A novel αIIbβ3 antagonist TFV-1 derived from snake venom prevents thrombosis without causing bleeding

Abstract

Current alphaIIbbeta3 antagonists are efficacious anti-thrombotics but have significant bleeding risk. The life-threatening thrombocytopenia and bleeding, common side effects of clinically available alphaIIbbeta3 antagonists, are associated with induction of ligand-induced integrin conformational changes and inhibition of inside-out signaling. Emerging evidence suggests that selective inhibition of outside-in signaling has potent antithrombotic effects without causing bleeding. To address this issue, we examined whether two purified disintegrins TFV-1 and TFV-3, from Trimeresurus flavoviridis snake venom, with distinct binding motifs that have different effects on the induction of alphaIIbbeta3 conformational change and integrin bidirectional signaling. Experiments were performed to explore either the mechanisms of intrinsic platelet activation of disintegrins in the presence of AP2 by examining the signal cascade or the selectively inhibitory activity of disintegrin on Galpha13 binding to integrin beta3 using co-immunoprecipitation between Galpha13-beta3 and talin-beta3. TFV-1 with different binding epitopes of alphaIIbbeta3 from that of TFV-3 and 7E3 could decelerate alphaIIbbeta3 ligation without causing conformational change of integrin and triggering FcgammaRII-mediating platelet aggregation via recruitment of AP2, an inhibitory monoclonal antibody raised against alphaIIbbeta3. Moreover, TFV-1 neither increased AP2 binding nor primed the platelets to bind fibrinogen or PAC-1, whereas TFV-3 did. Our study also revealed that TFV-1 selectively inhibits Galpha13 binding to integrin beta3 without inhibiting talin binding to beta3 in thrombin-stimulated human and mouse platelets, in contrast to TFV-3 and eptifibatide. Furthermore, TFV-3 inhibited thrombin-induced clot retraction of platelet-rich plasma whereas TFV-1 did not. Notably, TFV-1 and TFV-3 potently inhibited FeCl3-induced occlusive thrombosis in vivo, but TFV-1 did not cause severe thrombocytopenia mediated by FcgammaRIIa and prolonged tail bleeding time, compared to eptifibatide and TFV-3. To mimic hemorrhage during surgical procedures, we used ROTEM method of surgical hemorrhage. TFV-3 and eptifibatide repressed human whole blood coagulation-indexes, whereas TFV-1 did not impair hemostatic capacity as they were administered at efficacious antithrombotic doses. These findings highlight the alphaIIbbeta3 antagonist TFV-1 with minimal conformational effects and selective inhibition of outside-in signaling may potentially help resolve the major problem associated with the current integrin antagonists.