STRUCTURAL DOMAINS OF THROMBIN INVOLVED IN THE INDUCTION OF MITOGENESIS IN CULTURED HUMAN VASCULAR SMOOTH-MUSCLE CELLS

Human alpha-thrombin is a known human vascular smooth muscle cell (HVS MC) mitogen. We have previously reported that gamma-thrombin, in which the anion binding exosite is disrupted, but not catalytically inactiv ated PPACK-thrombin is mitogenic on HVSMC. Here, the structural requir ements for thrombin's mitogenic activity on HVSMC were further investi gated Total inhibition of thrombin-induced DNA synthesis was achieved by AT-III and hirudin AT-III, added 1 h after exposure of cells to thr ombin, failed to alter thrombin-induced mitogenicity. Modification of thrombin's anion binding exosite with peptides derived from the C-term inal sequence of hirudin resulted in a partial loss of thrombin's mito genic activity. PPACK-inactivated thrombin failed to induce a signific ant expression of the immediate early gene, c-fos. Unlike PPACK-inacti vated thrombin, alpha-thrombin and gamma-thrombin induced a significan t increase in the level of the PDGF-A gene expression. A correlation b etween PDGF-A gene induction and thrombin-induced mitogenicity was sug gested by the fact that the mitogenic forms of thrombin also stimulate d PDGF-A gene expression. Together, these results indicate that the mi togenic activity of thrombin on HVSMC requires the integrity of the en zyme's active site and is altered by modifications of its anion bindin g exosite.