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 (HVSMC) mitogen. We have previously reported that gamma-thrombin, in which the anion binding exosite is disrupted, but not catalytically inactivated PPACK-thrombin is mitogenic on HVSMC. Here, the structural requirements for thrombin's mitogenic activity on HVSMC were further investigated. Total inhibition of thrombin-induced DNA synthesis was achieved by AT-III and hirudin. AT-III, added 1 h after exposure of cells to thrombin, failed to alter thrombin-induced mitogenicity. Modification of thrombin's anion binding exosite with peptides derived from the C-terminal sequence of hirudin resulted in a partial loss of thrombin's mitogenic activity. PPACK-inactivated thrombin failed to induce a significant expression of the immediate early gene, c-fos. Unlike PPACK-inactivated thrombin, alpha-thrombin and gamma-thrombin induced a significant increase in the level of the PDGF-A gene expression. A correlation between PDGF-A gene induction and thrombin-induced mitogenicity was suggested by the fact that the mitogenic forms of thrombin also stimulated PDGF-A gene expression. Together, these results indicate that the mitogenic activity of thrombin on HVSMC requires the integrity of the enzyme's active site and is altered by modifications of its anion binding exosite.