PROTEIN-KINASE-C INHIBITORS ENHANCE G-PROTEIN INDUCED PHOSPHOLIPASE A(2) ACTIVATION IN INTACT HUMAN PLATELETS

Washed intact human platelets were prelabelled with [H-3]arachidonic a cid ([H-3]AA) and stimulated with thrombin or with AlF4-, a known unsp ecific activator of G-proteins. Both stimuli induced the liberation of [H-3]AA, the release of beta-thromboglobulin (beta-TG) and platelet a ggregation. PMA did not induce liberation of [H-3]AA although it induc ed beta-TG release and aggregation; preincubation with PMA did not mod ify significantly the amounts of [H-3]AA and beta-TG released by throm bin or AlF4-. Different inhibitors of PKC (staurosporine, H-7 and 4 ca lphostin C) increased the release of [H-3]AA and inhibited beta-TG rel ease and aggregation induced by AlF4- but they had no effect when plat elets were stimulated with thrombin (0.5 U/ml). Calphostin C was able to release [H-3]AA by itself without inducing aggregation or beta-TG r elease. Okadaic acid (a serine/threonine phosphoprotein phosphatase in hibitor) greatly inhibited the release of [H-3]AA, beta-TG and aggrega tion in AlF4--stimulated platelets. These results indicate the presenc e of a G-protein mediated mechanism for the activation of a platelet p hospholipase A(2) which is negatively affected by a protein kinase, se nsible to putative inhibitors of protein kinase C, and it is activated by a protein phosphatase, sensible to okadaic acid.