Sequential sodium-proton exchange in thrombin-induced human platelets

Abstract

Thrombin stimulation of human platelets initiates a membrane depolarization attributable to a Na + influx into, and an alkalinization of, the cytoplasm, both of which follow a similar rapid time scale and thrombin-dose dependence. These responses precede secretion of the contents of the dense granules (serotonin) and, after 1 minute, of lysosomes (β-glucuronidase). We have evaluated these parameters in the presence of 2H 2O in order to determine if the Na + influx and H + efflux are sequential or simultaneous. NMR evidence indicates that 2H 2O equilibration in rapid, and virtually complete within the 3 min prestimulation platelet equilibration period. In response to an 0.05 U/ml addition of thrombin, the rate of depolarization is 70–80% slower in 2H 2O than in H 2O. The time to reach maximal depolarization is 5 to 10 seconds longer in 2H 2O, the extent of depolarization 60% inhibited, and the pH change 85% inhibited. The serotonin secretion is unaltered, while the β-glucuronidase secretion is 130–180% enhanced. Dimethylamiloride inhibits the Na + influx and the pH change completely. These results suggest that the Na + and H + fluxes across the plasma membrane are interdependent but neither simultaneous nor electroneutral. Furthermore, granule secretion, previously shown by us to be independent of the existent Na + gradient, depends on the cytoplasmic K + and H + concentrations.