In vitro characterization of a novel factor Xa inhibitor, RPR 130737

RPR 130737 inhibited factor Xa (FXa) with a K-i of 2.4 nM and also displaye d excellent specificity toward FXa relative to other serine proteases. it s howed selectivity of more than 1000-fold over thrombin, activated protein C , plasmin, tissue-plasminogen activator, and trypsin. RPR 130737 prolonged plasma activated partial thromboplastin time and prothrombin time in a dose -dependent fashion. In the activated partial thromboplastin time assay, the concentrations required for doubling coagulation time were 0.32 mu M (huma n), 0.61 mu M (monkey), 0.44 mu M (dog), 0.15 mu M (rabbit), and 0.82 mu M (rat). The concentrations required to double prothrombin time were 0.86 mu M (human), 1.26 mu M (monkey). 1.15 mu M (dog), 0.39 mu M (rabbit), and 7.3 1 mu M (rat). Kinetic studies revealed that RPR 130737 was a fast-binding, reversible, and competitive inhibitor for FXa when Spectrozyme FXa, a chrom ogenic substrate, was used. A coupled-enzyme assay measuring thrombin activ ity following prothrombinase conversion of prothrombin to thrombin indicate d that RPR 130737 was a potent inhibitor for prothrombinase-bound FXa. In t his assay, RPR 130737 showed IC(50)s of 17 nM and 35.9 nM, respectively, wh en artificial phosphatidylserine/phosphatidylcholine (PS/PC) liposomes or g el-filtered platelets were used as the phospholipid source. An FX-deficient plasma clotting-time correction assay further demonstrated that RPR 130737 was a specific inhibitor of FXa. RPR 130737 showed no effect on platelet a ggregation in vitro. These results indicate that RPR 130737 has the potenti al to be developed as an antithrombotic agent based on its potent and selec tive inhibitory effect against FXa. (C) 2000 Elsevier Science Ltd. All righ ts reserved.