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1 The e ects of YM-60828, a newly synthesized factor Xa inhibitor, were investigated to analyse the relationship between its antithrombotic e ects and its prolongation of template bleeding time in rats. YM-60828 was compared with argatroban, heparin and dalteparin. All agents were intravenously administered as a bolus. 2 In ex vivo studies, YM-60828 and argatroban prolonged both prothrombin time and activated partial thromboplastin time in a dose-dependent manner, while heparin and dalteparin prolonged only activated partial thromboplastin time.3 In a venous thrombosis model, all agents exerted antithrombotic e ects in a dose-dependent manner. The ID 50 values of YM-60828, argatroban, heparin and dalteparin were 0.0081 mg kg 71 , 0.011 mg kg 71 , 6.3 iu kg 71 and 4.7 iu kg 71 , respectively. 4 In an arterio-venous shunt model, all agents exerted antithrombotic e ects in a dose-dependent manner. The ID 50 values of YM-60828, argatroban, heparin and dalteparin were 0.010 mg kg 71 , 0.011 mg kg 71 , 10 iu kg 71 and 4.2 iu kg 71 , respectively. 5 In bleeding time studies, all agents prolonged template bleeding time in a dose-dependent manner. ED 2 values, the doses causing a 2 fold prolongation of bleeding time in the saline group, of YM-60828, argatroban, heparin and dalteparin were 0.76 mg kg 71 , 0.081 mg kg 71 , 18 iu kg 71 and 25 iu kg 71 , respectively. 6 The ratio (ED 2 /ID 50 ) of YM-60828 was more than 30 fold greater than that of heparin and more than 10 fold greater than those of argatroban and dalteparin. 7 These data show that YM-60828 can exert its antithrombotic e ects with little prolongation of bleeding time compared with the other currently used anticoagulant agents.

A new platelet antagonist, tokaracetin, was isolated from the venom of Trimeresurus tokarensis by ion-exchange chromatography, heparin-Sepharose chromatography and hydrophobic HPLC. The purified protein showed an apparent molecular mass on SDS/PAGE of 28.9 kDa under non-reducing conditions. On reduction, 16.1 and 15.4 kDa subunits were observed, suggesting that the molecule is a heterodimer. Tokaracetin inhibited the binding of 125I-labelled bovine von Willebrand factor (vWF) and 125I-labelled human vWF in the presence of botrocetin to fixed human platelets. It did not block ADP-, collagen- or thrombin receptor agonist peptide-induced platelet aggregation in human platelet-rich plasma (PRP), or induce platelet agglutination in PRP. On reduction, tokaracetin lost its inhibitory activity on the agglutination of fixed human platelets by bovine vWF. 125I-Tokaracetin specifically bound to washed human platelets with high affinity (Kd 3.9 +/- 1.4 nM) at 47,440 +/- 2780 binding sites per platelet. Binding of tokaracetin to fixed human platelets was reversible, and was inhibited by monoclonal antibody GUR83-35, which is directed against the N-terminal vWF-binding domain of human glycoprotein Ib (GPIb). Tokaracetin completely inhibited vWF-dependent shear-induced platelet aggregation in PRP at 3 micrograms/ml. The N-terminal amino acid sequences of tokaracetin subunits showed a high degree of identity with those of alboaggregin-B. These results suggest that this new platelet antagonist may be a useful tool in the development of specific inhibitors of the vWF-GPIb interaction.

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SummaryThe antiplatelet and antithrombotic effects of the Fab fragment of the humanized antiplatelet glycoprotein (GP) IIb/IIIa monoclonal antibody C4G1 (YM337) were investigated in monkeys. First, the relationship between the inhibition of platelet aggregation and the prolongation of bleeding time was studied in rhesus monkeys. YM337 dose-dependently inhibited ex vivo platelet aggregation, with complete inhibition at doses higher than 0.25 mg/kg intravenous injection or 1.5 μg/kg/min infusion. At 0.25 mg/kg bolus injection followed by 1.5 μg/kg/min infusion, YM337 immediately and continuously inhibited platelet aggregation during the 6-h infusion period with platelet aggregation rapidly returning to over 50% of baseline within 1 h after the cessation of infusion. Template-bleeding time was significantly prolonged during the period of complete inhibition of platelet aggregation.Second, the antithrombotic effects of YM337 were investigated in a photochemically-induced thrombosis model in squirrel monkeys. YM337 at a dose of 1 mg/kg intravenous injection followed by 6 μg/kg/min infusion for 60 min prevented occlusive thrombus formation in all 4 monkeys. In contrast, time to occlusive thrombus formation did not change on intravenous bolus injection of aspirin 17 mg/kg (11.3 ± 5.2 min) or sodium ozagrel (9.4 ± 3.0 min) compared with saline (13.3 ± 4.0 min). YM337 but not aspirin or sodium ozagrel significantly inhibited ex vivo ADP-induced platelet aggregation, while all drugs completely inhibited arachidonic acid-induced platelet aggregation. However, while aspirin and sodium ozagrel inhibited the thromboxane B2 generation accompanying arachidonic acid-induced platelet aggregation, YM337 had no effect on this variable. Platelet counts and bleeding time showed no significant change in any group in this squirrel monkey model.These results indicate that YM337, with a short half-life, may be a useful therapeutic agent in patients with thrombotic disorders.