GTPγS and phorbol ester act synergistically to stimulate both Ca²⁺‐independent secretion and phospholipase D activity in permeabilized human platelets

Abstract

We have tested the hypothesis that phospholipase D (PLD) is the effector of the unidentified G protein (GE) mediating Ca"-independent exocytosis in platelets. Although GTPyS, and to a lesser extent phorbol 12-myristate 13-acetate (PMA), caused some secretion of 5-HT from electropermeabilized human platelets in the effective absence of Ca*' (pCa > 9) these stimuli had much more potent synergistic effects when added together. In all cases, secretion of 5-HT was closely correlated to the stimulus-induced formation of [3H]phosphatidic acid (['HIPA) from ['Hlarachidonatelabelled phospholipids. Addition of ethanol inhibited both secretion and [jH]PA formation and led to the accumulation of ['Hlphosphatidylethanol (['HIPEt). indicating that ['HIPA was formed largely by activation of PLD. BAPTA and analogues caused dose-dependent inhibitions of both GTPyS-induced secretion and PLD activity in the permeabilized platelets. This action of BAPTA did not appear to be mediated by chelation of Ca" or by direct inhibition of protein kinase C (PKC). The results suggest that PLD is the target of G, in platelets and that BAPTA can block PLD activation.

Protein kinase C-dependent and Ca2+-dependent mechanisms of secretion from streptolysin O-permeabilized platelets: effects of leakage of cytosolic proteins

“…Comparison of the initial part of the present study with previous work using electropermeabilized platelets [5,8,9,23,26] suggested that similar regulatory mechanisms were operating in the two permeabilized platelet preparations, but with some quantitative differences. The sensitivities of dense-granule secretion to Ca# + in the two preparations were comparable and, in both, addition of PMA or GTP[S] enhanced Ca# + responsiveness, the latter more effectively than the former.…”

“…Our observation that GTP[S] stimulated secretion in the presence of 10-100 nM PMA, which induces a marked to maximal phosphorylation of pleckstrin, confirms our previous conclusion [5][6][7][8] that GTP[S] acts by mechanisms additional to stimulation of the phospholipase C-PKC axis. We have previously provided evidence that GTP[S] may exert some of its effects by stimulating phospholipase D activity [8,9], but the present study suggests an additional action. Thus GTP [S] induced major changes in the ultrastructure of SLO-permeabilized platelets in the absence of Ca# + or of any activation of PKC.…”

“…Thus GTP or its more stable analogue, guanosine 5h-[γ-thio]-triphosphate (GTP [S]) is required for the G-protein-and Ca# + -dependent activation of phospholipase C in permeabilized platelets [7], which results in the stimulation of PKC by 1,2-diacylglycerol [8]. Secondly, experiments with electropermeabilized platelets have shown that GTP[S] acts synergistically with PKC when this enzyme is stimulated by phorbol 12-myristate 13-acetate (PMA), to induce a Ca# + -independent secretion of granule constituents [5] ; this effect is associated with the activation of phospholipase D, but not of phospholipase C [8,9]. GTP[S] also potentiated Ca# + -induced secretion from permeabilized platelets after protein kinase activities had been Abbreviations used : PKC, protein kinase C ; SLO, streptolysin O ; 5-HT, 5-hydroxytryptamine ; PMA, phorbol 12-myristate 13-acetate ; GTP [S], guanosine 5h-[γ-thio]triphosphate ; ECL, enhanced chemiluminescence.…”

A novel role for phospholipase D as an endogenous negative regulator of platelet sensitivity

“…In contrast to FIPI, ethanol and especially 1-butanol displayed distinct inhibitory effects on PAR-1 and PAR-4 activating peptide-induced Ca 2+ mobilization, dense granule secretion and aggregation of human platelets indicating that prior studies that rely on PLD inhibition by primary alcohols must be re-evaluated. In the nineties Coorssen and Haslam proposed that PLD is important for human platelet secretion by using ethanol to block PLD mediated effects on degranulation [45]. In a recent study it was shown that 1-butanol abrogated platelet aggregation by PAR-1 but not PAR-4 activating peptide [46].…”

Activation of phospholipase D by growth factors and oncogenes in murine fibroblasts follow alternative but cross-talking pathways

“…The ras-transformed cell lines used here were obtained from independent transfections and have been described previously [6,22], or were freshly generated using an LTR-driven vector (LP14-8) or simian virus 40 ), and butan-1-ol was from Merck. Bisindolylmaleimide was purchased from Calbiochem.…”