Snake venoms affect blood coagulation and platelet function in a complex manner. However, two classes of venom proteins, snaclecs and disintegrins have been shown to specifically target receptors including GPIb, α2β1, GPVI, CLEC-2 and integrins αIIbβ3, αvβ3, α5β1 expressed on platelets, endothelial cells, phagocytes, tumor cells, thus affecting cell-matrices and cell-cell interactions. Here, we focus on disintegrins, a class of low molecular mass Arg-Gly-Asp(RGD)/Lys-Gly-Asp(KGD)-containing, cysteine-rich polypeptide derived from various viper snake venoms. This review describes the potential applications of disintegrins in field of integrin-related diseases, especially arterial thrombosis, angiogenesis, tumor progression and septic inflammation. In addition, a novel RGD-containing disintegrin TMV-7 is being developed as a safer antithrombotic agent with minimal side effects, such as thrombocytopenia and bleeding.

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.

Snake venoms affect blood coagulation and platelet function in diverse ways. Some venom components inhibit platelet function, while other components induce platelet aggregation. Among the platelet aggregation inhibitors, disintegrins have been recognized as unique and potentially valuable tools for examining cell–matrix and cell–cell interactions and for the development of antithrombotic and antiangiogenic agents according to their anti-adhesive and anti-migration effect on tumor cells and antiangiogenesis activities. Disintegrins represent a family of low molecular weight, cysteine-rich, Arg-Gly-Asp(RGD)/Lys-Gly-Asp(KGD)-containing polypeptides, which inhibit fibrinogen binding to integrin αIIbβ3 (i.e., platelet glycoprotein IIb/IIIa), as well as ligand binding to integrins αvβ3, and α5β1 expressed on cells (i.e., fibroblasts, tumor cells, and endothelial cells). This review focuses on the current efforts attained from studies using disintegrins as a tool in the field of arterial thrombosis, angiogenesis, inflammation, and tumor metastasis, and briefly describes their potential therapeutic applications and side effects in integrin-related diseases. Additionally, novel R(K)GD-containing disintegrin TMV-7 mutants are being designed as safer antithrombotics without causing thrombocytopenia and bleeding.

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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.