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, synthetic, 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 thrombin generation was followed with a chromogenic substrate assay. The total amount of active thrombin formed was quantified by calculating the area under the thrombin generation curve. Ro 46-6240 and r-hirudin delayed thrombin formation in a concentration-dependent manner in both activation systems whereas heparin showed this effect only in the intrinsic system. Heparin was the most potent inhibitor of extrinsic and intrinsic thrombin generation with IC50 values of 20 and 27 nM, respectively. Ro 46-6240 was nearly as potent as r-hirudin for inhibiting extrinsic thrombin generation (IC50 418 vs 229 nM) and intrinsic thrombin generation (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. The similar potency of the small active-site thrombin inhibitor compared to the larger hirudin may be explained by different kinetic mechanisms for inhibition of thrombin and by a higher accessibility to the phospholipid surface where thrombin generation takes place. In conclusion, our results show that a specific small thrombin inhibitor efficiently inhibits and delays thrombin generation in human coagulating plasma. This reduced thrombin generation might be caused by inhibition of thrombin-mediated feedback reactions during blood coagulation.
Gast
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Preclinical Research Department, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Tschopp
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TB
D000991:Antithrombins / Q000494:pharmacology*; D001665:Binding Sites; D002863:Chromogenic Compounds / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology*; D006629:Hirudins / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D009281:Naphthalenes / Q000494:pharmacology*; D010880:Piperidines / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism
D000991:Antithrombins; D002863:Chromogenic Compounds; D004151:Dipeptides; D006629:Hirudins; D009281:Naphthalenes; D010880:Piperidines; D011994:Recombinant Proteins; C021129:S 2238; D006493:Heparin; D013917:Thrombin; C087198:napsagatran