Human platelet phospholipase A2 activity is responsive in vitro to pH and Ca2+ variations which parallel those occurring after platelet activation in vivo.

Secretion of human platelet dense granule contents in response to epinephrine and other weak agonists requires the prior liberation of membrane-esterified arachidonic acid by a phospholipase A2 enzyme species whose activity is regulated by Na+/H+ exchange (e.g., Sweatt et al. (1986) J. Biol. Chem. 261, 8660-8673 and Banga et al. (1986) Proc. Natl. Acad. Sci. USA 83, (197-9201). Based on our earlier findings in intact platelets, we postulated that the alkalinization of the platelet interior that accompanies accelerated activity of the Na+/H+ antiporter enables the phospholipase A2 enzyme to function at ambient or low concentrations of intraplatelet Ca2+. To test the hypothesis that the Ca2+ dependence of platelet phospholipase A2 activity is influenced by changes in intraplatelet pH that occur following platelet activation, we characterized the Ca2+ dependence of this enzyme as a function of changes in pH (from pH 6.8-8.0), since it is within this range that intraplatelet pH changes occur following platelet activation. Phospholipase A2 enzymatic activity in platelet particulate preparations was detectable in the presence of micromolar concentrations of Ca2+ (EC50 1-2 microM) and plateaued above 10 microM Ca2+. Enzymatic activity measured at 4.8 microM Ca2+ was increased by raising the pH from 5.5 to 8.0 (EC50 7.4), was optimal at pH 8.0 and declined at more alkaline values. Furthermore, increases in pH from pH 6.8 to pH 8.0 not only increased maximal enzymatic activity but also enabled detection of enzymatic activity at lower Ca2+ concentrations. The interdependent regulation of phospholipase A2 activity by changes in pH and Ca2+ suggests that phospholipase A2 could serve to integrate changes in intracellular pH and available Ca2+ that occur subsequent to activation of human platelets by epinephrine and other weak agonists.