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Using intracellularly-trapped, fluorescent Ca2+ indicator quin 2, we measured directly cytoplasmic Ca2+ in washed human platelets stimulated by thrombin and examined the effects of cyclic AMP and cyclic GMP on cytoplasmic Ca2+ levels and 14C-serotonin (5-HT) release. Thrombin (0.2U/ml) evoked a rise in cytoplasmic Ca2+ from the basal level of about 0.1μM to 1-3μM which was fast enough to trigger 5-HT release. This rise was inhibited in a dose-dependent manner by preincubation with prostaglandin E1 (PGE1) (0.1-10μM) or dibutyrylcyclic AMP (0.01-1mM). Parallel to this, 5-HT release was also inhibited by these drugs. When added to platelets after stimulation by thrombin, PGE1 caused the rapid decrease of elevated cytoplasmic Ca2+. Sodium nitroprusside (1-100μM) and SIN (0.1-10μM), both of which are potent stimulators of platelet guanylate cyclase, or 8-bromo-cyclic GMP (0.01-1mM) inhibited thrombin-induced increase of cytoplasmic Ca2+ and 5-HT release in a dose-dependent manner. These results provide direct evidence that cytoplasmic Ca2+ levels in platelets are regulated by cyclic AMP and that an increase in platelet cyclic GMP also inhibits Ca2+ mobilization and prevents platelet activation.

Effects of cyclic nucleotides on cytoplasmic free calcium in human platelets stimulated by thrombin: direct measurement with quin 2.