Sustained generation of alpha-thrombin and its breakdown forms at sites of thromboses has focused attention on the roles thrombin may play in vascular responses to thrombosis and injury. We have previously shown that alpha-thrombin stimulates many growth signals in cultured rat aortic smooth muscle cells (VSMC). To characterize thrombin growth mechanisms, we studied the effects on cultured VSMC of gamma-thrombin (catalytically active with obstructed anion-binding site required for clotting activity) and D-phenylalanyl-L-prolyl-L-arginine chloromethylketone-alpha-thrombin (catalytically inactive with intact anion-binding exosite) on cultured VSMC. Either derivative alone failed to increase growth, but in combination at 130 nM each, they caused a 75 +/- 5% increase in protein synthesis, similar to that observed with alpha-thrombin. This increase in protein synthesis was related to activation of protein kinase C (PKC) and Na+/H+ exchange, because only in combination could the derivatives increase phosphorylation of a 76,000-dalton PKC substrate and alkalinize the cells. Activation of PKC was correlated with a synergistic effect of the derivatives on diacylglycerol formation at 2 min (maximum, 55 +/- 1% combined increase vs. 24 +/- 9% and 4 +/- 4% individual increases with gamma- and D-phenylalanyl-L-prolyl-L-arginine chloromethylketone-alpha-thrombin alone, respectively, p less than 0.05). The derivatives stimulated PKC without increasing inositol trisphosphate, intracellular Ca2+, or expression of the protooncogene, c-fos. Thus, thrombin stimulation of Na+/H+ exchange, diacylglycerol formation, and growth of VSMC can be distinguished from thrombin mobilization of [Ca2+]i and induction of c-fos mRNA. These data indicate the presence of more than one mechanism for thrombin-mediated signaling events in cultured VSMC. Our results also suggest that various thrombin forms retained in clots may have significant effects on VSMC growth and function.