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

Abstract Introduction Diadenosine 5',5'''-P 1 ,P 4 - tetraphosphate (Ap 4 A) is stored in platelet dense granules, but its effects on platelet function are not well understood. Methods and Results We examined the effects of Ap 4 A on platelet purinergic receptors P2Y 1 , P2Y 12 and P2X 1 . Flow cytometry was used to measure the effects of Ap 4 A in the presence or absence of ADP on: a) P2Y 12 -mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y 1 -mediated increase in platelet cytosolic Ca 2+ , and c) P2X 1 -mediated intraplatelet entry of extracellular Ca 2+ . ADP-stimulated platelet shape change (P2Y 1 -mediated) and aggregation (P2Y 1 - and P2Y 12 -mediated) were measured optically. Ap 4 A inhibited 3 μM ADP-induced: a) platelet aggregation (IC 50 9.8 ± 2.8 μM), b) P2Y 1 -mediated shape change, c) P2Y 1 -mediated increase in platelet cytosolic Ca 2+ (IC 50 40.8 ± 12.3 μM), and d) P2Y 12 -mediated decrease in VASP phosphorylation (IC 50  > 250 μM). In the absence of added ADP, Ap 4 A had agonist effects on platelet P2X 1 and P2Y 12 , but not P2Y 1 , receptors. Conclusion Ap 4 A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y 1 and P2Y 12 receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X 1 and P2Y 12 receptors.