PDF herunterladen
CC BY 4.0 · Thromb Haemost 2019; 119(01): 128-139
DOI: 10.1055/s-0038-1676589
Cellular Haemostasis and Platelets
Georg Thieme Verlag KG Stuttgart · New York

Agonist-Evoked Increases in Intra-Platelet Zinc Couple to Functional Responses

Niaz S. Ahmed
1   School of Life Sciences, Anglia Ruskin University, Cambridge, United Kingdom
,
Maria E. Lopes Pires
1   School of Life Sciences, Anglia Ruskin University, Cambridge, United Kingdom
,
Kirk A. Taylor
2   Cardio-Respiratory Interface Section, National Heart and Lung Institute, Imperial College London, London, United Kingdom
,
Nicholas Pugh
1   School of Life Sciences, Anglia Ruskin University, Cambridge, United Kingdom
› Institutsangaben Funding This work was supported by British Heart Foundation project grants (PG/14/47/30912 and PG/18/64/33922).
Weitere Informationen

Publikationsverlauf

26. Juni 2018

31. Oktober 2018

Publikationsdatum:
31. Dezember 2018 (online)

   Lizenzen und Reprints

Abstract

Background Zinc (Zn2+) is an essential trace element that regulates intracellular processes in multiple cell types. While the role of Zn2+ as a platelet agonist is known, its secondary messenger activity in platelets has not been demonstrated.

Objectives This article determines whether cytosolic Zn2+ concentrations ([Zn2+]i) change in platelets in response to agonist stimulation, in a manner consistent with a secondary messenger, and correlates the effects of [Zn2+]i changes on activation markers.

Methods Changes in [Zn2+]i were quantified in Fluozin-3 (Fz-3)-loaded washed, human platelets using fluorometry. Increases in [Zn2+]i were modelled using Zn2+-specific chelators and ionophores. The influence of [Zn2+]i on platelet function was assessed using platelet aggregometry, flow cytometry and Western blotting.

Results Increases of intra-platelet Fluozin-3 (Fz-3) fluorescence occurred in response to stimulation by cross-linked collagen-related peptide (CRP-XL) or U46619, consistent with a rise of [Zn2+]i. Fluoresence increases were blocked by Zn2+ chelators and modulators of the platelet redox state, and were distinct from agonist-evoked [Ca2+]i signals. Stimulation of platelets with the Zn2+ ionophores clioquinol (Cq) or pyrithione (Py) caused sustained increases of [Zn2+]i, resulting in myosin light chain phosphorylation, and cytoskeletal re-arrangements which were sensitive to cytochalasin-D treatment. Cq stimulation resulted in integrin αIIbβ3 activation and release of dense, but not α, granules. Furthermore, Zn2+-ionophores induced externalization of phosphatidylserine.

Conclusion These data suggest that agonist-evoked fluctuations in intra-platelet Zn2+ couple to functional responses, in a manner that is consistent with a role as a secondary messenger. Increased intra-platelet Zn2+ regulates signalling processes, including shape change, αIIbβ3 up-regulation and dense granule release, in a redox-sensitive manner.

Keywords

platelets - zinc - platelet activation - signal transduction - secretory vesicles - granule release

Authors' Contributions

N.S.A., M.L.P. and N.P. designed and conducted experiments, and wrote the manuscript. N.S.A., M.L.P., K.T. and N.P. designed experiments and wrote the manuscript.


Supplementary Material

 

