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CC BY 4.0 · Thromb Haemost 2018; 118(02): 369-380
DOI: 10.1160/TH17-07-0530
Cellular Haemostasis and Platelets
Schattauer GmbH Stuttgart

P2X1 Receptors Amplify FcγRIIa-Induced Ca2+ Increases and Functional Responses in Human Platelets

Zeki Ilkan
1   Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, Leicester, United Kingdom
,
Stephanie Watson
2   Institute of Cardiovascular Sciences, Institute of Biomedical Research Building, University of Birmingham, Birmingham, United Kingdom
,
Steve P. Watson
2   Institute of Cardiovascular Sciences, Institute of Biomedical Research Building, University of Birmingham, Birmingham, United Kingdom
3   Centre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, Midlands, UK
,
Martyn P. Mahaut-Smith
1   Department of Molecular and Cell Biology, Henry Wellcome Building, University of Leicester, Leicester, United Kingdom
› Author Affiliations Funding This work was supported by the British Heart Foundation grants PG/11/56, PG/05/014 and RG/13/18/30563. Z.I. was supported by a Medical Research Council Doctoral Training Award.
Further Information

Publication History

31 July 2017

18 November 2017

Publication Date:
29 January 2018 (online)

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Abstract

Platelets express key receptors of the innate immune system such as FcγRIIa and Toll-like receptors (TLR). P2X1 cation channels amplify the platelet responses to several major platelet stimuli, particularly glycoprotein (GP)VI and TLR2/1, whereas their contribution to Src tyrosine kinase-dependent FcγRIIa receptors remains unknown. We investigated the role of P2X1 receptors during activation of FcγRIIa in human platelets, following stimulation by cross-linking of an anti-FcγRIIa monoclonal antibody (mAb) IV.3, or bacterial stimulation with Streptococcus sanguinis. Activation was assessed in washed platelet suspensions via measurement of intracellular Ca2+ ([Ca2+]i) increases, ATP release and aggregation. P2X1 activity was abolished by pre-addition of α,β-meATP, exclusion of apyrase or the antagonist NF449. FcγRIIa activation evoked a robust increase in [Ca2+]i (441 ± 33 nM at 30 μg/mL mAb), which was reduced to a similar extent (to 66–70% of control) by NF449, pre-exposure to α,β-meATP or apyrase omission, demonstrating a significant P2X1 receptor contribution. FcγRIIa activation-dependent P2X1 responses were partially resistant to nitric oxide (NO), but abrogated by 500 nM prostacyclin (PGI2). Aggregation responses to bacteria and FcγRIIa activation were also inhibited by P2X1 receptor desensitization (to 66 and 42% of control, respectively). However, FcγRIIa-mediated tyrosine phosphorylation and ATP release were not significantly altered by the loss of P2X1 activity. In conclusion, we show that P2X1 receptors enhance platelet FcγRIIa receptor-evoked aggregation through an increase in [Ca2+]i downstream of the initial tyrosine phosphorylation events and early dense granule release. This represents a further route whereby ATP-gated cation channels can contribute to platelet-dependent immune responses in vivo.

