Role of thrombomodulin in the inacttvation of single-chain urokinase-type plasminogen activator by thrombin in a plasma milieu

Thrombin cleaves single-chain urokinase-type plasminogen activator (scu-PA) into a virtually inactive two-chain form (tcu-PA/T), which can be detected in human body fluids under certain pathological conditions. The mechanism of this inactivation has been described in detail in a purified system. The aim of this study was to examine the inactivation of scu-PA by thrombin in a plasma milieu and the role of thrombomodulin (TM) on this process to get more insight in the physiological relevance of this phenomenon. Citrated pooled normal plasma supplemented with scu-PA was treated with thrombin in the absence and presence of TM. The amount of scu-PA and tcu-PA/T was determined in the plasma-derived serum using sensitive bioimmunoassays. Inactivation of scu-PA induced by plasma recalcification was studied by incubating normal, factor XIand factor XII-deficient plasma with calcium and measuring the generation of tcu-PA/T. Generation of tcu-PA/T was also measured in normal, factor V'4' and protein C-deficient plasma recalcified in the absence and presence of TM. TM enhanced the inactivation of scu-PA by thrombin in plasma at least 5-fold. Inactivation of scu-PA by thrombin in the absence and presence of TM was found to occur rapidly and declined within a few minutes. Recalcification of plasma resulted in considerable inactivation of scu-PA due to endogenous thrombin generation through the contact activation system. Calcium-induced generation of tcu-PA/T could be completely inhibited by TM. In plasmas with a disturbed protein C pathway, the inhibiting effect of TM was partially (factor yLeuknj Qr COInpiejeiy (protein C deficiency) abolished. This indicates that the inhibition by TM was most likely due to activation of protein C by the complex formed between TM and endogenously generated thrombin. TM thus appeared to play a dual role by both stimulating the inactivation of scu-PA by thrombin in plasma and inhibiting calcium-induced inactivation of scu-PA. This study shows that the inactivation of scu-PA in plasma depends mainly on the generation of thrombin and on TM. Inactivation of scu-PA-induced fibrinolysis appears to be regulated by activation of the coagulation system, providing a link between fibrinolysis and coagulation.