Role of Na +/H + exchange in thrombin- and arachidonic acid-induced Ca 2+ influx in platelets

Abstract

Platelet activation is accompanied by an increase of cytosolic free Ca 2+ concentration [Ca 2+] i, (due to both extracellular Ca 2+ influx and Ca 2+ movements from the dense tubular system) and an Na + influx associated with H + extrusion. The latter event is attributable to the activation of Na +/H + exchange, which requires Na + in the extracellular medium and is inhibited by amiloride and its analogs. The present study was carried out to determine whether a link exists between Ca 2+ transients (measured by the quin2 method and the 45CaCl 2 technique) and Na +/H + exchange activation (studied with the pH-sensitive intracellular probe, 6-carboxyfluorescein) during platelet stimulation. Washed human platelets, stimulated with thrombin and arachidonic acid, showed: (1) a large and rapid [Ca 2+] i rise, mostly due to a Ca 2+ influx through the plasma membrane; (2) a marked intracellular alkalinization. Both phenomena were markedly inhibited in the absence of extracellular Na + or in the pesence of an amiloride analog (EIPA). Monensin, a cation exchanger which elicits Na + influx and alkalinization, and NH 4Cl, which induces alkanization only, were able to evoke an increase in [Ca 2+] i, mostly as an influx from the extracellular medium. Our results suggest that Ca 2+ influx induced by thrombin and arachidonic acid in human platelets is strictly dependent on Na +/H +-exchange activation.