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The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range

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Abstract

The role of ATP-stimulated P2X1 receptors in human platelets is still unclear. They may act alone or in synergy with other pathways, such as P2Y1 or P2Y12 receptors, to accelerate and enhance calcium mobilisation, shape change and aggregation. To date very few pharmacological means of selectively inhibiting platelet P2X1 receptors have been described, although recent work has shown that suramin is a useful lead compound for the development of high-affinity P2X1 antagonists. We therefore investigated the effects of a series of bivalent and tetravalent suramin analogues on αβmeATP (P2X1 receptors)-induced or ADP (P2Y1 receptors)-induced intracellular calcium increases and shape change, as well as on ADP-induced aggregation (P2Y1 & P2Y12 receptors) in human platelets. Changes in intracellular calcium were measured using standard fluorescence techniques, while shape change and aggregation were determined by turbidimetry. The novel tetravalent compound NF864 (8,8′,8″,8′″-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt) proved to be the most potent platelet P2X1 antagonist reported to date, blocking αβmeATP-induced Ca2+ increases and shape change in a concentration-dependent manner, with a pA2 of 8.17 and 8.49, respectively. The ability to inhibit the platelet P2X1 receptor displayed the following order : NF864>NF449≥NF110>NF023=MK-HU1=suramin. A different antagonistic profile was observed for ADP-induced Ca2+ increases, shape change and aggregation; however, overall four compounds showed sufficient ability to selectively inhibit P2X1 responses, with the order NF110>NF449≥NF864≥MK-HU1. Therefore, these compounds should prove useful tools for investigating the functional significance of platelet P2X1 receptors in thrombosis and haemostasis, NF864 being the most promising compound.

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Abbreviations

αβmeATP:

α,β-methylene-adenosine-5′-triphosphate

NF864:

8,8′,8″,8‴-(carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino)))tetrakis-naphthalene-1,3,5-trisulfonic acid-dodecasodium salt

NF449:

4,4′,4″,4‴-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzene-1,3-disulfonic acid-octasodium salt

NF110:

4,4′,4″,4‴-(carbonylbis(imino-5,1,3-benzenetriylbis(carbonylimino)))tetrakis-benzenesulfonic acid-tetrasodium salt

NF023:

8,8′-(carbonyl-bis(imino-3,1-phenylenecarbonylimino))bis(naphthalene-1,3,5-trisulfonic acid)-hexasodium salt

MK-HU1:

8,8′-(carbonylbis(imino-3,1-phenylenecarbonylimino))bis-benzene-1,3-disulfonic acid-tetrasodium salt

MRS2179:

N6-methyl-2′-deoxy-adenosine-3′,5′‐bisphosphate

2-meSAMP:

2-methylthio-adenosine-monophosphate

pCMBS:

p-chloromercuribenzene sulfonic acid

pHMBS:

p-hydroxymercuribenzene sulfonic acid

MRS2159:

pyridoxal-α5-phosphate-6-phenylazo-4′-carboxylic acid

[Ca2+]i:

intracellular free calcium concentration

s.c.:

shape change

References

  • Abbracchio MP, Boeynaems JM, Barnard EA, Boyer JL, Kennedy C, Miras-Portugal MT, King BF, Gachet C, Jacobson KA, Weisman GA, Burnstock G (2003) Characterization of the UDP-glucose receptor (re-named here the P2Y14 receptor) adds diversity to the P2Y receptor family. Trends Pharmacol Sci 24:52–55

    Article  PubMed  Google Scholar 

  • Baurand A, Raboisson P, Freund M, Leon C, Cazenave JP, Bourguignon JJ, Gachet C (2001) Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist. Eur J Pharmacol 412:213–221

    Article  PubMed  Google Scholar 

  • Boyer JL, Mohanram A, Camaioni E, Jacobson KA, Harden TK (1998) Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2′-deoxyadenosine 3′,5′-bisphosphate. Br J Pharmacol 124:1–3

    Article  PubMed  Google Scholar 

  • Braun K, Rettinger J, Ganso M, Kassack M, Hildebrandt C, Ullmann H, Nickel P, Schmalzing G, Lambrecht G (2001) NF449: a subnanomolar potency antagonist at recombinant rat P2X1 receptors. Naunyn-Schmiedebergs Arch Pharmacol 364:285–290

