The effect of bovine thrombomodulin on the specificity of bovine thrombin.
Bovine lung thrombomodulin is purified and used to investigate the basis of the change in substrate specificity of bovine thrombin when bound to thrombomodulin. Bovine thrombomodulin is a single polypeptide having an apparent molecular weight of 84,000 and associates with thrombin with high affinity and rapid equilibrium, to act as a potent cofactor for protein C activation and antagonist of reactions of thrombin with fibrinogen, heparin cofactor 2, and hirudin. Bovine thrombomodulin inhibits the clotting activity of thrombin with Kd less than 2.5 nM. Kinetic analysis of the effect of bovine thrombomodulin on fibrinopeptide A hydrolysis by thrombin indicates competitive inhibition with Kis = 0.5 nM. The active site of thrombin is little perturbed by thrombomodulin, as tosyl-Gly-Pro-Arg-p-nitroanilide hydrolysis and inhibition by antithrombin III are unaffected. Insensitivity of the reaction with antithrombin III is likewise observed with thrombin bound to thrombomodulin on intact endothelium. Antithrombin III-heparin, human heparin cofactor 2, and hirudin inhibit thrombin-thrombomodulin more slowly than thrombin. These effects may arise from a decrease in Ki of the inhibitors for thrombin-thrombomodulin or from changes in the active site not detected by tosyl-Gly-Pro-Arg-p-nitroanilide or antithrombin III. Bovine prothrombin fragment 2 inhibits thrombin clotting activity (Kd less than 7.5 microM) and acts as a competitive inhibitor of protein C activation (Kis = 2.1 microM). The data are consistent with a mechanism whereby thrombomodulin alters thrombin specificity by either binding to or allosterically altering a site on thrombin distinct from the catalytic center required for binding or steric accommodation of fibrinogen, prothrombin fragment 2, heparin cofactor 2, and hirudin.
D000818:Animals; D000990:Antithrombin III / Q000494:pharmacology; D001665:Binding Sites; D002417:Cattle; D005340:Fibrinogen / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism / Q000494:pharmacology; D015844:Heparin Cofactor II; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008970:Molecular Weight; D011486:Protein C; D011487:Protein Conformation; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000494:pharmacology*; D018179:Receptors, Thrombin; D013379:Substrate Specificity; D013917:Thrombin / Q000037:antagonists & inhibitors*
D006023:Glycoproteins; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D015844:Heparin Cofactor II; D000990:Antithrombin III; D005340:Fibrinogen; D013917:Thrombin