The antiaggregatory properties of trimetazidine were investigated further by analyzing its effects on cytosolic calcium and proton concentrations, well-known regulators of platelet reactivity. Aggregatory responses of washed platelets were assessed by turbidometry, and cytosolic Ca2+ concentration ([Ca2+]i) and pH (pHi) were determined by their respective fluorescent probes: Fura-2 and BCECF. Preincubation with trimetazidine dose-dependently inhibited platelet aggregation induced by 0.05 U/ml thrombin (p < 0.001). At concentrations < or = 1 mM, trimetazidine did not affect the resting [Ca2+]i value but slightly alkalinized the cytosol by 0.05 +/- 0.03 pH units (p < 0.02, n = 11). In platelets stimulated by 0.05 U/ml thrombin, 0.1 mM trimetazidine did not modify pHi variations but decreased [Ca2+]i variations (p < 0.003, n = 16), blunting by 28 +/- 6% the transient peak of [Ca2+]i (p < 0.006) and decreasing by 6 +/- 2% the equilibrium value (p < 0.005). These inhibitory effects were inversely dependent on thrombin concentrations (p < 0.004, n = 21) and were abolished in the virtual absence of external Ca2+. Trimetazidine therefore attenuates the Ca2+ influx evoked by thrombin, thereby limiting Ca2+ accumulation in stimulated platelets. Such a protective effect may participate in the antiaggregatory properties of trimetazidine.