Acceleration of the thrombin inactivation of single chain urokinase-type plasminogen activator (pro-urokinase) by thrombomodulin.
The in vitro effects of thrombomodulin on the inactivation of single chain urokinase-type plasminogen activator (scu-PA) by thrombin were investigated by incubating scu-PA with varying concentrations of human thrombin, in both the absence and presence of soluble rabbit thrombomodulin. 50% inactivation of scu-PA occurred in 45 min at 160 ng/ml thrombin in the absence of thrombomodulin and at 4.6 ng/ml thrombin in the presence of thrombomodulin. No difference was found in either the absence or the presence of thrombomodulin between the inactivation rates of high molecular weight scu-PA, and a low molecular weight scu-PA which lacked the growth factor and kringle domains. Enzyme kinetic experiments with varying concentrations of scu-PA showed that thrombomodulin decreased the Km of thrombin for scu-PA from 7.8 to 0.43 microM and increased the kcat from 0.30 to 1.2 s-1, corresponding to a 70-fold increase in the second-order rate constant kcat/Km. SDS-polyacrylamide gel electrophoresis showed that scu-PA was cleaved into two chains upon inactivation by thrombin, and confirmed the acceleration effect of thrombomodulin on inactivation of scu-PA. Thrombomodulin thus not only has anticoagulant properties but is also antifibrinolytic. The acceleration may imply a new mechanism for the regulation of local plasminogen activator activity on the cell surface.
de Munk
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GA
Gaubius Institute TNO, Leiden, The Netherlands.
Groeneveld
E
E
Rijken
D C
DC
D002118:Calcium / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000502:physiology*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000494:pharmacology*; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors* / Q000502:physiology
D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator; D002118:Calcium