THROMBIN-STIMULATED EVENTS IN CULTURED VASCULAR SMOOTH-MUSCLE CELLS

Thrombin is present in high concentrations at sites of clots and may have important post-clotting effects on adjacent vascular tissue. This may be particularly important for vascular smooth-muscle cells (VSMC), whose growth and contractility are altered following atherosclerotic-associated thromboses. To study the cellular signal events by which thrombin exerts its actions, the effects of purified human alpha-thrombin were examined in cultured rat aortic VSMC. alpha-Thrombin stimulated a biphasic change in intracellular pH (pH(i)), causing an early rapid acidification, followed by a sustained alkalinization. The increase in pH(i) was dependent on extracellular Na+ and inhibited by 5'-(NN-dimethyl)amiloride, consistent with mediation by Na+/H+ exchange. alpha-Thrombin rapidly increased free intracellular [Ca2+] ([Ca2+]i). The increase in [Ca2+]i was secondary to activation of phospholipase C, as demonstrated by increases in InsP3 (226%) and InsP2 (387%) and decreases in polyphosphoinositides at 15 s. Expression of the mRNA for the proto-oncogene c-fos was induced by alpha-thrombin. Stimulation of c-fos mRNA was not dependent on alterations in pH(i), but required a rise in [Ca2+]i. Despite many growth-related signals shared by alpha-thrombin with platelet-derived growth factor, alpha-thrombin failed to stimulate [H-3]thymidine incorporation or cell division, although there was a maximal increase of 52% in protein synthesis. The data suggest that there are cellular signal events not activated by alpha-thrombin which are required for proliferation of these aortic VSMC.