Keywords

bacteria - immunity - inflammation - thrombosis - platelet
 

  • References

  • 1 Arman M, Krauel K. Human platelet IgG Fc receptor FcγRIIA in immunity and thrombosis. J Thromb Haemost 2015; 13 (06) 893-908
    CrossrefPubMedGoogle Scholar
  • 2 Watson SP, Gibbins J. Collagen receptor signalling in platelets: extending the role of the ITAM. Immunol Today 1998; 19 (06) 260-264
    CrossrefPubMedGoogle Scholar
  • 3 Zhi H, Rauova L, Hayes V. , et al. Cooperative integrin/ITAM signaling in platelets enhances thrombus formation in vitro and in vivo. Blood 2013; 121 (10) 1858-1867
    CrossrefPubMedGoogle Scholar
  • 4 Kerrigan SW, Clarke N, Loughman A, Meade G, Foster TJ, Cox D. Molecular basis for Staphylococcus aureus-mediated platelet aggregate formation under arterial shear in vitro. Arterioscler Thromb Vasc Biol 2008; 28 (02) 335-340
    CrossrefPubMedGoogle Scholar
  • 5 Arman M, Krauel K, Tilley DO. , et al. Amplification of bacteria-induced platelet activation is triggered by FcγRIIA, integrin αIIbβ3, and platelet factor 4. Blood 2014; 123 (20) 3166-3174
    CrossrefPubMedGoogle Scholar
  • 6 Fitzgerald JR, Foster TJ, Cox D. The interaction of bacterial pathogens with platelets. Nat Rev Microbiol 2006; 4 (06) 445-457
    CrossrefPubMedGoogle Scholar
  • 7 Mahaut-Smith MP. The unique contribution of ion channels to platelet and megakaryocyte function. J Thromb Haemost 2012; 10 (09) 1722-1732
    CrossrefPubMedGoogle Scholar
  • 8 Fung CYE, Cendana C, Farndale RW, Mahaut-Smith MP. Primary and secondary agonists can use P2X1 receptors as a major pathway to increase intracellular Ca2+ in the human platelet. J Thromb Haemost 2007; 5 (05) 910-917
    CrossrefPubMedGoogle Scholar
  • 9 Hechler B, Lenain N, Marchese P. , et al. A role of the fast ATP-gated P2X1 cation channel in thrombosis of small arteries in vivo. J Exp Med 2003; 198 (04) 661-667
    CrossrefPubMedGoogle Scholar
  • 10 Fung CY, Jones S, Ntrakwah A, Naseem KM, Farndale RW, Mahaut-Smith MP. Platelet Ca2+ responses coupled to glycoprotein VI and Toll-like receptors persist in the presence of endothelial-derived inhibitors: roles for secondary activation of P2X1 receptors and release from intracellular Ca2+ stores. Blood 2012; 119 (15) 3613-3621
    CrossrefPubMedGoogle Scholar
  • 11 Erhardt JA, Toomey JR, Douglas SA, Johns DG. P2X1 stimulation promotes thrombin receptor-mediated platelet aggregation. J Thromb Haemost 2006; 4 (04) 882-890
    CrossrefPubMedGoogle Scholar
  • 12 Oury C, Toth-Zsamboki E, Thys C, Tytgat J, Vermylen J, Hoylaerts MF. The ATP-gated P2X1 ion channel acts as a positive regulator of platelet responses to collagen. Thromb Haemost 2001; 86 (05) 1264-1271
    Article in Thieme ConnectPubMedGoogle Scholar
  • 13 Pampolina C, McNicol A. Streptococcus sanguis-induced platelet activation involves two waves of tyrosine phosphorylation mediated by FcgammaRIIA and alphaIIbbeta3. Thromb Haemost 2005; 93 (05) 932-939
    Article in Thieme ConnectPubMedGoogle Scholar
  • 14 Jones S, Evans RJ, Mahaut-Smith MP. Extracellular Ca2 modulates ADP-evoked aggregation through altered agonist degradation: implications for conditions used to study P2Y receptor activation. Br J Haematol 2011; 153 (01) 83-91
    CrossrefPubMedGoogle Scholar
  • 15 Tilley DO, Arman M, Smolenski A. , et al. Glycoprotein Ibα and FcγRIIa play key roles in platelet activation by the colonizing bacterium, Streptococcus oralis. J Thromb Haemost 2013; 11 (05) 941-950
    CrossrefPubMedGoogle Scholar
  • 16 Gibbins J, Asselin J, Farndale R, Barnes M, Law CL, Watson SP. Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets. J Biol Chem 1996; 271 (30) 18095-18099
