A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor gamma-chain complex.

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor gamma-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific auto-Abs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.

Pharmacological characterization of the active synthetic factor Xa inhibitors M55551 and M55165.

Factor Xa plays an important role in blood coagulation and is widely regarded as an attractive target for antithrombotic drug development. M55551 and M55165 (1-arylsulfonyl-3-piperazinone derivatives) are novel synthetic factor Xa inhibitors. In vitro, M55551 and M55165 competitively inhibited factor Xa with K(i) values of 3.2 nM and 2.3 nM, respectively, and prolonged clotting time in human and rat plasma. Pharmacokinetic analysis of these compounds revealed that M55551 was intravenously active with a short half-life (0.2 h) and that M55165 exhibited good bioavailability (31%) with a long half-life (3.9 h). Therefore, the antithrombotic effects of M55551 and M55165 were compared with those of the intravenous anticoagulant argatroban and the oral anticoagulant warfarin. Intravenous administration of M55551 and oral administration of M55165 inhibited thrombus formation at 0.3 mg/kg and 10 mg/kg, respectively, without significant prolongation of bleeding time. In contrast, although argatroban (0.3 mg/kg) and warfarin (1 mg/kg) also inhibited thrombus formation, significant prolongation of bleeding time was observed at dosages of 3 mg/kg and 1 mg/kg, respectively. These results suggest that M55551 and M55165 are potent factor Xa inhibitors that are active upon intravenous and oral administration, respectively, and that may prove clinically useful for the treatment of thrombosis while minimizing bleeding risks.