Modes of inhibitory action of 4β-phorbol 12-myristate 13-acetate in thrombin-stimulated arachidonic acid release in intact and permeabilized platelets

Abstract

The tumor-promoting phorbol ester 4β-phorbol 12-myristate 13-acetate (PMA) inhibited thrombin-stimulated arachidonic acid (AA) release in rabbit and human platelets. PMA was effective over the same concentration range that activates protein kinase C in intact rabbit platelets: IC 50 vs thrombin = 0.5 n m, >90% inhibition at 10 n m. Suppression of thrombin-stimulated AA release was evident within 5 min of pretreatment with 1 n m PMA. A non-tumor-promoting phorbol ester, 4- O-methyl PMA, showed a very weak ability to inhibit AA release. Thrombin-stimulated serotonin secretion was progressively inhibited by PMA pretreatment in platelets, while PMA was a stimulus for secretion at higher concentrations. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a selective inhibitor of protein kinase C, blocked PMA-induced inhibition of AA release. Furthermore, H-7 enhanced the effect of thrombin on AA release. PMA pretreatment reduced the inhibitory effect of thrombin on forskolin-stimulated cAMP accumulation, but had no effect on nonstimulated cAMP metabolism in the presence of thrombin. PMA did not inhibit AA release caused by A23187 or melittin. In digitonin-permeabilized platelets, thrombin plus guanosine 5′-(3- O-thio)triphosphate (GTPγS)-stimulated AA release, but not GTPγS- and AlF 4 −-stimulated AA release, was abolished by PMA pretreatment. These results suggest that activation of protein kinase C may exert negative feedback on the receptor-mediated activation of phospholipase A 2. A possible uncoupling of thrombin receptor to GTP-binding protein leading to activation of phospholipase A 2 by PMA pretreatment is discussed.