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

• Published in: Biochimica et biophysica acta 2000-09, Vol.1497 (3), p.351-358
• Main Authors: Braat, Ellen A.M, Collen, Annemie, Jie, Annie F.H, Grimbergen, Jos M, Rijken, Dingeman C
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - pharmacology; Cell Membrane - metabolism; Cells, Cultured; Down-Regulation; Endothelial cells; Endothelium, Vascular - drug effects; Endothelium, Vascular - metabolism; Fibrinolysis; Humans; HUVEC; Receptors, Cell Surface - immunology; Receptors, Cell Surface - metabolism; Receptors, Urokinase Plasminogen Activator; scu-PA; Thrombin; Thrombin - pharmacology; Urokinase; Urokinase receptor; Urokinase-Type Plasminogen Activator - chemistry; Urokinase-Type Plasminogen Activator - metabolism
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/S0167-4889(00)00073-2

Single-chain urokinase-type plasminogen activator (scu-PA) is cleaved by thrombin, resulting in an inactive molecule called thrombin-cleaved two-chain urokinase-type plasminogen activator (tcu-PA/T). There is no knowledge about cell-mediated inactivation of scu-PA. We have studied whether scu-PA bound to cultured human umbilical vein endothelial cells (HUVEC) could be inactivated by thrombin. High molecular weight scu-PA was bound to HUVEC and incubated with increasing amounts of thrombin for 30 min at 37°C. Cell-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 sensitive bioimmunoassays. Cell-bound scu-PA was efficiently inactivated by thrombin. 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 antibody. These results indicated that scu-PA bound to HUVEC via the urokinase receptor can be inactivated by thrombin. The efficient inactivation of cell-bound scu-PA suggests that a cofactor for thrombin may be involved, like thrombomodulin 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 extracellular proteolysis during processes in which thrombin is also involved.