THE EFFLUX OF RB-86 AND [H-3] 5-HT FROM HUMAN PLATELETS DURING CONTINUOUS PERFUSION - EFFECTS OF POTASSIUM-INDUCED MEMBRANE DEPOLARIZATION AND THROMBIN STIMULATION

In order to study the ionic efflux or granule release from human platelets following pulse exposure to various stimuli, a method for continuous perfusion of platelets was developed. The method was applied to compare the effects of membrane depolarization and thrombin stimulation on the release of Rb-86 and [H-3]5-HT. Washed and preloaded human platelets were placed on a membrane filter in a temperature controlled polypropylene chamber, and subsequently perfused with buffer. After an initial washout period the efflux of Rb-86 or [H-3]5-HT reached steady, low levels. K+ induced concentration dependent increases in Rb-86 efflux, corresponding to a depolarization of the membrane potential, whereas the efflux of [H-3]5-HT was unaltered. Thrombin induced concentration dependent increases in the efflux of both Rb-86 and [H-3]5-HT. Pretreatment with K+ 12 or 30 mM did not alter the [H-3]5-HT efflux induced by thrombin 0.1 U ml-1. Scanning electron micrographs of platelets on the filter showed that the unstimulated platelets had regular shape, whereas after addition of thrombin there was formation of pseudopods and minor aggregates. The effect of potassium-induced membrane depolarization on platelet aggregation was also studied. High concentration of K+ did not induce aggregation or shape change during 2 or 10 minutes of incubation. K+ had little or no effect on aggregation induced by ADP 2-mu-M or thrombin 0.4 U ml-1. The results from release experiments and aggregation tests argue against an immediate coupling between membrane potential and platelet reactivity.