    Article  PubMed  Google Scholar 

  • Burnstock G (2004) Introduction: P2 receptors. Curr Top Med Chem 4:793–803

    Article  PubMed  Google Scholar 

  • Cheng HC (2002) The power issue: determination of KB or Ki from IC50. A closer look at the Cheng-Prussoff equation, the Schild plot and related power equations. J Pharmacol Toxicol Methods 46:61–71

    Article  Google Scholar 

  • Cheng Y, Prusoff WH (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 22:3099–3108

    PubMed  Google Scholar 

  • Conley PB, Delaney SM (2003) Scientific and therapeutic insights into the role of the platelet P2Y12 receptor in thrombosis. Curr Opin Hematol 10:333–338

    Article  PubMed  Google Scholar 

  • Cusack NJ, Hourani SMO (1982) Specific but noncompetitive inhibition by 2-alkylthio analogues of adenosine 5′-monophosphate and adenosine 5′-triphosphate fo human platelet aggregation induced by adenosine 5′-diphosphate. Br J Pharmacol 75:397–400

    PubMed  Google Scholar 

  • Ding Z, Kim S, Dorsam RT, Jin J, Kunapuli SP (2003) Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270. Blood 101:3908–3914

    Article  PubMed  Google Scholar 

  • Erhardt JA, Pillarisetti K, Toomey JR (2003) Potentiation of platelet activation through the stimulation of P2X1 receptors. J Thromb Haemost 1:2626–2635

    Article  PubMed  Google Scholar 

  • Fabre JE, Nguyen M, Latour A, Keifer JA, Audoly LP, Coffman TM, Koller BH (1999) Decreased platelet aggregation, increased bleeding time and resistance to thromboembolism in P2Y1-deficient mice. Nat Med 5:1199–1202

    Article  PubMed  Google Scholar 

  • Fiske CH, Subbarow Y (1925) The colorimetric determination of phosphorus. J Biol Chem 66:375–400

    Google Scholar 

  • Frolenkov GI, Belyantseva IA, Kurc M, Mastroianni MA, Kachar B (1998) Cochlear outer hair cell electromotility can provide force for both low and high intensity distortion product otoacoustic emissions. Hear Res 126:67–74

    Article  PubMed  Google Scholar 

  • Gachet C (2001) ADP receptors of platelets and their inhibition. Thromb Haemost 86:222–232

    PubMed  Google Scholar 

  • Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450

    PubMed  Google Scholar 

  • Hausmann R, Rettinger J, Kassack M, Lambrecht G, Schmalzing G (2004) Characterization of the novel suramin analogue NF110 as a potent P2X3 receptor-selective antagonist. Naunyn-Schmiedebergs Arch Pharmacol 369(Suppl 1):R28

    Google Scholar 

  • Hechler B, Leon C, Vial C, Vigne P, Frelin C, Cazenave JP, Gachet C (1998) The P2Y1 receptor is necessary for adenosine 3′,5-diphosphate-induced platelet aggregation. Blood 92:152–159

    PubMed  Google Scholar 

  • Hechler B, Lenain N, Marchese P, Vial C, Heim V, Freund M, Cazenave JP, Cattaneo M, Ruggeri ZM, Evans R, Gachet C (2003) A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo. J Exp Med 198:661–667

    Article  Google Scholar 

  • Hechler B, Magnenat S, Zighetti ML, Kassack MU, Ullmann H, Cazenave JP, Evans R, Cattaneo M, Gachet C (2005) Inhibition of platelet functions and thrombosis through selective or non selective inhibition of the platelet P2 receptors with increasing doses of NF449. J Pharmacol Exp Ther 314:232-243

    Article  PubMed  Google Scholar 

  • Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D, Conley PB (2001) Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409:202–207

    Article  PubMed  Google Scholar 

  • Horner S, Ganso M, Braun K, Hildebrandt C, Lambrecht G (2002) Is MRS2179 P2Y1-selective in whole tissues? Drug Dev Res 56:A153

    Google Scholar 

  • Horner S, Hildebrandt C, Menke K, Lambrecht G (2004a) Pharmacological properties of the homomeric bivalent ligand CLII and its monomer, MRS2159, at native P2 receptor subtypes. Naunyn-Schmiedebergs Arch Pharmacol 369 (Suppl 1):R30

