Urokinase-type plasminogen activator (uPA) binds with high affinity to a specific cell surface glycosyl phosphatidyl-inositol (GPI)-anchored receptor, the urokinase receptor (uPAR). Pro-uPA, the enzymatically inactive single-chain form of uPA after having been activated by certain proteases, converts plasminogen into plasmin. This activation of pro-u PA to enzymatically active uPA can be prevented by the action of thrombin on pro-uPA. This inactivation process is accelerated in the presence of thrombomodulin (TM). The present study investigated whether pro-uPA bound to uPAR is still susceptible to inactivation by thrombin in the presence or absence of TM. A truncated soluble form of the uPAR lacking the GPI-anchor was cloned and expressed in CHO-cells (rec-uPAR27r). Rec-uPAR277 efficiently inhibited the thrombin-mediated inactivation of pro-uPA up to 90% in a concentration dependent manner. The protective effect of rec-uPAR277 was far less pronounced when thrombin was complexed withTM. Enzyme kinetic experiments with varying concentrations of pro-uPA showed that in the presence of TM the catalytic efficiency (kcat/Km) of thrombin-mediated inactivation raised from 0.010 μΜ" 1 s" 1 to 0.50 μΜ" 1 s" 1 corresponding to a fifty-fold increase. In the presence of rec-uPAR277, however, the catalytic efficiency dropped by 4.1-fold (0.5 μΜ~ 18~ 1 to 0.122 μΜ~ 18~ 1). The inactivation kinetics of pro-uPA by thrombin (no TM added) in the presence of an excess of rec-uPAR277 could not be determined since virtually no inactivation occurred. Our data suggest that prouPA once bound to uPAR, is significantly protected from inactivation by thrombin. In the presence of TM, however, receptor-bound pro-uPA is still susceptible to inactivation by thrombin. Thus, the receptors TM and uPAR could play an important role in thrombinmediated inactivation of pro-uPA.