The influence of Na+ on the alpha 2-adrenergic receptor system of human platelets. A method for removal of extraplatelet Na+. Effect of Na+ removal on aggregation, secretion, and cAMP accumulation.

Abstract

A method is described for the preparation of functionally responsive human platelets which were suspended in Na+-containing and "Na+-free" buffer.The platelets in plasma were isolated and exchanged into buffer by centrifugation through a stepwise albumin gradient followed by gel filtration through a Sepharose 2B column.Sodium was replaced by the inert cation Nmethyl-D-glucamine and removal of Na+ was confirmed by atomic absorption spectrophotometry.The gel-filtered platelets were utilized to evaluate the ability of epinephrine, acting via az-adrenergic receptors, to induce platelet aggregation and secretion of dense, serotonin-containing granules in the absence and presence of Na'.Sodium has previously been shown to modulate a-receptor-ligand interactions and to enhance a-adrenergic-mediated attenuation of adenylate cyclase activity in broken cell preparations of human platelets.Both the rate and extent of aggregation of the platelets in Na+-free medium were diminished in response to a maximal dose of epinephrine.Similarly, epinephrine-stimulated [3H]serotonin release was markedly diminished in platelets prepared in Na+-free buffer as compared to platelets in Na+-containing buffer.In contrast to these results, a maximal dose of thrombin, a stimulus that acts via a distinct receptor and a presumably independent mechanism, produced comparable aggregation and secretion responses in the platelets from both groups.These results suggest that Na+ plays an important role in a-adrenergic mediated platelet function, and results presented in the accompanying manuscript suggest that the locus of the "Na' effect'' may be intraplatelet.Epinephrine attenuation of prostaglandin El-stimulated cAMP accumulation was similar in control and Na+-free platelets, in contrast to reported effects of Na+ on adenylate cyclase activity in washed platelet particulate preparations.The apparent dissociation of the effects of Na+ on a-adrenergic-induced human platelet functional responses and a-adrenergic attenuation of cAMP levels may indicate that 1) decreases in cAMP alone do not mediate epinephrine-induced aggregation and serotonin release; 2) changes in cAMP occurring in a particular microenvironment are obscured by measuring total CAMP levels; or 3) Na+ effects on epineph-HL 25182, AM 20593, GM 07628, and HL 12486.The costs of publi-