Differential effects of calmodulin antagonists on phospholipases A2 and C in thrombin-stimulated platelets.

The calmodulin antagonist trifluoperazine inhibits many responses of human platelets to stimuli such as thrombin. We have now demonstrated that trifluoperazine inhibits the production of all arachidonic acid metabolites, by both cyclooxygenase and lipoxygenase pathways, in thrombin-stimulated platelets. The metabolism of exogenously supplied arachidonlc acid was not affected by trifluoperazine. Rather trifluoperazine inhibited the mobilization of arachidonic acid from platelet phospholipids. In platelets prelabeled with [l%]arachidonic acid, inhibition of the release of radioactivity from phosphatidylcholine closely paralleled inhibition of metabolite production; both were inhibited over 60% by 50 pM trifluoperazine and over 90% by 100 PM. In contrast, release of radioactivity from phosphatidylinositol was stimulated by 25-50 pM trifluoperazine and only partially (25%) inhibited by 100 pM. While the release of arachidonic acid from phosphatidylcholine presumably results solely from the action of phospholipase AZ, phosphatidylinositol can also be degraded by a phospholipase C specific for that lipid. The diglyceride produced by that enzyme can be converted to phosphatidic acid. The appearance of either radioactive arachidonic acid or phosphate in phosphatidic acid in response to thrombin was stimulated by 50 pM trifluoperazine and only partially inhibited by 100 pM. Similar results were seen with other phenylthiazines (chlorpromazine and fluphenazine) and with nonphenylthiazine antagonists of calmodulin. At all concentrations of trifluoperazine studied, the loss of radioactive arachidonic acid from phosphatidylinositol was virtually identical with the appearance of radioactivity in phosphatidic acid. These results demonstrate that in intact platelets, phospholipase AZ may require calmodulin for stimulus activation, while phospholipase C may not. Furthermore, they suggest that phospholipase AZ, acting on phosphatidylcholine, accounts for the bulk of arachidonic acid released and metabolized, while the phosphatidyllnositol degraded by phospholipase C is largely converted to phosphatidic acid.