Diadenosine 5,5ٟ-P1,P2-diphosphate (Ap2A) is one of the adenylic dinucleotides stored in platelet granules. Along with proaggregant ADP, it is released upon platelet activation and is known to stimulate myocyte proliferation. We have previously demonstrated synthesis of Ap2A and of two isomers thereof, called P18 and P24, from their high pressure liquid chromatography retention time, by the ADP-ribosyl cyclase CD38 in mammalian cells. Here we show that Ap2A and its isomers are present in resting human platelets and are released during thrombin-induced platelet activation. The three adenylic dinucleotides were identified by high pressure liquid chromatography through a comparison with the retention times and the absorption spectra of purified standards. Ap2A, P18, and P24 had no direct effect on platelet aggregation, but they inhibited platelet aggregation induced by physiological agonists (thrombin, ADP, and collagen), with mean IC 50 values ranging between 5 and 15 M. Moreover, the three dinucleotides did not modify the intracellular calcium concentration in resting platelets, whereas they significantly reduced the thrombin-induced intracellular calcium increase. Through binding to the purinergic receptor P2Y 11 , exogenously applied Ap2A, P18, and P24 increased the intracellular cAMP concentration and stimulated platelet production of nitric oxide, the most important endogenous antiaggregant. The presence of Ap2A, P18, and P24 in resting platelets and their release during thrombin-induced platelet activation at concentrations equal to or higher than the respective IC 50 value on platelet aggregation suggest a role of these dinucleotides as endogenous negative modulators of aggregation.The dinucleoside diphosphates diadenosine 5Ј,5ٞ-P1,P2-diphosphate (Ap2A), 2 adenosine guanosine diphosphate (Ap2G), and diguanosine diphosphate (Gp2G) represent a new class of growth-promoting extracellular mediators present in platelet secretory granules (1) and in cardiac myocytes (2), capable of stimulating cardiac myocyte proliferation (1) and believed to play a role in the control of cardiovascular tone (3). The intraplatelet concentration of each one of these dinucleotides has been estimated to be in the range between 30 and 100 M (1). It has also been shown that the concentration of Ap2A, Ap2G, and Gp2G in the supernatant of platelets stimulated with 0.05 units/ml thrombin is ϳ60% of the total intraplatelet amount of each dinucleoside diphosphate, suggesting that their primary function is extracellular (1). The enzyme responsible for their synthesis has not been identified, and the effect of these dinucleotides on platelet function has not yet been investigated.We have recently demonstrated that ADP-ribosyl cyclases from Axinella polypoides, (Porifera, Demospongiae), Aplysia californica (Molluscs), and human CD38 can synthesize three adenylic dinucleotides from cyclic ADP-ribose and adenine (Ade): Ap2A and two isomers thereof, called P18 and P24, which are characterized by an unusual N-glycosidic bond between one adenine a...