    Google Scholar 

  • Horner S, Menke K, Hildebrandt C, Kassack M, Nickel P, Ullmann H, Lambrecht G (2004b) The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with nanomolar potency. Purines 2004, Chapel Hill, NC, USA, Abstract Book: 74W

    Google Scholar 

  • Hourani, SMO (2001) Discovery and recognition of purine receptor subtypes on platelets. Drug Dev Res 52:140–149

    Article  Google Scholar 

  • Hourani SM, Hall DA, Nieman CJ (1992) Effects of the P2-purinoceptor antagonist, suramin, on human platelet aggregation induced by adenosine 5′-diphosphate. Br J Pharmacol 105:453–457

    PubMed  Google Scholar 

  • Jacobson KA, Jarvis MF, Williams M (2002) Purine and pyrimidine P2 receptors as drug targets. J Med Chem 45:4057–4093

    Article  PubMed  Google Scholar 

  • Jacobson KA, Costanzi S, Ohno M, Joshi BV, Besada P, Xu B, Tchilibon S (2004) Molecular recognition at purine and pyrimidine nucleotide (P2) receptors. Curr Top Med Chem 4:805–819

    Article  PubMed  Google Scholar 

  • Jantzen HM, Gousset L, Bhaskar V, Vincent D, Tai A, Reynolds EE, Conley PB (1999) Evidence for two distinct G-protein-coupled ADP receptors mediating platelet activation. Thromb Haemost 81:111–117

    PubMed  Google Scholar 

  • Jin J, Daniel JL, Kunapuli SP (1998) Molecular basis for ADP-induced platelet activation. II. The P2Y1 receptor mediates ADP-induced intracellular calcium mobilization and shape change in platelets. J Biol Chem 273:2030–2034

    Article  PubMed  Google Scholar 

  • Kassack MU, Braun K, Ganso M, Ullmann H, Nickel P, Böing B, Müller G, Lambrecht G (2004) Structure-activity relationships of analogues of NF449 confirm NF449 as the most potent and selective known P2X1 receptor antagonist. Eur J Med Chem 39:345–357

    Article  PubMed  Google Scholar 

  • Kauffenstein G, Bergmeier W, Eckly A, Ohlmann P, Leon C, Cazenave JP, Nieswandt B, Gachet C (2001) The P2Y12 receptor induces platelet aggregation through weak activation of the αIIbβ3 integrin-a phosphoinositide 3-kinase-dependent mechanism. FEBS Lett 505:281–290

    Article  PubMed  Google Scholar 

  • Kettlun AM, Leyton M, Valenzuela MA, Mancilla M, Traverso-Cori A (1992) Identification and subcellular localization of two isoenzymes of apyrase from solanum tuberosum. Phytochemistry 31:1889–1894

    Article  Google Scholar 

  • Khakh BS, Burnstock G, Kennedy C, King BF, North RA, Séguéla P, Voigt M, Humphrey PPA (2001) International Union of Pharmacology. XXIV. Current status of the nomenclature and properties of P2X receptors and their subunits. Pharmacol Rev 53:107–118

    PubMed  Google Scholar 

  • Kim YC, Brown SG, Harden TK, Boyer JL, Dubyak G, King BF, Burnstock G, Jacobson KA (2001) Structure-activity relationships of pyridoxal phosphate derivatives as potent and selective antagonists of P2X1 receptors. J Med Chem 44:340–349

    Article  PubMed  Google Scholar 

  • Klages B, Brandt U, Simon MI, Schultz G, Offermanns S (1999) Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets. J Cell Biol 144:745–754

    Article  PubMed  Google Scholar 

  • Kubista H, Lechner SG, Wolf AM, Boehm S (2003) Attenuation of the P2Y receptor-mediated control of neuronal Ca2+ channels in PC12 cells by antithrombotic drugs. Br J Pharmacol 138:343–350

    Article  PubMed  Google Scholar 

  • Kunapuli SP, Ding Z, Dorsam RT, Kim S, Murugappan S, Quinton TM (2003) ADP receptors-targets for developing antithrombotic agents. Curr Pharm Des 9:2303–2316

