GTPγS and phorbol ester act synergistically to stimulate both Ca2+‐independent secretion and phospholipase D activity in permeabilized human platelets

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Abstract
We have tested the hypothesis that phospholipase D (PLD) is the effector of the unidentified G protein (GE) mediating Ca2+‐independent exocytosis in platelets. Although GTPγS, and to a lesser extent phorbol 12‐myristate 13‐acetate (PMA), caused some secretion of 5‐HT from electropermeabilized human platelets in the effective absence of Ca2+ (pCa > 9), these stimuli had much more potent synergistic effects when added together. In all cases, secretion of 5‐HT was closely correlated to the stimulus‐induced formation of [3H]phosphatidic acid ([3H]PA) from [3H]arachidonatelabelled phospholipids. Addition of ethanol inhibited both secretion and [3H]PA formation and led to the accumulation of [3H]phosphatidylethanol ([3H]PEt), indicating that [3H]PA was formed largely by activation of PLD. BAPTA and analogues caused dose‐dependent inhibitions of both GTPγS‐induced secretion and PLD activity in the permeabilized platelets. This action of BAPTA did not appear to be mediated by chelation of Ca2+ or by direct inhibition of protein kinase C (PKC). The results suggest that PLD is the target of GE in platelets and that BAPTA can block PLD activation.