DIFFERENTIAL-EFFECTS OF PHORBOL 12-MYRISTATE 13-ACETATE AND DIACYLGLYCEROLS ON THROMBOXANE A(2)-INDEPENDENT PHOSPHOLIPASE A(2) ACTIVATION IN COLLAGEN-STIMULATED HUMAN PLATELETS

We investigated the priming effects of protein kinase C (PKC) activato rs such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC(8) and OAG, and 1,3-DiC(8) (a poor activator of PKC) on thromboxane A(2) (TxA(2))- independent phospholipase A(2) (PLA(2)) activation in human platelets using collagen and A23187 as agonists. We measured PLA(2) activation i n collagen-stimulated platelets in the presence of BW755C, which aboli shed TxA(2) synthesis, rise in cytosolic Ca2+, and aggregation. In the presence of PMA (50 nM), the amount of arachidonic acid (AA) released in platelets stimulated with collagen and A23187 represented 300% (13 .85 nmol versus 4.5 nmol) and 400% (28 nmol versus 7 nmol) of controls (without PMA), respectively, while 1,2-DiC(8), OAG, and 1,3-DiC(8) in creased TxA(2)-independent AA release by 50% in A23187-stimulated plat elets and had no effect on the release of AA in collagen-stimulated pl atelets. Interestingly, 1,3-DiC(8), which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC(8)) in priming TxA(2)-independent PLA(2) activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the T XA(2)-dependent IP3-mediated rise in cytosolic Ca2+ may not be obligat ory for priming PLA(2) activation in the presence of PMA in collagen-s timulated platelets. In contrast, 1,2-DiC(8), OAG, and 1,3-DiC(8) like ly enhance PLA(2) activation via intracellular Ca2+ as they selectivel y affect this enzyme only in A23187-stimulated platelets. We also obse rved a significant increase in both saturated (palmitic and stearic ac ids) and unsaturated fatty acids (oleic and linoleic acids) in platele ts stimulated by collagen or A23187 in the presence of PMA (50 nM), bu t not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA(1), or nonspecific PLA(2 ). Since both 1,2-DiC(8) and OAG exert no significant effect on the re lease of these fatty acids, the effects observed with PMA on DAG lipas e/PLA(1) may not involve a PKC dependent mechanism. We, therefore, con clude that the mechanisms by which PMA and DAGs prime PLA(2) activatio n are different and that the priming mechanism by DAGs may not involve PKC, but may require a rise in intracellular Ca2+. (C) 1994 Academic Press, Inc.