Identification of diadenosine hexaphosphate in human erythrocytes

Diadenosine polyphosphates have been identified as important regulators of vascular tone and blood pressure. In reference to the background of the well-known vasoconstriction induced by hemolysate, we questioned whether this action may be due in part to the presence of diadenosine polyphosphates in human erythrocytes. Therefore, erythrocytes were separated from other blood cells and deproteinated. To concentrate and purify nucleotides, the extract was chromatographed by anion exchange, affinity, and reversed-phase columns. In one of the purified fractions, diadenosine hexaphosphate (diadenosine 5', 5'-P(1), P(6) hexaphosphate [AP(6)A]) was identified by matrix-assisted laser desorption/ionization mass spectrometry, ultraviolet spectroscopy, and enzymatic analysis. Hemolysate of erythrocytes injected into the isolated perfused rat kidney induced a dose-dependent vasoconstriction, which was partially inhibited by P(2)-purinoceptor antagonist. The data document the existence of AP(6)A in erythrocytes. AP(6)A may be involved in the pathogenesis of vasospasm induced by free hemoglobin.