Activation of phospholipase A2 and β-thromboglobulin release in human platelets: Comparative effects of thrombin and fluoroaluminate stimulation

Protein kinase C inhibitors enhance G‐protein induced phospholipase A2 activation in intact human platelets

“…Washed labelled platelets (WLP) were prepared as previously reported [7]. Briefly, blood (50-80 ml) was drawn from healthy volunteers in citrate-dextrose as anticoagulant (1:6 volumes of blood) and washed platelets were prepared in the presence of 0.2 ~tM PGI2, 50 U/ ml heparin and 0.01 mg/ml apyrase and incubated with [3H]AA (1 ~Ci/ml of platelet suspension) at 37°C for 1 h. The unincorporated precursor was removed by further washings.…”

“…0.5 ml WLP suspension was stimulated and with thrombin 0.5 U/ml for 3 rain or with 21 mM A1F~-(NaF and A1C13 solution in a 5000:1 molar ratio) for 20 min under continuous stirring at 1,100 rpm [7]. When indicated, WLP were preincubated with PMA (100 nM for 5 rain) or staurosporine (200 nM for 4 min) or H-7 (200 p.M for 4 min) or calphostin C (2 IzM for 4 min) or okadaic acid (1 I.tM for 4 min or for 10 min) before stimulation with A1F~-or with thrombin.…”

“…Lipids were extracted and AA and phosphatidic acid (PA) were isolated by TLC as previously described [7]. The corresponding areas were scraped off and their radioactivity determined by liquid scintillation counting (Emulsifier-safe, Packard Instrument Co., IL) and quenching corrected by external standardization.…”

“…We have demonstrated that PLA2 activation and the release of 13-TG can be induced directly by a G-protein dependent mechanism, which does not require a previous activation of phospholipase C (PLC) [7], differently from what happens when thrombin is the stimulus.…”

“…This compound (10 mM or less) produces diacylglycerol and TxB2, without activating PLC, and increases PKC activity without an apparent increase of intracellular Ca 2+ [15]. At higher concentrations (20 mM or more), A1F 4 activates PLC and liberates TxB2 being the two events independent [7,14].…”