  • References

  • 1 Andreini C, Bertini I. A bioinformatics view of zinc enzymes. J Inorg Biochem 2012; 111: 150-156
    CrossrefPubMedGoogle Scholar
  • 2 Blakemore LJ, Trombley PQ. Zinc as a neuromodulator in the central nervous system with a focus on the olfactory bulb. Front Cell Neurosci 2017; 11: 297
    CrossrefPubMedGoogle Scholar
  • 3 Maret W. Zinc in cellular regulation: the nature and significance of “zinc signals”. Int J Mol Sci 2017; 18 (11) E2285
    CrossrefPubMedGoogle Scholar
  • 4 Taylor KA, Pugh N. The contribution of zinc to platelet behaviour during haemostasis and thrombosis. Metallomics 2016; 8 (02) 144-155
    CrossrefPubMedGoogle Scholar
  • 5 Sharir H, Zinger A, Nevo A, Sekler I, Hershfinkel M. Zinc released from injured cells is acting via the Zn2+-sensing receptor, ZnR, to trigger signaling leading to epithelial repair. J Biol Chem 2010; 285 (34) 26097-26106
    CrossrefPubMedGoogle Scholar
  • 6 Bernardo MM, Day DE, Olson ST, Shore JD. Surface-independent acceleration of factor XII activation by zinc ions. I. Kinetic characterization of the metal ion rate enhancement. J Biol Chem 1993; 268 (17) 12468-12476
    CrossrefPubMedGoogle Scholar
  • 7 Watson BR, White NA, Taylor KA. , et al. Zinc is a transmembrane agonist that induces platelet activation in a tyrosine phosphorylation-dependent manner. Metallomics 2016; 8 (01) 91-100
    CrossrefPubMedGoogle Scholar
  • 8 Yamasaki S, Sakata-Sogawa K, Hasegawa A. , et al. Zinc is a novel intracellular second messenger. J Cell Biol 2007; 177 (04) 637-645
    CrossrefPubMedGoogle Scholar
  • 9 Haase H, Ober-Blöbaum JL, Engelhardt G. , et al. Zinc signals are essential for lipopolysaccharide-induced signal transduction in monocytes. J Immunol 2008; 181 (09) 6491-6502
    CrossrefPubMedGoogle Scholar
  • 10 Haase H, Maret W. Fluctuations of cellular, available zinc modulate insulin signaling via inhibition of protein tyrosine phosphatases. J Trace Elem Med Biol 2005; 19 (01) 37-42
    CrossrefPubMedGoogle Scholar
  • 11 Arslan P, Di Virgilio F, Beltrame M, Tsien RY, Pozzan T. Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+. J Biol Chem 1985; 260 (05) 2719-2727
    CrossrefPubMedGoogle Scholar
  • 12 Hyun TH, Barrett-Connor E, Milne DB. Zinc intakes and plasma concentrations in men with osteoporosis: the Rancho Bernardo Study. Am J Clin Nutr 2004; 80 (03) 715-721
    CrossrefPubMedGoogle Scholar
  • 13 Matias CM, Sousa JM, Quinta-Ferreira ME, Arif M, Burrows HD. Validation of TPEN as a zinc chelator in fluorescence probing of calcium in cells with the indicator Fura-2. J Fluoresc 2010; 20 (01) 377-380
    CrossrefPubMedGoogle Scholar
  • 14 Qian C, Colvin RA. Zinc flexes its muscle: correcting a novel analysis of calcium for zinc interference uncovers a method to measure zinc. J Gen Physiol 2016; 147 (01) 95-102
    CrossrefPubMedGoogle Scholar
  • 15 Zhao J, Bertoglio BA, Gee KR, Kay AR. The zinc indicator FluoZin-3 is not perturbed significantly by physiological levels of calcium or magnesium. Cell Calcium 2008; 44 (04) 422-426
    CrossrefPubMedGoogle Scholar
  • 16 Maret W. Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc. Neurochem Int 1995; 27 (01) 111-117
    CrossrefPubMedGoogle Scholar
  • 17 Beckman JS, Minor Jr RL, White CW, Repine JE, Rosen GM, Freeman BA. Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance. J Biol Chem 1988; 263 (14) 6884-6892
    CrossrefPubMedGoogle Scholar
