Summary. Background: Factor Xa (FXa), a key serine protease that converts prothrombin to thrombin in the coagulation cascade, is a promising target enzyme for the prophylaxis and treatment of thromboembolic diseases. DU-176b is a novel antithrombotic agent that directly inhibits FXa activity. Objective: To evaluate the in vitro pharmacological profiles and in vivo effects of DU-176b in animal models of thrombosis and bleeding. Methods: In vitro, FXa inhibition, specificity and anticoagulant activities were examined. Oral absorption was studied in rats and cynomolgus monkeys. In vivo effects were studied in rat and rabbit models of venous thrombosis and tail bleeding. Results: DU-176b inhibited FXa with Ki values of 0.561 nM for free FXa, 2.98 nM for prothrombinase, and exhibited >10 000-fold selectivity for FXa. In human plasma, DU-176b doubled prothrombin time and activated partial thromboplastin time at concentrations of 0.256 and 0.508 lM, respectively. DU-176b did not impair platelet aggregation by ADP, collagen or U46619. DU-176b was highly absorbed in rats and monkeys, as demonstrated by more potent anti-Xa activity and higher drug concentration in plasma following oral administration than a prototype FXa inhibitor, DX-9065a. In vivo, DU-176b dose-dependently inhibited thrombus formation in rat and rabbit thrombosis models, although bleeding time in rats was not significantly prolonged at an antithrombotic dose. Conclusions: DU-176b is a more potent and selective FXa inhibitor with high oral bioavailability compared with its prototype, DX-9065a. DU-176b represents a promising new anticoagulant for the prophylaxis and treatment of thromboembolic diseases.

Edoxaban, an oral, direct factor Xa inhibitor, has a similar or low incidence of bleeding events compared with other anticoagulants in clinical trials. Therefore, agents to reverse the anticoagulant effects of edoxaban could be desirable in emergency situations. In this study, the reversal effects of haemostatic agents were determined on prothrombin time (PT) prolongation in vitro and bleeding time prolongation in vivo by edoxaban. PT using human plasma was measured in the presence of edoxaban at therapeutic and excess concentrations with the haemostatic agents, prothrombin complex concentrate (PPSB-HT), activated prothrombin complex concentrate (Feiba), and recombinant factor VIIa (rFVIIa). In rats, rFVIIa and Feiba was given during intensive anticoagulation with edoxaban. The haemostatic effect was evaluated in a model of planta template bleeding and a potential prothrombotic effect was evaluated in a venous thrombosis model. PPSB-HT, Feiba, and rFVIIa concentration-dependently shortened PT prolonged by edoxaban. Among these, rFVIIa and Feiba showed potent activities in reversing the PT prolongation by edoxaban. rFVIIa (1 and 3 mg/kg, i.v.) and Feiba (100 U/kg, i.v.) significantly reversed edoxaban (1 mg/kg/h)-induced prolongation of bleeding time in rats. In a rat venous thrombosis model, no potentiation of thrombus formation was observed when the highest dose (3 mg/kg) of rFVIIa was added to edoxaban (0.3 and 1 mg/kg/h) compared with the control. The present study indicated that rFVIIa, Feiba, and PPSB-HT have the potential to be reversal agents for edoxaban.

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Congenital thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) is associated with an inherited von Willebrand factor-cleaving protease (ADAMTS13 [a disintegrin and metalloprotease with thrombospondin type I domains 13]) deficiency. In this study, we identified novel mutations in the ADAMTS13 gene in a patient with TTP. The patient was a 51-year-old Japanese male who exhibited TTP symptoms at frequent intervals. The ADAMTS13 activity during acute episodes was less than 3% that of normal. The enzyme activities of the patient's father and mother were both 46%, and both parents were asymptomatic. Genetic analysis revealed that the patient was a compound heterozygote for 2 mutations. One mutation was a missense mutation in the metalloprotease domain (A250V, exon 7), and the other was a guanine to adenine substitution at the 5 end of intron 3 (intron 3 G3A). In vitro expression studies revealed that the A250V mutation markedly reduced ADAMTS13 activity and the intron 3 G3A mutation caused abnormal mRNA