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

Abstract

Life-threatening thrombocytopenia and bleeding, common side effects of clinically available α IIb β 3 antagonists, are associated with the induction of ligand-induced integrin conformational changes and exposure of ligand-induced binding sites (LIBSs). To address this issue, we examined intrinsic mechanisms and structure-activity relationships of purified disintegrins, from Protobothrops flavoviridis venom (i.e., Trimeresurus flavoviridis), TFV-1 and TFV-3 with distinctly different pro-hemorrhagic tendencies. TFV-1 with a different α IIb β 3 binding epitope from that of TFV-3 and chimeric 7E3 Fab, i.e., Abciximab, decelerates α IIb β 3 ligation without causing a conformational change in α IIb β 3 , as determined with the LIBS antibody, AP5, and the mimetic, drug-dependent antibody (DDAb), AP2, an inhibitory monoclonal antibody raised against α IIb β 3 . Consistent with their different binding epitopes, a combination of TFV-1 and AP2 did not induce FcγRIIa-mediated activation of the ITAM-Syk-PLCγ2 pathway and platelet aggregation, in contrast to the clinical antithrombotics, abciximab, eptifibatide, and disintegrin TFV-3. Furthermore, TFV-1 selectively inhibits Gα 13 -mediated platelet aggregation without affecting talin-driven clot firmness, which is responsible for physiological hemostatic processes. At equally efficacious antithrombotic dosages, TFV-1 caused neither severe thrombocytopenia nor bleeding in FcγRIIa-transgenic mice. Likewise, it did not induce hypocoagulation in human whole blood in the rotational thromboelastometry (ROTEM) assay used in perioperative situations. In contrast, TFV-3 and eptifibatide exhibited all of these hemostatic effects. Thus, the α IIb β 3 antagonist, TFV-1, efficaciously prevents arterial thrombosis without adversely affecting hemostasis. Keywords: arterial thrombosis; antiplatelet agent; integrin α IIb β 3 ; bleeding side effect; snake venom proteins; disintegrins Key Contribution: Current α IIb β 3 antagonists are efficacious anti-thrombotics, but have significant adverse bleeding effects. This study clarifies a pathologically intrinsic mechanism in drug-induced thrombocytopenia and identifies a new candidate that may lead to development of safer anti-thrombotics with significantly reduced bleeding risk.

Exogenous Integrin αIIbβ3 Inhibitors Revisited: Past, Present and Future Applications

“…We can speculate that disintegrins have evolved into forms that escape the human immune system. A previous study revealed a disintegrin that did not cause a conformational change of integrin αIIbβ3, resulting in antiplatelet activity without causing bleeding [ 138 ]. More research into disintegrins, including structure-activity relationships, may provide more insight into their biological activity and mechanism of platelet inhibition.…”

Improved Antithrombotic Activity and Diminished Bleeding Side Effect of a PEGylated αIIbβ3 Antagonist, Disintegrin

“…AP2 is an inhibitory monoclonal antibody raised against α IIb β 3 . In combination with AP2 (4 μg/mL), the lowest concentration of the agent to activate platelets/the IC 50 of the agent on collagen-induced platelet aggregation was defined as the safety index [ 8 , 15 ].…”

“…ROTEM assay (Tem International, Munich, Germany) was performed according to the ROTEM ® delta system’s instructions as previously described [ 8 , 15 ]. Four variables the including maximum clot firmness (MCF, mm), clotting time (CT, sec), clot formation time (CFT, sec), and alpha angle (α, o) were incorporated into a coagulation index (CI) as defined by the equation: CI= −0.6516CT − 0.3772CFT + 0.1224MCF + 0.0759α − 7.7922.…”

“…In the present study, we compared pharmacokinetic characterization between native RR and PEGylated RR (PEG-RR) and investigated their in vivo anti-thrombosis efficacy in vessel injury-induced thrombosis model. In addition, we used monoclonal AP2 as a platform [ 8 , 15 ] to evaluate the adverse reaction of bleeding upon administration of RR and PEG-RR in vitro, and further investigated their tendency to cause thrombocytopenia in FcγRIIA-transgenic mouse model ex vivo. In conclusion, this study provides a picture of how to achieve a stable formulation with minimal bleeding side effects and long-circulating properties.…”

Introduction to the Toxins Special Issue on Identification and Functional Characterization of Novel Venom Components

“…Kuo et al [7] examine mechanisms and structure-activity relationships of disintegrins TFV-1 and TFV-3, from Protobothrops flavoviridis venom, which have very different pro-hemorrhagic tendencies. The find that TFV-1 selectively inhibits Gα13-mediated platelet aggregation without affecting physiological hemostatic processes.…”