J Biol Chem, Vol. 274, Issue 34, 23926-23931, August 20, 1999

Identification and Characterization of P¹,P⁷-Di(adenosine-5')-heptaphosphate from Human Platelets

Joachim Jankowski, Martin Tepel, Markus van der Giet, Ilva Maria Tente, Lars Henning, Ralf Junker, Walter Zidek, and Hartmut Schlüter

From the Medizinische Klinik I, Universitäts-Klinik Marienhospital, Ruhr University of Bochum, 44625 Herne, Germany and  Institut für Klinische Chemie und Laboratoriumsmedizin and Institut für Arterioskleroseforschung, Westfälische Wilhelms-University of Münster, 48149 Münster, Germany

Diadenosine pentaphosphate and diadenosine hexaphosphate have been isolated in human platelets and have been postulated to play an important role in the control of vascular tone. Here we describe the isolation and identification of diadenosine heptaphosphate from human platelets. Dinucleoside polyphosphates were concentrated by affinity chromatography from a nucleotide-containing fraction from deproteinated human platelets. Dinucleoside polyphosphates were purified by anion-exchange and reversed phase high performance liquid chromatography to homogeneity. Analysis of one of these fractions with matrix-assisted laser desorption/ionization mass spectrometry revealed a molecular mass of 1076.4 (1077.4 = [M + H]⁺) Da. UV spectroscopic analysis of this fraction showed the spectrum of an adenosine derivative. Comparison of the postsource decay matrix-assisted laser desorption/ionization mass spectrum of the fraction minus that of diadenosine heptaphosphate (Ap₇A) demonstrated that the isolated substance was identical to Ap₇A. The identity of the retention times of the authentic and the isolated compound confirmed this result. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5'-positions of the riboses. With thrombin-induced platelet aggregation, Ap₇A is released from the platelets into the extracellular space. The vasoconstrictive action of Ap₇A on the vasculature of the isolated perfused rat kidney Ap₇A was slightly less than that of Ap₆A. The threshold of the vasoconstrictive action of Ap₇A was 10⁵ mol/liter. The vasoconstrictive effect was abolished by suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid, suggesting an activation of P_2x receptors. Furthermore, Ap₇A inhibits ADP-induced platelet aggregation. Thus, the potent vasoconstrictor Ap₇A derived from human platelets, like other diadenosine polyphosphates, may play a role in the regulation of vascular tone and hemostasis.