Inhibition of platelet functions and thrombosis through selective or nonselective inhibition of the platelet P2 receptors with increasing doses of NF449 [4,4′,4=,4=′-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt]

Our aim was to determine whether the newly described P2X(1) antagonist NF449 [ 4,4', 4", 4"'-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid octasodium salt] could selectively antagonize the platelet P2X(1) receptor and how it affected platelet function. NF449 inhibited alpha,beta-methyleneadenosine 5'- triphosphate-induced shape change (IC50 = 83 +/- 13 nM; n = 3) and calcium influx (pA(2) = 7.2 +/- 0.1; n = 3) (pIC(50) = 6.95) in washed human platelets treated with apyrase to prevent desensitization of the P2X(1) receptor. NF449 also antagonized the calcium rise mediated by the P2Y(1) receptor, but with lower potency (IC50 = 5.8 +/- 2.2 mu M; n = 3). In contrast, it was a very weak antagonist of the P2Y(12)-mediated inhibition of adenylyl cyclase activity. Selective blockade of the P2X(1) receptor with NF449 led to reduced collagen-induced aggregation, confirming a role of this receptor in platelet activation induced by collagen. Intravenous injection of 10 mg/kg NF449 into mice resulted in selective inhibition of the P2X(1) receptor and decreased intravascular platelet aggregation in a model of systemic thromboembolism (35 +/- 4 versus 51 +/- 3%) (P = 0.0061; n = 10) but without prolongation of the bleeding time (106 +/- 16 versus 78 +/- 7s; n = 10) (N. S.; P = 0.1209). At a higher dose (50 mg/kg), NF449 inhibited the three platelet P2 receptors. This led to a further reduction in platelet consumption compared with mice injected with saline (13 +/- 4 versus 42 +/- 3%) (P = 0.0002; n = 5). NF449 also reduced dose-dependently the size of thrombi formed after laser-induced injury of mesenteric arterioles. Overall, our results indicate that NF449 constitutes a new tool to investigate the functions of the P2X(1) receptor and could be a starting compound in the search for new antithrombotic drugs targeting the platelet P2 receptors.