  • 18 Bakdash N, Williams MS. Spatially distinct production of reactive oxygen species regulates platelet activation. Free Radic Biol Med 2008; 45 (02) 158-166
    CrossrefPubMedGoogle Scholar
  • 19 Rosado JA, Redondo PC, Salido GM, Gómez-Arteta E, Sage SO, Pariente JA. Hydrogen peroxide generation induces pp60src activation in human platelets: evidence for the involvement of this pathway in store-mediated calcium entry. J Biol Chem 2004; 279 (03) 1665-1675
    CrossrefPubMedGoogle Scholar
  • 20 Marx G, Korner G, Mou X, Gorodetsky R. Packaging zinc, fibrinogen, and factor XIII in platelet alpha-granules. J Cell Physiol 1993; 156 (03) 437-442
    CrossrefPubMedGoogle Scholar
  • 21 White JG, Rao GH, Gerrard JM. Effects of the lonophore A23187 on blood platelets I. Influence on aggregation and secretion. Am J Pathol 1974; 77 (02) 135-149
    PubMedGoogle Scholar
  • 22 Fujikawa K, Fukumori R, Nakamura S, Kutsukake T, Takarada T, Yoneda Y. Potential interactions of calcium-sensitive reagents with zinc ion in different cultured cells. PLoS One 2015; 10 (05) e0127421
    CrossrefPubMedGoogle Scholar
  • 23 Aiba I, West AK, Sheline CT, Shuttleworth CW. Intracellular dialysis disrupts Zn2+ dynamics and enables selective detection of Zn2+ influx in brain slice preparations. J Neurochem 2013; 125 (06) 822-831
    CrossrefPubMedGoogle Scholar
  • 24 Park M-H, Lee S-J, Byun HR. , et al. Clioquinol induces autophagy in cultured astrocytes and neurons by acting as a zinc ionophore. Neurobiol Dis 2011; 42 (03) 242-251
    CrossrefPubMedGoogle Scholar
  • 25 Pula G, Schuh K, Nakayama K, Nakayama KI, Walter U, Poole AW. PKCdelta regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation. Blood 2006; 108 (13) 4035-4044
    CrossrefPubMedGoogle Scholar
  • 26 Getz TM, Dangelmaier CA, Jin J, Daniel JL, Kunapuli SP. Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in platelets. J Thromb Haemost 2010; 8 (10) 2283-2293
    CrossrefPubMedGoogle Scholar
  • 27 Smolenski A. Novel roles of cAMP/cGMP-dependent signaling in platelets. J Thromb Haemost 2012; 10 (02) 167-176
    CrossrefPubMedGoogle Scholar
  • 28 Iitaka M, Kakinuma S, Fujimaki S. , et al. Induction of apoptosis and necrosis by zinc in human thyroid cancer cell lines. J Endocrinol 2001; 169 (02) 417-424
    CrossrefPubMedGoogle Scholar
  • 29 Eron SJ, MacPherson DJ, Dagbay KB, Hardy JA. Multiple mechanisms of zinc-mediated inhibition for the apoptotic caspases -3, -6, -7, & -8. ACS Chem Biol 2018; 13 (05) 1279-1290
    CrossrefPubMedGoogle Scholar
  • 30 Treves S, Trentini PL, Ascanelli M, Bucci G, Di Virgilio F. Apoptosis is dependent on intracellular zinc and independent of intracellular calcium in lymphocytes. Exp Cell Res 1994; 211 (02) 339-343
    CrossrefPubMedGoogle Scholar
  • 31 Hamatake M, Iguchi K, Hirano K, Ishida R. Zinc induces mixed types of cell death, necrosis, and apoptosis, in molt-4 cells. J Biochem 2000; 128 (06) 933-939
    CrossrefPubMedGoogle Scholar
  • 32 Cohen Z, Wilson J, Ritter L, McDonagh P. Caspase inhibition decreases both platelet phosphatidylserine exposure and aggregation: caspase inhibition of platelets. Thromb Res 2004; 113 (06) 387-393
    CrossrefPubMedGoogle Scholar
  • 33 Sugiura T, Nakamura H. Metallothionein in platelets. Int Arch Allergy Immunol 1994; 103 (04) 341-348
    CrossrefPubMedGoogle Scholar
  • 34 Meeusen JW, Nowakowski A, Petering DH. Reaction of metal-binding ligands with the zinc proteome: zinc sensors and N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine. Inorg Chem 2012; 51 (06) 3625-3632
