PHORBOL ESTER TREATMENT OF INTACT RABBIT PLATELETS GREATLY ENHANCES BOTH THE BASAL AND GUANOSINE 5'-[GAMMA-THIO]TRIPHOSPHATE-STIMULATED PHOSPHOLIPASE-D ACTIVITIES OF ISOLATED PLATELET MEMBRANES - PHYSIOLOGICAL ACTIVATION OF PHOSPHOLIPASE-D MAY BE SECONDARY TO ACTIVATION OF PHOSPHOLIPASE-C

Rabbit platelets were labelled with [3H]glycerol and incubated with or without phorol 12-myristate 13-acetate (PMA). Membranes were then isolated and assayed for phospholipase D (PLD) activity by monitoring [3H]phosphatidylethanol formation in the presence of 300 mM-ethanol. At a [Ca2+(free)] of 1 μM, PLD activity was detected in control membranes, but was 5.4 ± 0.8-fold (mean ± S.E.M.) greater in membranes from PMA-treated platelets. Under the same conditions, 10 μM-guanosine 5'-[γ-thio]triphosphate (GTP[S]) stimulated PLD by 18 ± 3-fold in control membranes, whereas PMA treatment and GTP[S] interacted synergistically to increase PLD activity by 62 ± 12-fold. GTP[S]-stimulated PLD activity was observed in the absence of Ca2+, but was increased by 1 μM-Ca2+ (3.5 ± 0.2-fold and 1.8 ± 0.1-fold in membranes from control and PMA-treated platelets respectively). GTP exerted effects almost as great as those of GTP[S], but 20-30-fold higher concentrations were required. Guanosine 5'-[β-thio]diphosphate inhibited the effects of GTP[S] or GTP, suggesting a role for a GTP-binding protein in activation of PLD. Thrombin (2 units/ml) stimulated the PLD activity of platelet membranes only very weakly and in a GTP-independent manner. The actions of PMA and analogues on PLD activity correlated with their ability to stimulate protein kinase C in intact platelets. Staurosporine, a potent protein kinase inhibitor, had both inhibitory and, at higher concentrations, stimulatory effects on the activation of PLD by PMA. The a results suggest that PMA not only stimulates PLD via activation of protein kinase C but can also activate the enzyme by a phosphorylation-independent mechanism in the presence of staurosporine. However, under physiological conditions, full activation of platelet PLD may require the interplay of protein kinase C, increased Ca2+ and a GTP-binding protein, and may occur as a secondary effect of the activation of phospholipase C.