Effects of prostaglandins, cAMP, and changes in cytosolic calcium on platelet aggregation induced by a thromboxane A2 mimic (U46619)

Abstract

The effects of U46619, a thromboxane mimic, on cytosolic Ca2+ concentration and platelet aggregation were determined in human platelets. Cytosoiic Ca2+ concentration was determined by Quin-2 fluorescence and platelet aggregation quantitated with an aggregometer. Addition of U46619 (1 × 10−7 M) to the platelet suspension produced a rapid increase in cytosolic Ca2+ and platelet aggregation. Pretreatment of platelets with EGTA (3 × 10−3 M), verapamil (5 × 10−4 M), a calcium entry blocker, or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (1 × 10−3 M), an inhibitor of intracellular Ca2+ release, either blunted or markedly delayed the rate, but not the magnitude, of increase in cytosolic Ca2+ and prevented platelet aggregation by U46619. Pretreatment of platelets with prostaglandin I2 (PGI2) (5 × 10−8 M), PGD2 (5 × 10−8 M), PGE1 (5 × 10−8 M), PGF2α (1 × 10−5 M), dibutyryl cAMP (5 × 10−3 M), or forskolin (1 × 10−6 M) prevented both the increase in cytosolic Ca2+ and the associated platelet aggregation induced by U46619. These data suggest that U46619 may induce platelet aggregation through an increase in cytosolic Ca2+, and that both Ca2+ entry and its release from intracellular storage sites probably contribute to the increase in cytosolic Ca2+. Furthermore, the rate of the increase in cytosolic Ca2+ concentration, as well as the magnitude of the increase, appear to be critical for platelet aggregation induced by U46619. Our data are consistent with the hypothesis that PGs inhibit U46619-induced platelet aggregation by preventing the increase in cytosolic Ca2+, and that these effects may be mediated via an increase in cAMP, since they were induced by PGs and cAMP.Key words: thromboxane, cytosolic calcium, platelet aggregation, prostaglandins, EGTA, verapamil, 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride.