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CARDIOVASCULAR

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]

Institut National de la Santé et de la Recherche Médicale U.311, Etablissement Français du Sang-Alsace, Strasbourg, France (B.H., S.M., J.-P.C., C.G.); Centro Emofilia e Trombosi A. Bianchi Bonomi, Istituto di Ricovero e Cura a Carattere Scientifico Ospedale Maggiore, Università di Milano, Milan, Italy (M.L.Z.); Pharmazeutisches Institut, Universität Bonn, Bonn, Germany (M.U.K., H.U.); Department of Cell Physiology and Pharmacology, University of Leicester, Leicester, United Kingdom (R.E.); and Unità di Ematologia e Trombosi, Ospedale San Paolo, Dipartimento di Medicina Chirurgia e Odontoiatria, Università di Milano, Milan, Italy (M.C.)

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 ,-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 µM; 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.

Address correspondence to: Dr. C. Gachet, INSERM U. 311, Etablissement Français du Sang-Alsace, 10, rue Spielmann, BP N° 36, 67065 Strasbourg Cédex, France. E-mail: christian.gachet{at}efs-alsace.fr

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