Diacylglycerol mediates the thrombin-induced, protein kinase C and Ca2+ independent activation of the Na+/H+ exchanger in platelets

Treatment of aspirinated platelets with the electroneutral K+/H+ exchanger nigericin induces a decrease in intraplatelet pH as measured with the intracellular fluorescent indicator BCECF. Under these conditions, the proton permeability of the plasma membrane is unaffected. The addition of thrombin induces a rapid partial recovery of pH(i), which is completely abolished by the Na+/H+ exchanger inhibitor NHA. The effect is also evident in the presence of the PKC inhibitors GF 109203X or staurosporine and in the absence of both external (EGTA-chelated) and internal (BAPTA-chelated) Ca2+. This makes the thrombin-induced activation of the exchanger independent of the involvement of the hitherto described activators, namely PKC and the increase in [Ca2+](i), as well of the recently reported activator arachidonic acid [Cavallini, L., Coassin, M., Borean, A., and Alexandre, A. (1996) Biochem. J. 319, 567-574], whose production requires a high [Ca2+](i). The thrombin-dependent recovery of pH(i) is prevented by the phospholipase C inhibitor ET 18 O-CH3 and is mimicked by the addition of the permeable diglyceride dioctanoyl glycerol (DiC(8)) exogenously supplied. The effect of thrombin and DiC(8) is unaffected by inhibition of diacylglycerol lipase and diacylglycerol kinase. These experiments identify diglyceride as a novel activator of the Na+/H+ exchanger in platelets.