The inactivation of single-chain urokinase-type plasminogen activator by thrombin on cultured human endothelial cells

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by;th rombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge abo ut cell-mediated inactivation of scu-PA. We have studied whether scu-PA bou nd to cultured human umbilical vein endothelial cells (HUVEC) could be inac tivated by thrombin. High molecular weight scu-PA was bound to HUVEC and in cubated with increasing amounts of thrombin for 30 min at 37 degrees C. Cel l-bound urokinase-type plasminogen activator (u-PA) was released and levels of scu-PA, tcu-PA/T and active two-chain u-PA were measured using sensitiv e bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombi n. Fifty percent inactivation of scu-PA occurred at about 0.2 nM thrombin. In the presence of monoclonal anti-urokinase receptor IgG, at least 50% of the binding of scu-PA to HUVEC was inhibited. The relative amount of tcu-PA /T that was generated by thrombin was not affected by the monoclonal antibo dy. These results indicated that scu-PA bound to HUVEC via the urokinase re ceptor can be inactivated by thrombin. The efficient inactivation of cell-b ound scu-PA suggests that a cofactor for thrombin may be involved, like thr ombomodulin or glycosaminoglycans. It is concluded that scu-PA bound to the urokinase receptor on a cell surface can be inactivated by thrombin, which may have profound effects on u-PA-mediated local fibrinolysis and extracel lular proteolysis during processes in which thrombin is also involved. (C) 2000 Published by Elsevier Science B.V.