ENDOTOXIC LIPID-A INDUCES INTRACELLULAR CA2+ INCREASE IN HUMAN PLATELETS

The activation of protein kinase C by endotoxic lipid A was observed with both intact platelets and in a cell-free system [Romano & Hawiger (1990) J. Biol. Chem. 265, 1765-1770]. We have now studied the action of lipid A on intracellular Ca2+ concentration ([Ca2+]i). Lipid A induced a concentration-dependent rise in [Ca2+]i in human platelets loaded with fura-2, which reached a maximum at 37.1 +/- 3.8 s (t(max.)). Maximum [Ca2+]i levels, observed at 30-mu-M lipid A, were 432 +/- 60 nm. EGTA (2 mM) or NiCl2 (1 mM) each decreased the lipid A-dependent elevation of [Ca2+]i by 50-60% without significant modification of t(max.), but shortening the time for 50% recovery (t(50)) from > 400 s to 113.1 +/- 29.1 s and 54 +/- 2.1 s, respectively. Quenching of the fura-2 signal was also observed in lipid A-stimulated platelets resuspended with MnCl2 (1 mM), suggesting that both mobilization and external influx of Ca2+ occur. Intracellular Ca2+ mobilization depended on release from Ins(1,4,5)P3-sensitive stores, since Ins(1,4,5)P3 accumulation was detected in lipid A-activated platelets. Staurosporine, an inhibitor of protein kinase C, blocked the [Ca2+]i rise generated by lipid A in platelets [concn. giving 50% inhibition (IC50) = 0.1-mu-M], prolonging the t(max.) to 54.7 +/- 5.1 s, but decreasing the t(50) to 157.5 +/- 31.8 s. Staurosporine also suppressed InsP3 accumulation (IC50 = 0. 1 5-mu-M). These results suggest that platelet activation by lipid A involves an interaction between [Ca 2+]i elevation and protein kinase C activation.