    CrossrefPubMedGoogle Scholar
  • 17 Hechler B, Magnenat S, Zighetti ML. , et al. 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]. J Pharmacol Exp Ther 2005; 314 (01) 232-243
    CrossrefPubMedGoogle Scholar
  • 18 Fung CY, Brearley CA, Farndale RW, Mahaut-Smith MP. A major role for P2X1 receptors in the early collagen-evoked intracellular Ca2+ responses of human platelets. Thromb Haemost 2005; 94 (01) 37-40
    Article in Thieme ConnectPubMedGoogle Scholar
  • 19 Schwarz UR, Walter U, Eigenthaler M. Taming platelets with cyclic nucleotides. Biochem Pharmacol 2001; 62 (09) 1153-1161
    CrossrefPubMedGoogle Scholar
  • 20 Rink TJ, Sanchez A. Effects of prostaglandin I2 and forskolin on the secretion from platelets evoked at basal concentrations of cytoplasmic free calcium by thrombin, collagen, phorbol ester and exogenous diacylglycerol. Biochem J 1984; 222 (03) 833-836
    CrossrefPubMedGoogle Scholar
  • 21 Wang L, Ostberg O, Wihlborg AK, Brogren H, Jern S, Erlinge D. Quantification of ADP and ATP receptor expression in human platelets. J Thromb Haemost 2003; 1 (02) 330-336
    CrossrefPubMedGoogle Scholar
  • 22 MacKenzie AB, Mahaut-Smith MP, Sage SO. Activation of receptor-operated cation channels via P2X1 not P2T purinoceptors in human platelets. J Biol Chem 1996; 271 (06) 2879-2881
    CrossrefPubMedGoogle Scholar
  • 23 Semple JW, Aslam R, Kim M, Speck ER, Freedman J. Platelet-bound lipopolysaccharide enhances Fc receptor-mediated phagocytosis of IgG-opsonized platelets. Blood 2007; 109 (11) 4803-4805
    CrossrefPubMedGoogle Scholar
  • 24 Maugeri N, Rovere-Querini P, Evangelista V. , et al. Neutrophils phagocytose activated platelets in vivo: a phosphatidylserine, P-selectin, and beta2 integrin-dependent cell clearance program. Blood 2009; 113 (21) 5254-5265
    CrossrefPubMedGoogle Scholar
  • 25 Huang ZY, Chien P, Indik ZK, Schreiber AD. Human platelet FcγRIIA and phagocytes in immune-complex clearance. Mol Immunol 2011; 48 (04) 691-696
    CrossrefPubMedGoogle Scholar
  • 26 Cox D, Kerrigan SW, Watson SP. Platelets and the innate immune system: mechanisms of bacterial-induced platelet activation. J Thromb Haemost 2011; 9 (06) 1097-1107
    CrossrefPubMedGoogle Scholar
  • 27 Kapur R, Zufferey A, Boilard E, Semple JW. Nouvelle cuisine: platelets served with inflammation. J Immunol 2015; 194 (12) 5579-5587
    CrossrefPubMedGoogle Scholar
  • 28 Kraemer BF, Campbell RA, Schwertz H. , et al. Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation. PLoS Pathog 2011; 7 (11) e1002355
    CrossrefPubMedGoogle Scholar
  • 29 Crittenden JR, Bergmeier W, Zhang Y. , et al. CalDAG-GEFI integrates signaling for platelet aggregation and thrombus formation. Nat Med 2004; 10 (09) 982-986
    CrossrefPubMedGoogle Scholar
  • 30 Stefanini L, Roden RC, Bergmeier W. CalDAG-GEFI is at the nexus of calcium-dependent platelet activation. Blood 2009; 114 (12) 2506-2514
    CrossrefPubMedGoogle Scholar
  • 31 Eto K, Murphy R, Kerrigan SW. , et al. Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. Proc Natl Acad Sci U S A 2002; 99 (20) 12819-12824
    CrossrefPubMedGoogle Scholar
  • 32 Rolf MG, Brearley CA, Mahaut-Smith MP. Platelet shape change evoked by selective activation of P2X1 purinoceptors with alpha, beta-methylene ATP. Thromb Haemost 2001; 85 (02) 303-308
    Article in Thieme ConnectPubMedGoogle Scholar
  • 33 Tolhurst G, Vial C, Léon C, Gachet C, Evans RJ, Mahaut-Smith MP. Interplay between P2Y1, P2Y12, and P2X1 receptors in the activation of megakaryocyte cation influx currents by ADP: evidence that the primary megakaryocyte represents a fully functional model of platelet P2 receptor signaling. Blood 2005; 106 (05) 1644-1651
