Objective:In order to elucidate the role of Na+—Ca2 +exchange in regulating cytosolic free Ca2+concentration in human platelets, we investigated the relationship between cytosolic free Na+and Ca2+concentrations in human platelets.Methods:Sodium-binding benzofuran isophthalate and fura-2 were used to monitor cytosolic free Na+and Ca2+concentrations, respectively.Results:Ouabain at a concentration of 100 µmol/l induced an increase in cytosolic free Na+concentration within 5 min, followed by increases in resting cytosolic free Ca2+concentration and intracellular Ca2+store. These three parameters were increased in a time-dependent manner significantly above the timed control over a period of 60 min. Pre-incubation of platelets with 100 µmol/l ouabain for 30 min significantly enhanced the cytosolic free Ca2+response to thrombin and arginine vasopressin in the absence of extracellular Ca2+. The decrease from peak cytosolic free Ca2+concentration in response to ionomycin in the absence of extracellular Ca2+was significantly slower in low-Na+buffer than in standard buffer. In addition, 5 µmol/l ionomycin increased the cytosolic free Na+concentration markedly in the presence of 0.1 mmol/l extracellular Ca2+, but the rise in cytosolic free Na+concentration was suppressed by 2 mmol/l Ni2+(NiCl2) or by removal of extracellular Ca2+.Conclusions:These results suggest that Na+—Ca2+exchange is important in extruding Ca2+from the cytosol in human platelets, and inhibition of this exchange leads to the accumulation of intracellular Ca2+store, which may be responsible for the enhanced responsiveness of cytosolic free Ca2+to agonists.