Regulation of plasma membrane Ca2+-ATPase by small GTPases and phosphoinositides in human platelets

Abstract

We have investigated the restoration of [Ca2+]i in human platelets following the discharge of the intracellular Ca2+ stores. We found that the plasma membrane Ca2+-ATPase is the main mechanism involved in Ca2+ extrusion in human platelets. Treatment of platelets with the farnesylcysteine analogs, farnesylthioacetic acid and N-acetyl-S- geranylgeranyl-L-cysteine, inhibitors of activation of Ras proteins, accelerated the rate of decay of [Ca2+](i) to basal levels after activation with thapsigargin combined with a low concentration of ionomycin, indicating that Ras proteins are involved in the negative regulation of Ca2+ extrusion. Rho A, which is involved in actin polymerization, was not responsible for this effect. Consistent with this, the actin polymerization inhibitors, cytochalasin D and latrunculin A, did not alter the recovery of [Ca2+](i). Activation of human platelets with thapsigargin and ionomycin stimulated the tyrosine phosphorylation of the plasma membrane Ca2+-ATPase, a mechanism that was inhibited by farnesylcysteine analogs, suggesting that Ras proteins could regulate Ca2+ extrusion by mediating tyrosine phosphorylation of the plasma membrane Ca2+-ATPase. Treatment of platelets with LY294002, a specific inhibitor of phosphatidylinositol 3- and phosphatidylinositol 4-kinase, resulted in a reduction in the rate of recovery of [Ca2+](i) to basal levels, suggesting that the products of these kinases are involved in stimulating Ca2+ extrusion in human platelets.