    Article  PubMed  Google Scholar 

  • Lambrecht G (2000) Agonists and antagonists acting at P2X receptors: selectivity profiles and functional implications. Naunyn-Schmiedebergs Arch Pharmacol 362:340–350

    Article  PubMed  Google Scholar 

  • Lambrecht G, Braun K, Damer S, Ganso M, Hildebrandt C, Ullmann H, Kassack MU, Nickel P (2002) Structure-activity relationships of suramin and pyridoxal-5′-phosphate derivatives as P2 receptor antagonists. Curr Pharm Des 8:2371–2399

    Article  PubMed  Google Scholar 

  • Leon C, Hechler B, Vial C, Leray C, Cazenave JP, Gachet C (1997) The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells. FEBS Lett 403:26–30

    Article  PubMed  Google Scholar 

  • Leon C, Hechler B, Freund M, Eckly A, Vial C, Ohlmann P, Dietrich A, LeMeur M, Cazenave JP, Gachet C (1999) Defective platelet aggregation and increased resistance to thrombosis in purinergic P2X1 receptor-null mice. J Clin Invest 104:1731–1737

    PubMed  Google Scholar 

  • Mahaut-Smith MP, Tolhurst G, Evans RJ (2004) Emerging roles for P2X1 receptors in platelet activation. Platelets 15:131–144

    Article  PubMed  Google Scholar 

  • Menke K, Horner S, Mahaut-Smith MP, Hildebrandt C, Kassack M, Nickel P, Lambrecht G (2003) NF449 and MRS2179 are useful tools to distinguish between P2X1 and P2Y1 receptors in human platelets. Naunyn-Schmiedebergs Arch Pharmacol 367(Suppl 1):R35

    Google Scholar 

  • Michal F, Maguire MH, Gough G (1969) 2-Methylthioadenosine-5′-phosphate: a specific inhibitor of platelet aggregation. Nature 222:1073–1074

    PubMed  Google Scholar 

  • Mills DC (1996) ADP receptors on platelets. Thromb Haemost 76:835–856

    PubMed  Google Scholar 

  • Moro S, Guo D, Camaioni E, Boyer JL, Harden TK, Jacobson KA (1998) Human P2Y1 receptor: molecular modeling and site-directed mutagenesis as tools to identify agonist and antagonist recognition sites. J Med Chem 41:1456–1466

    Article  PubMed  Google Scholar 

  • Müller W, Wollert U (1976) Spectroscopic studies on the complex formation of suramin with bovine and human serum albumin. Biochem Biophys Acta 427:465–480

    PubMed  Google Scholar 

  • North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82:1013–1067

    PubMed  Google Scholar 

  • Ohlmann P, Eckly A, Freund M, Cazenave JP, Offermanns S, Gachet C (2000) ADP induces partial platelet aggregation without shape change and potentiates collagen-induced aggregation in the absence of Gαq. Blood 96:2134–2139

    PubMed  Google Scholar 

  • Oury C, Toth-Zsamboki E, Thys C, Tytgat J, Vermylen J, Hoylaerts MF (2001) The ATP-gated P2X1 ion channel acts as a positive regulator of platelet responses to collagen. Thromb Haemost 86:1264–1271

    PubMed  Google Scholar 

  • Oury C, Kuijpers MJ, Toth-Zsamboki E, Bonnefoy A, Danloy S, Vreys I, Feijge MA, De Vos R, Vermylen J, Heemskerk JW, Hoylaerts MF (2003) Overexpression of the platelet P2X1 ion channel in transgenic mice generates a novel prothombotic phenotype. Blood 101:3969–3976

    Article  PubMed  Google Scholar 

  • Paul BZ, Daniel JL, Kunapuli SP (1999) Platelet shape change is mediated by both calcium- dependent and -independent signaling pathways. Role of p160 Rho-associated coiled-coil-containing protein kinase in platelet shape change. J Biol Chem 274:28293–28300

    Article  PubMed  Google Scholar 

  • Paul BZ, Kim S, Dangelmaier C, Nagaswami C, Jin J, Hartwig JH, Weisel JW, Daniel JL, Kunapuli SP (2003) Dynamic regulation of microtubule coils in ADP-induced platelet shape change by p160ROCK (Rho-kinase). Platelets 14:159–169

