An αIIbβ3 antagonist prevents thrombosis without causing Fc receptor γ‐chain IIa‐mediated thrombocytopenia

Abstract

Background Thrombocytopenia, a common side effect of Arg-Gly-Asp-mimetic antiplatelet drugs, is associated with drug-dependent antibodies (DDAbs) that recognize conformation-altered integrin α β . Objective To explore the correlation between α β binding epitopes and induction of DDAb binding to conformation-altered α β , we examined whether two purified disintegrins, TMV-2 and TMV-7, with distinct binding motifs have different effects on induction of α β conformational change and platelet aggregation in the presence of AP2, an IgG inhibitory mAb raised against α β . Methods We investigated the possible mechanisms of intrinsic platelet activation of TMV-2 and TMV-7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ-chain IIa (FcγRIIa) transgenic mice. Results TMV-7 has a binding motif that recognizes the α β-propeller domain of α β , unlike that of TMV-2. TMV-7 neither primed the platelets to bind ligand, nor caused a conformational change of α β as identified with the ligand-induced binding site mAb AP5. In contrast to eptifibatide and TMV-2, cotreatment of TMV-7 with AP2 did not induce FcγRIIa-mediated platelet aggregation and the downstream activation cascade. Both TMV-2 and TMV-7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV-2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV-7 did not impair hemostatic capacity. Conclusions TMV-7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested α β antagonists, and may offer advantages as a therapeutic agent with a better safety profile.

A Novel αIIbβ3 Antagonist from Snake Venom Prevents Thrombosis without Causing Bleeding

“…Thrombocytopenia can occur on first exposure to RGD mimetic drugs and the platelet count declines abruptly within hours of commencement of drug administration [45], suggesting the presence of naturally occurring antiplatelet antibody [46]. Eptifibatide-or tirofiban-dependent antibodies (i.e., DDAbs) recognize ligand-induced binding sites (LIBS) in a drug-dependent manner [12,45,47], and subsequently recruit FcγRIIa and trigger a FcγRIIa-mediated platelet aggregation and consumption [19]. It has been reported that these intrinsic antibodies are capable of directly increasing in circulating procoagulant, suggesting that platelet activation might, in some instances, contribute to occasional thrombocytopenia [14].…”

“…We previously reported that upon drug-induced LIBS exposure, the mimetic drug-dependent antibody (DDAb) mAb, AP2, which was raised against the epitopes of IIb3, recruited FcRIIa [19]…”

“…We previously reported that upon drug-induced LIBS exposure, the mimetic drug-dependent antibody (DDAb) mAb, AP2, which was raised against the epitopes of α IIb β 3 , recruited FcγRIIa [19] and elicited FcγRIIa-mediated platelet aggregation [29] and platelet consumption [11]. The recent report also indicated that eptifibatide-induced thrombocytopenia is associated with an increase in circulating procoagulant platelet-derived microparticles [14].…”

“…Insoluble materials were removed by centrifugation at 14,000 rpm for 15 min at 4 • C. Aliquots of cell lysates were resolved on 10% SDS-PAGE under reducing conditions and electrotransferred to Immobilon-PVDF membrane (Millipore). Western blotting and immunoprecipitation were conducted as described previously [19].…”

“…To address this issue, two disintegrins with different hemorrhagic properties, TFV-1 and TFV-3, were purified from Protobothrops flavoviridis venom. Previously, we found that eptifibatide or disintegrin TMV-2, in combination with 10E5 or AP2, the inhibitory monoclonal antibodies (mAbs) raised against α IIb β 3 , specifically triggered platelet activation [19]. Because of the unique properties of these mAbs, we used them as probes to clarify binding to α IIb β 3 and the pro-hemorrhagic tendencies of disintegrins.…”