    CrossrefPubMedGoogle Scholar
  • 35 Li F, Abuarab N, Sivaprasadarao A. Reciprocal regulation of actin cytoskeleton remodelling and cell migration by Ca2+ and Zn2+: role of TRPM2 channels. J Cell Sci 2016; 129 (10) 2016-2029
    CrossrefPubMedGoogle Scholar
  • 36 Krezel A, Hao Q, Maret W. The zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signaling. Arch Biochem Biophys 2007; 463 (02) 188-200
    CrossrefPubMedGoogle Scholar
  • 37 Krężel A, Maret W. The functions of metamorphic metallothioneins in zinc and copper metabolism. Int J Mol Sci 2017; 18 (06) E1237
    CrossrefPubMedGoogle Scholar
  • 38 Begonja AJ, Gambaryan S, Geiger J. , et al. Platelet NAD(P)H-oxidase-generated ROS production regulates alphaIIbbeta3-integrin activation independent of the NO/cGMP pathway. Blood 2005; 106 (08) 2757-2760
    CrossrefPubMedGoogle Scholar
  • 39 Rudolf E, Klvacová L, John S, Cervinka M. Zinc alters cytoskeletal integrity and migration in colon cancer cells. Acta Med (Hradec Kralove) 2008; 51 (01) 51-57
    CrossrefPubMedGoogle Scholar
  • 40 Perrin L, Roudeau S, Carmona A. , et al. Zinc and copper effects on stability of tubulin and actin networks in dendrites and spines of hippocampal neurons. ACS Chem Neurosci 2017; 8 (07) 1490-1499
    CrossrefPubMedGoogle Scholar
  • 41 Mackenzie GG, Oteiza PI. Zinc and the cytoskeleton in the neuronal modulation of transcription factor NFAT. J Cell Physiol 2007; 210 (01) 246-256
    CrossrefPubMedGoogle Scholar
  • 42 Hathaway DR, Adelstein RS. Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity. Proc Natl Acad Sci U S A 1979; 76 (04) 1653-1657
    CrossrefPubMedGoogle Scholar
  • 43 Mills JS, Johnson JD. Metal ions as allosteric regulators of calmodulin. J Biol Chem 1985; 260 (28) 15100-15105
    CrossrefPubMedGoogle Scholar
  • 44 Heijnen H, van der Sluijs P. Platelet secretory behaviour: as diverse as the granules … or not?. J Thromb Haemost 2015; 13 (12) 2141-2151
    CrossrefPubMedGoogle Scholar
  • 45 Golebiewska EM, Poole AW. Platelet secretion: from haemostasis to wound healing and beyond. Blood Rev 2015; 29 (03) 153-162
    CrossrefPubMedGoogle Scholar
  • 46 Mann JJ, Fraker PJ. Zinc pyrithione induces apoptosis and increases expression of Bim. Apoptosis 2005; 10 (02) 369-379
    CrossrefPubMedGoogle Scholar
  • 47 Rudolf E, Červinka M. Zinc pyrithione induces cellular stress signaling and apoptosis in Hep-2 cervical tumor cells: the role of mitochondria and lysosomes. Biometals 2010; 23 (02) 339-354
    CrossrefPubMedGoogle Scholar
  • 48 Truong-Tran AQ, Ho LH, Chai F, Zalewski PD. Cellular zinc fluxes and the regulation of apoptosis/gene-directed cell death. J Nutr 2000; 130 (5S, Suppl): 1459S-1466S
    CrossrefPubMedGoogle Scholar
  • 49 Kondoh M, Tasaki E, Takiguchi M, Higashimoto M, Watanabe Y, Sato M. Activation of caspase-3 in HL-60 cells treated with pyrithione and zinc. Biol Pharm Bull 2005; 28 (04) 757-759
    CrossrefPubMedGoogle Scholar
  • 50 Zhu B, Wang J, Zhou F. , et al. Zinc depletion by TPEN induces apoptosis in human acute promyelocytic NB4 cells. Cell Physiol Biochem 2017; 42 (05) 1822-1836
    CrossrefPubMedGoogle Scholar
  • 51 Gyulkhandanyan AV, Mutlu A, Freedman J, Leytin V. Markers of platelet apoptosis: methodology and applications. J Thromb Thrombolysis 2012; 33 (04) 397-411
    CrossrefPubMedGoogle Scholar
  • 52 Leytin V. Apoptosis in the anucleate platelet. Blood Rev 2012; 26 (02) 51-63
    CrossrefPubMedGoogle Scholar