Adenylic Dinucleotides Produced by CD38 Are Negative Endogenous Modulators of Platelet Aggregation

Abstract

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...

Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins

“…Intriguingly, PAR4-stimulated Akt signaling was not reliably detected in HeLa cells. This may result from low or absent P2Y12 expression, as HeLa cells are not normally responsive to ADP stimulation (50).…”

Diadenosine Homodinucleotide Products of ADP-ribosyl Cyclases Behave as Modulators of the Purinergic Receptor P2X7

“…Admittedly, selectivity of these dinucleotides on the other members of the purinergic receptor family requires further investigation, although it is interesting to underline that P24, P18, and Ap2A, are all agonists of the P2Y 11 purinergic receptor (13).…”

“…P18 and P24 were also identified in resting platelets and were shown to be released together with Ap2A following addition of thrombin (13). Ap2A, P18, and P24 inhibit platelet aggregation induced by thrombin, ADP, or collagen, through the activation of the purinergic receptor P2Y 11 , resulting in an increase of the intraplatelet cyclic AMP (cAMP) concentration and subsequent overproduction of nitric oxide, the most potent endogenous antagonist of aggregation (13).…”

A critical look at the function of the P2Y11 receptor

“…Many other nucleotide compounds target the P2Y 11 receptor, including diadenosine polyphosphate Ap2A and its isomers P18 and P24 [56,57]. Nicotinamide adenine dinucleotide (NAD + ) is also capable of activating P2Y 11 and causing an increase in intracellular Ca 2+ , IP 3 , and cAMP in P2Y 11 transfected 1321N1 cells [58,59].…”

“…C-terminal eGFP-tagged human and canine P2Y 11 receptors both showed similar signalling properties to the respective non-tagged receptors [6,51]. Nearly all vectors expressing human P2Y 11 described in published studies were created from the initial P2RY11 sequence arising from the fusion transcript (AF030335) [2,34,45,47,48,52,54,56,58,59,[62][63][64][65][66][67]. The sequence difference results in a slightly altered N-terminal of the P2Y 11 protein from its rightful MAANVSGAK to MDRGAK that originates from the transgenic splicing with PPAN.…”