Effect of thrombomodulin on the kinetics of the interaction of thrombin with substrates and inhibitors.
Thrombomodulin decreased by 20-30% the Michaelis constant of two tripeptidyl p-nitroanilide substrates of thrombin. Thrombomodulin increased the rate of inactivation of thrombin by two peptidyl chloromethane inhibitors by a similar amount. This effect appeared to be due to a decrease in the dissociation constants of the inhibitors. An improved method for the separation of fibrinopeptides A and B by h.p.l.c. was developed, and this method was used to study the effect of thrombomodulin on the thrombin-catalysed cleavage of fibrinogen. In this reaction, thrombomodulin was a competitive inhibitor with respect to the A alpha-chain of fibrinogen. The release of fibrinopeptide B was also inhibited by thrombomodulin. Analysis of the inhibition caused by thrombomodulin with respect to fibrinopeptides A and B yielded the same dissociation constant for the thrombin-thrombomodulin complex. In the presence of thrombomodulin, the rate of inactivation of thrombin by antithrombin III was stimulated 4-fold. This stimulation showed saturation kinetics with respect to thrombomodulin. Thrombomodulin was found to compete with hirudin for a binding site on thrombin. As a result of this competition, hirudin became a slow-binding inhibitor of thrombin at high thrombomodulin concentrations. Estimates of the dissociation constant for thrombomodulin were obtained in several of the above experiments, and the weighted mean value was 0.7 nM.
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000990:Antithrombin III / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D005344:Fibrinopeptide A / Q000378:metabolism; D005345:Fibrinopeptide B / Q000378:metabolism; D006629:Hirudins / Q000378:metabolism; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000494:pharmacology*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*
D000590:Amino Acid Chloromethyl Ketones; D006629:Hirudins; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; C020924:phenylalanyl-prolyl-arginine methyl chloride; C027587:phenylalanyl-phenylalanyl-arginine chloromethyl ketone; D000990:Antithrombin III; D005340:Fibrinogen; D013917:Thrombin