Differential Effects of Phorbol 12-Myristate 13-Acetate and Diacylglycerols on Thromboxane A2-Independent Phospholipase A2 Activation in Collagen-Stimulated Human Platelets

We investigated the priming effects of protein kinase C (PKC) activators such as phorbol 12-myristate 13-acetate (PMA), 1,2-DiC₈ and OAG, and 1,3-DiC₈ (a poor activator of PKC) on thromboxane A₂ (TxA₂)-independent phospholipase A₂ (PLA₂) activation in human platelets using collagen and A23187 as agonists. We measured PLA₂ activation in collagen-stimulated platelets in the presence of BW755C, which abolished TxA₂ 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-DiC₈, OAG, and 1,3-DiC₈ increased TxA₂-independent AA release by 50% in A23187-stimulated platelets and had no effect on the release of AA in collagen-stimulated platelets. Interestingly, 1,3-DiC₈, which is a poor activator of PKC, was as effective as the other two DAGs (OAG and 1,2-DiC₈) in priming TxA₂-independent PLA₂ activation, but was less effective than PMA in platelets stimulated with A23187. These results suggest that the TXA₂-dependent IP₃-mediated rise in cytosolic Ca²⁺ may not be obligatory for priming PLA₂ activation in the presence of PMA in collagen-stimulated platelets. In contrast, 1,2-DiC₈, OAG, and 1,3-DiC₈ likely enhance PLA₂ activation via intracellular Ca²⁺ as they selectively affect this enzyme only in A23187-stimulated platelets. We also observed a significant increase in both saturated (palmitic and stearic acids) and unsaturated fatty acids (oleic and linoleic acids) in platelets stimulated by collagen or A23187 in the presence of PMA (50 nM), but not in the presence of DAGs. These findings imply that PMA may also affect the activation of DAG/MAG lipases, PLA₁, or nonspecific PLA₂. Since both 1,2-DiC₈ and OAG exert no significant effect on the release of these fatty acids, the effects observed with PMA on DAG lipase/PLA₁ may not involve a PKC dependent mechanism. We, therefore, conclude that the mechanisms by which PMA and DAGs prime PLA₂ activation are different and that the priming mechanism by DAGs may not involve PKC, but may require a rise in intracellular Ca²⁺.