Agonist and antagonist effects of diadenosine tetraphosphate, a platelet dense granule constituent, on platelet P2Y1, P2Y12 and P2X1 receptors

IntroductionDiadenosine 5',5'''-P1,P4- tetraphosphate (Ap4A) is stored in platelet dense granules, but its effects on platelet function are not well understood.Methods and ResultsWe examined the effects of Ap4A on platelet purinergic receptors P2Y1, P2Y12 and P2X1. Flow cytometry was used to measure the effects of Ap4A in the presence or absence of ADP on: a) P2Y12-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y1-mediated increase in platelet cytosolic Ca2+, and c) P2X1-mediated intraplatelet entry of extracellular Ca2+. ADP-stimulated platelet shape change (P2Y1-mediated) and aggregation (P2Y1- and P2Y12-mediated) were measured optically. Ap4A inhibited 3 μM ADP-induced: a) platelet aggregation (IC50 9.8 ± 2.8 μM), b) P2Y1-mediated shape change, c) P2Y1-mediated increase in platelet cytosolic Ca2+ (IC50 40.8 ± 12.3 μM), and d) P2Y12-mediated decrease in VASP phosphorylation (IC50 > 250 μM). In the absence of added ADP, Ap4A had agonist effects on platelet P2X1 and P2Y12, but not P2Y1, receptors.ConclusionAp4A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y1 and P2Y12 receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X1 and P2Y12 receptors.