisting drug-dependent antibodies to platelet glycoprotein IIb/IIIa. Blood 1999; 94: Schwarz M, et al. Single-chain antibodies for the conformation-specific blockade of activated platelet integrin alphaiibbeta3 designed by subtractive selection from naive human phage libraries. FASEB J 2004; 18: Hantgan RR, Stahle MC. Integrin priming dynamics: mechanisms of integrin antagonist-promoted alphaiibbeta3: PAC-1 molecular recognition. Biochemistry 2009; 48: Hantgan RR, et al. αiibβ3 priming and clustering by orally active and intravenous integrin antagonists. J Thromb Haemost 2007; 5: Reynolds AR, et al. Stimulation of tumor growth and angiogenesis by low concentrations of RGD-mimetic integrin inhibitors. Nat Med 2009; 15: Lele M, et al. Eptifibatide and 7E3, but not tirofiban, inhibit alpha(v)beta(3) integrin-mediated binding of smooth muscle cells to thrombospondin and prothrombin. Circulation 2001; 104: Schwarz M, et al. The GP IIb/IIIa inhibitor abciximab (c7e3) inhibits the binding of various ligands to the leukocyte integrin Mac-1 (CD11b/CD18, alphambeta2). Thromb Res 2002; 107: Coller BS. Anti-GPIIb/IIIa drugs: current strategies and future directions. Thromb Haemost 2001; 86: Peter K, et al. Flow cytometric monitoring of glycoprotein IIb/IIIa blockade and platelet function in patients with acute myocardial infarction receiving reteplase, abciximab, and ticlopidine: continuous platelet inhibition by the combination of abciximab and ticlopidine. Circulation 2000; 102: Schwarz M, et al. Reversibility versus persistence of GPIIb/IIIa blocker-induced conformational change of GPIIb/IIIa (alphaiibbeta3, CD41/CD61). J Pharmacol Exp Ther 2004; 308: Moliterno DJ, et al. Outcomes at 6 months for the direct comparison of tirofiban and abciximab during percutaneous coronary revascularisation with stent placement: the TARGET follow-up study. Lancet 2002; 360: Van't Hof AW, et al. Prehospital initiation of tirofiban in patients with ST-elevation myocardial infarction undergoing primary angioplasty (On-TIME 2): a multicentre, double-blind, randomised controlled trial. Lancet 2008; 372: Huber K, et al. Use of glycoprotein IIb/IIIa inhibitors in primary percutaneous coronary intervention: insights from the APEX-AMI trial. Eur Heart J 2010; 31: Blue R, et al. Structural and therapeutic insights from the species specificity and in vivo antithrombotic activity of a novel alphaiib-specific alphaiibbeta3 antagonist. Blood 2009; 114: Blue R, et al. Application of high-throughput screening to identify a novel αiibspecific small- molecule inhibitor of αiibβ3-mediated platelet interaction with fibrinogen. Blood 2008; 111: Schwarz M, et al. Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets. Circ Res 2006; 99: Hagemeyer CE, et al. Single-chain antibodies as diagnostic tools and therapeutic agents. Thromb Haemost 2009; 101: Stoll P, et al. Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation. Arterioscler Thromb Vasc Biol 2007; 27: Topcic D, et al. An activation-specific platelet inhibitor that can be turned on/off by medically used hypothermia. Arterioscler Thromb Vasc Biol 2011; 31: Weitz-Schmidt G, et al. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat Med 2001; 7: Ahrens I, Peter K. Therapeutic integrin inhibition: allosteric and activation-specific inhibition strategies may surpass the initial ligand-mimetic strategies. Thromb Haemost 2008; 99: Haas TA. Allosteric regulation of alpha(iib)beta(3) by beta(3) Thromb Haemost 2008; 99: Thrombosis and Haemostasis 107.5/2012 Schattauer 2012