    CrossrefPubMedGoogle Scholar
  • 34 Salaün C, James DJ, Chamberlain LH. Lipid rafts and the regulation of exocytosis. Traffic 2004; 5 (04) 255-264
    CrossrefPubMedGoogle Scholar
  • 35 Bodin S, Viala C, Ragab A, Payrastre B. A critical role of lipid rafts in the organization of a key FcgammaRIIa-mediated signaling pathway in human platelets. Thromb Haemost 2003; 89 (02) 318-330
    Article in Thieme ConnectPubMedGoogle Scholar
  • 36 Vial C, Fung CY, Goodall AH, Mahaut-Smith MP, Evans RJ. Differential sensitivity of human platelet P2X1 and P2Y1 receptors to disruption of lipid rafts. Biochem Biophys Res Commun 2006; 343 (02) 415-419
    CrossrefPubMedGoogle Scholar
  • 37 Rolf MG, Mahaut-Smith MP. Effects of enhanced P2X1 receptor Ca2+ influx on functional responses in human platelets. Thromb Haemost 2002; 88 (03) 495-502
    Article in Thieme ConnectPubMedGoogle Scholar
  • 38 Jones S, Evans RJ, Mahaut-Smith MP. Ca2+ influx through P2X1 receptors amplifies P2Y1 receptor-evoked Ca2+ signaling and ADP-evoked platelet aggregation. Mol Pharmacol 2014; 86 (03) 243-251
    CrossrefPubMedGoogle Scholar
  • 39 Braun A, Varga-Szabo D, Kleinschnitz C. , et al. Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation. Blood 2009; 113 (09) 2056-2063
    CrossrefPubMedGoogle Scholar
  • 40 Tolhurst G, Carter RN, Amisten S, Holdich JP, Erlinge D, Mahaut-Smith MP. Expression profiling and electrophysiological studies suggest a major role for Orai1 in the store-operated Ca2+ influx pathway of platelets and megakaryocytes. Platelets 2008; 19 (04) 308-313
    CrossrefPubMedGoogle Scholar
  • 41 Hassock SR, Zhu MX, Trost C, Flockerzi V, Authi KS. Expression and role of TRPC proteins in human platelets: evidence that TRPC6 forms the store-independent calcium entry channel. Blood 2002; 100 (08) 2801-2811
    CrossrefPubMedGoogle Scholar
  • 42 Karas SP, Rosse WF, Kurlander RJ. Characterization of the IgG-Fc receptor on human platelets. Blood 1982; 60 (06) 1277-1282
    PubMedGoogle Scholar
  • 43 Tomiyama Y, Kunicki TJ, Zipf TF, Ford SB, Aster RH. Response of human platelets to activating monoclonal antibodies: importance of Fc gamma RII (CD32) phenotype and level of expression. Blood 1992; 80 (09) 2261-2268
    PubMedGoogle Scholar
  • 44 Lu J, Marnell LL, Marjon KD, Mold C, Du Clos TW, Sun PD. Structural recognition and functional activation of FcgammaR by innate pentraxins. Nature 2008; 456 (7224): 989-992
    CrossrefPubMedGoogle Scholar
  • 45 Tutwiler V, Madeeva D, Ahn HS. , et al. Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia. Blood 2016; 127 (04) 464-472
    CrossrefPubMedGoogle Scholar
  • 46 Qiao J, Al-Tamimi M, Baker RI, Andrews RK, Gardiner EE. The platelet Fc receptor, FcγRIIa. Immunol Rev 2015; 268 (01) 241-252
    CrossrefPubMedGoogle Scholar
  • 47 Canobbio I, Stefanini L, Guidetti GF, Balduini C, Torti M. A new role for FcgammaRIIA in the potentiation of human platelet activation induced by weak stimulation. Cell Signal 2006; 18 (06) 861-870
    CrossrefPubMedGoogle Scholar
  • 48 Blake RA, Schieven GL, Watson SP. Collagen stimulates tyrosine phosphorylation of phospholipase C-gamma 2 but not phospholipase C-gamma 1 in human platelets. FEBS Lett 1994; 353 (02) 212-216
    CrossrefPubMedGoogle Scholar
  • 49 Eckly A, Rinckel JY, Proamer F. , et al. Respective contributions of single and compound granule fusion to secretion by activated platelets. Blood 2016; 128 (21) 2538-2549
    CrossrefPubMedGoogle Scholar
  • 50 Jonnalagadda D, Izu LT, Whiteheart SW. Platelet secretion is kinetically heterogeneous in an agonist-responsive manner. Blood 2012; 120 (26) 5209-5216
    CrossrefPubMedGoogle Scholar