    Article  PubMed  Google Scholar 

  • Rettinger J, Braun K, Hochmann H, Kassack MU, Ullmann H, Nickel P, Schmalzing G, Lambrecht G (2005) Profiling at recombinant homomeric and heteromeric rat P2X receptors identifies the suramin analogue NF449 as a highly potent P2X1 receptor antagonist. Neuropharmacology 48:461–468

    Article  PubMed  Google Scholar 

  • Rolf MG, Mahaut-Smith MP (2002) Effects of enhanced P2X1 receptor Ca2+ influx on functional responses in human platelets. Thromb Haemost 88:495–502

    PubMed  Google Scholar 

  • Rolf MG, Brearley CA, Mahaut-Smith MP (2001) Platelet shape change evoked by selective activation of P2X1 purinoceptors with α,β-methylene ATP. Thromb Haemost 85:303–308

    PubMed  Google Scholar 

  • Shankar H, Murugappan S, Kim S, Jin J, Ding Z, Wickman K, Kunapuli SP (2004) Role of G protein-gated inwardly rectifying potassium channels in P2Y12 receptor-mediated platelet functional responses. Blood 104:1335–1343

    Article  PubMed  Google Scholar 

  • Soto F, Lambrecht G, Nickel P, Stühmer W, Busch AE (1999) Antagonistic properties of the suramin analogue NF023 at heterologously expressed P2X receptors. Neuropharmacology 38:141–149

    Article  PubMed  Google Scholar 

  • Soulet C, Sauzeau V, Plantavid M, Herbert JM, Pacaud P, Payrastre B, Savi P (2004) Gi-dependent and -independent mechanisms downstream of the P2Y12 ADP-receptor. J Thromb Haemost 2:135–146

    Article  PubMed  Google Scholar 

  • Takasaki J, Kamohara M, Saito T, Matsumoto M, Matsumoto S, Ohishi T, Soga T, Matsushime H, Furuichi K (2001). Molecular cloning of the platelet P2TAC ADP receptor: pharmacological comparison with another ADP receptor, the P2Y1 receptor. Mol Pharmacol 60:432–439

    PubMed  Google Scholar 

  • Tallarida RJ, Murray RB (1986) Manual of pharmacologic calculations with computer programs. Springer, Berlin Heidelberg New York

    Google Scholar 

  • Von Kügelgen I, Wetter A (2000) Molecular pharmacology of P2Y receptors. Naunyn-Schmiedebergs Arch Pharmacol 362:310–323

    Article  PubMed  Google Scholar 

  • Wilde JI, Retzer M, Siess W, Watson SP (2000) ADP-induced platelet shape change: an investigation of the signalling pathways involved and their dependence on the method of platelet preparation. Platelets 11:286–295

    Article  PubMed  Google Scholar 

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Acknowledgements

We gratefully acknowledge the financial support of the Fonds der Chemischen Industrie (Germany) and of the Deutsche Forschungsgemeinschaft (grant numbers: La 350/7-3, GRK 677/1). We thank Dr. S. Harder and his team for providing us with blood.

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Authors and Affiliations

  1. Department of Pharmacology, University of Frankfurt, Marie-Curie-Strasse 9, 60439, Frankfurt (Main), Germany

    Susanne Horner, Kirsten Menke, Caren Hildebrandt & Günter Lambrecht

  2. Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121, Bonn, Germany

    Matthias U. Kassack, Peter Nickel & Heiko Ullmann

  3. Department of Physiology, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK

    Martyn P. Mahaut-Smith

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  1. Susanne Horner
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  2. Kirsten Menke
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  3. Caren Hildebrandt
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  4. Matthias U. Kassack
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  5. Peter Nickel
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  6. Heiko Ullmann
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  7. Martyn P. Mahaut-Smith
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  8. Günter Lambrecht
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Correspondence to Günter Lambrecht.

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Horner, S., Menke, K., Hildebrandt, C. et al. The novel suramin analogue NF864 selectively blocks P2X1 receptors in human platelets with potency in the low nanomolar range. Naunyn Schmied Arch Pharmacol 372, 1–13 (2005). https://doi.org/10.1007/s00210-005-1085-z

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