Identification and characterization of P-1,P-7-di(adenosine-5 ')-heptaphosphate from human platelets

Diadenosine pentaphosphate and diadenosine hexaphosphate have been isolated in human platelets and have been postulated to play an important role in t he control of vascular tone. Here we describe the isolation and identificat ion of diadenosine heptaphosphate from human platelets. Dinucleoside polyph osphates were concentrated by affinity chromatography from a nucleotide con taining fraction from deproteinated human platelets. Dinucleoside polyphosp hates were purified by anion-exchange and reversed phase high performance l iquid chromatography to homogeneity. Analysis of one of these fractions wit h matrix-assisted laser desorption/ionization mass spectrometry revealed a molecular mass of 1076.4 (1077.4 = [M + H](+)) Da. UV spectroscopic analysi s of this fraction showed the spectrum of an adenosine derivative. Comparis on of the postsource decay matrix-assisted laser desorption/ionization mass spectrum of the fraction minus that of diadenosine heptaphosphate (Ap(7)A) demonstrated that the isolated substance was identical to Ap(7)A. The iden tity of the retention times of the authentic and the isolated compound conf irmed this result. Enzymatic analysis demonstrated an interconnection of th e phosphate groups with the adenosines in the 5'-positions of the riboses. With thrombin-induced platelet aggregation, Ap(7)A is released from the pla telets into the extracellular space. The vasoconstrictive action of Ap(7)A on the vasculature of the isolated perfused rat kidney Ap(7)A was slightly less than that of Ap(6)A. The threshold of the vasoconstrictive action of A p(7)A was 10(-5) mol/liter. The vasoconstrictive effect was abolished by su ramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, suggesting an activation of P-2x receptors. Furthermore, Ap(7)A inhibits ADP-induced platelet aggregation. Thus, the potent vasoconstrictor Ap(7)A derived from human platelets, Like other diadenosine polyphosphates, may play a role in the regulation of vascular tone and hemostasis.