HEPSIN, A PUTATIVE MEMBRANE-ASSOCIATED SERINE-PROTEASE, ACTIVATES HUMAN FACTOR-VII AND INITIATES A PATHWAY OF BLOOD-COAGULATION ON THE CELL-SURFACE LEADING TO THROMBIN FORMATION

Previous studies have shown that hepsin is a putative membrane-associa ted serine protease that is required for cell growth (Torres-Rosado, A ., O'Shea, K. S., Tsuji, A., Chou, S.-H., and Kurachi, K. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 7181 7185). In the present study, we ha ve transfected baby hamster kidney (BHK) cells with a plasmid containi ng the cDNA for human hepsin and examined these cells for their abilit y to activate several blood coagulation factors including factors X, I X, VII, prothrombin, and protein C. Little, if any, proteolytic activa tion of factors X, IX, prothrombin, or protein C was observed when the se clotting factors were incubated with hepsin-transfected cells. On t he other hand, hepsin-transfected cells proteolytically activated sign ificant concentrations of human factor VII in a time- and calcium-depe ndent manner, whereas essentially no activation of factor VII was obse rved in BHK cells transfected with plasmid lacking the cDNA for hepsin . The factor VII activating activity in the hepsin-transfected BHK cel l line was confined exclusively to the total membrane fraction and was inhibited >95% by antibody raised against a fusion protein consisting of maltose-binding protein and the extracellular domain of human heps in. An active site factor VII mutant, S344A factor VII, was cleaved as readily as plasma-derived factor VII by hepsin-transfected cells, ind icating that factor VII was not converted to factor Wa autocatalytical ly on the cell surface. In contrast, an activation cleavage site facto r VII mutant, R152E factor VII, was not cleaved by hepsin-transfected cells, suggesting that factor VII and S344A factor VII were activated on these cells by cleavage of the Arg(152)-Ile(153) peptide bond. In t he copresence of factor VII and factor X, hepsin-transfected BHK cells supported the formation of factor Xa. In addition, in the copresence of factor VII, factor X, and prothrombin, hepsin-transfected BHK cells supported the formation of thrombin. These results strongly suggest t hat membrane associated hepsin converts zymogen factor VII to factor V IIa, which in turn, is capable of initiating a coagulation pathway on the cell surface that ultimately leads to thrombin formation.