THROMBIN RECEPTOR ACTIVATION ELICITS RAPID PROTEIN-TYROSINE PHOSPHORYLATION AND STIMULATION OF THE RAF-1 MAP KINASE PATHWAY PRECEDING DELAYED MITOGENESIS IN CULTURED RAT AORTIC SMOOTH-MUSCLE CELLS - EVIDENCE FOR AN OBLIGATE AUTOCRINE MECHANISM PROMOTING CELL-PROLIFERATION INDUCED BY G-PROTEIN-COUPLED RECEPTOR AGONISTS/

Treatment of quiescent rat aortic smooth muscle cells with either alph a-thrombin or a thrombin receptor-derived agonist peptide (SFLLRNP) re sulted in pronounced increases in [H-3]thymidine incorporation that we re concentration dependent and reached a maximum of similar to 15-fold above serum-starved controls, However, in contrast to FPS, PDGF-BB, o r basic fibroblast growth factor (bFGF), that initiated DNA synthesis promptly after 16-19 h, thymidine incorporation in response to thrombi n was delayed by an additional 3-6 h, Delayed mitogenesis correlated w ith the appearance of a potent mitogenic activity in conditioned media samples obtained from thrombin-stimulated rat aortic smooth muscle ce lls, as assayed using Swiss 3T3 fibroblasts, This activity was not inh ibited by neutralizing antibodies directed against PDGF or bFGF. Furth ermore, in the Swiss 3T3 cells, simple addition of either alpha-thromb in or SFLLRNP failed to elicit a significant mitogenic response. In si gnal transduction studies, both thrombin and SFLLRNP treatment led to rapid tyrosine phosphorylation of proteins with apparent molecular mas ses of 42, 44, 75, 120, and 190 kD, respectively, as assessed by antip hosphotyrosine immunoblotting. The overall pattern of protein tyrosine phosphorylation was distinct from that observed after PDGF-BB additio n, Activation of Raf-1 and the mitogen-activated protein (MAP) kinases p44(mapk) and p42(mapk) was also observed, However, the time course a nd duration of Raf-1/MAP kinase activation after thrombin stimulation were similar to those elicited by PDGF-BB, Taken together, our results indicate that thrombin-stimulated vascular smooth muscle proliferatio n is delayed and requires the de novo expression of one or more autocr ine mitogens, in addition, the rapid induction of discrete intracellul ar signaling mechanisms by thrombin, including the Raf-1/MAP kinase pa thway, appears to be insufficient alone to promote vascular smooth mus cle cell mitogenesis.