INHIBITION OF EXTRINSIC AND INTRINSIC THROMBIN GENERATION BY A NOVEL SYNTHETIC THROMBIN INHIBITOR (RO 46-6240), RECOMBINANT HIRUDIN AND HEPARIN IN HUMAN PLASMA

To further define the anticoagulant activity of Ro 46-6240, a novel, s ynthetic, thrombin inhibitor, we compared its effect on extrinsic and intrinsic thrombin generation in human platelet-poor plasma with that of recombinant hirudin and standard heparin. The time course of thromb in generation was followed with a chromogenic substrate assay. The tot al amount of active thrombin formed was quantified by calculating the area under the thrombin generation curve. Ro 46-6240 and r-hirudin del ayed thrombin formation in a concentration-dependent manner in both ac tivation systems whereas heparin showed this effect only in the intrin sic system. Heparin was the most potent inhibitor of extrinsic and int rinsic thrombin generation with IC50 values of 20 and 27 nM, respectiv ely. Ro 46-6240 was nearly as potent as r-hirudin for inhibiting extri nsic thrombin generation (IC50 418 vs 229 nM) and intrinsic thrombin g eneration (IC50 463 vs 343 nM) despite a much lower affinity of Ro 46- 6240 for thrombin (Ki apparent: 0.3 nM) in a purified buffer system. T he similar potency of the small active-site thrombin inhibitor compare d to the larger hirudin may be explained by different kinetic mechanis ms for inhibition of thrombin and by a higher accessibility to the pho spholipid surface where thrombin generation takes place. In conclusion , our results show that a specific small thrombin inhibitor efficientl y inhibits and delays thrombin generation in human coagulating plasma. This reduced thrombin generation might be caused by inhibition of thr ombin-mediated feedback reactions during blood coagulation.