Hechler Béatrice, Magnenat Stéphanie, Zighetti Maddalena L, Kassack Matthias U, Ullmann Heiko, Cazenave Jean-Pierre, Evans Richard, Cattaneo Marco, Gachet Christian
INSERM U. 311, Etablissement Français du Sang-Alsace, 10, rue Spielmann, Strasbourg Cédex, France.
J Pharmacol Exp Ther. 2005 Jul;314(1):232-43. doi: 10.1124/jpet.105.084673. Epub 2005 Mar 25.
Our aim was to determine whether the newly described P2X1 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 P2X1 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 (pA2 = 7.2 +/- 0.1; n = 3) (pIC50 = 6.95) in washed human platelets treated with apyrase to prevent desensitization of the P2X1 receptor. NF449 also antagonized the calcium rise mediated by the P2Y1 receptor, but with lower potency (IC50 = 5.8 +/- 2.2 microM; n = 3). In contrast, it was a very weak antagonist of the P2Y12-mediated inhibition of adenylyl cyclase activity. Selective blockade of the P2X1 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 P2X1 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 +/- 7 s; 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 P2X1 receptor and could be a starting compound in the search for new antithrombotic drugs targeting the platelet P2 receptors.
我们的目的是确定新描述的P2X1拮抗剂NF449 [4,4',4'',4'''-(羰基双(亚氨基-5,1,3-苯三基双(羰基亚氨基)))四苯-1,3-二磺酸八钠盐]是否能选择性拮抗血小板P2X1受体以及它如何影响血小板功能。在用腺苷双磷酸酶处理以防止P2X1受体脱敏的洗涤人血小板中，NF449抑制α,β-亚甲基腺苷5'-三磷酸诱导的形态变化（IC50 = 83±13 nM；n = 3）和钙内流（pA2 = 7.2±0.1；n = 3）（pIC50 = 6.95）。NF449也拮抗由P2Y1受体介导的钙升高，但效力较低（IC50 = 5.8±2.2 μM；n = 3）。相比之下，它是P2Y12介导的腺苷酸环化酶活性抑制的非常弱的拮抗剂。用NF449选择性阻断P2X1受体导致胶原诱导的聚集减少，证实该受体在胶原诱导的血小板活化中的作用。向小鼠静脉注射10 mg/kg NF449导致在全身血栓栓塞模型中P2X1受体的选择性抑制和血管内血小板聚集减少（35±4对51±3%）（P = 0.0061；n = 10），但不延长出血时间（106±16对78±7 s；n = 10）（无显著性差异；P = 0.1209）。在较高剂量（50 mg/kg）时，NF449抑制三种血小板P2受体。与注射生理盐水的小鼠相比，这导致血小板消耗进一步减少（13±4对42±3%）（P = 0.0002；n = 5）。NF449还剂量依赖性地减小了激光诱导肠系膜小动脉损伤后形成的血栓大小。总体而言，我们的结果表明NF449构成了研究P2X1受体功能的新工具，并且可能是寻找靶向血小板P2受体的新型抗血栓药物的起始化合物。
