Download PDF
Thromb Haemost 2021; 121(11): 1435-1447
DOI: 10.1055/a-1401-5014
Cellular Haemostasis and Platelets

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation

Joanne C. Clark 
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
,
Raluca A. I. Neagoe
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
3   Institute of Experimental Biomedicine I, University Hospital and Rudolf Virchow Center, University of Wurzburg, Wurzburg, Germany
,
Malou Zuidscherwoude
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
,
Deirdre M. Kavanagh
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
,
Alexandre Slater
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
,
Eleyna M. Martin
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
,
Mark Soave
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
4   Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom
,
David Stegner
3   Institute of Experimental Biomedicine I, University Hospital and Rudolf Virchow Center, University of Wurzburg, Wurzburg, Germany
,
Bernhard Nieswandt
3   Institute of Experimental Biomedicine I, University Hospital and Rudolf Virchow Center, University of Wurzburg, Wurzburg, Germany
,
Natalie S. Poulter
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
,
Johan Hummert
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
5   Department for Physical Chemistry, Heidelberg University, Heidelberg, Germany
,
Dirk-Peter Herten
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
5   Department for Physical Chemistry, Heidelberg University, Heidelberg, Germany
,
Michael G. Tomlinson
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
6   School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
,
Stephen J. Hill
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
4   Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Queen's Medical Centre, Nottingham, United Kingdom
,
Steve P. Watson
1   Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom
2   Centre of Membrane Proteins and Receptors, The Universities of Birmingham and Nottingham, The Midlands, United Kingdom
› Author Affiliations Funding This study was supported by a grant from the Centre of Membrane Proteins and Receptors, University of Birmingham and University of Nottingham, Midlands, United Kingdom and British Heart Foundation (RG/13/18/30563). R.A.I.N. receives funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement (No. 766118). S.P.W. is a British Heart Foundation Professor (CH 03/003).
› Further Information
    Permissions and Reprints

Abstract

Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.

Keywords

platelets - glycoprotein VI receptor - dimerization - collagen - single-molecule microscopy

Author Contributions

J.C. Clark performed experiments, generated constructs, analyzed data, wrote and edited the manuscript. R.A.I. Neagoe performed FRET experiments, analyzed data, wrote and edited the manuscript. M. Zuidscherwoude performed experiments, generated constructs, provided supervision and molecular biology training and analyzed data. D.M. Kavanagh designed experiments and performed FCS and photobleaching experiments, and analyzed data. A. Slater, E.M. Martin and M.G. Tomlinson designed constructs and experiments and edited the manuscript. M. Soave provided reagents, constructs and expertise for nanoBRET study design and analysis. D. Stegner, B. Nieswandt and N.S. Poulter have provided reagents, supervision and edited the manuscript. J. Hummert and D. Herten have analyzed data and developed the algorithms for photobleaching image analysis and edited the manuscript. S.J. Hill provided supervision, concept, funding and contributed to manuscript editing. S.P. Watson provided supervision, funding, study design and concept, reviewed data, wrote and edited manuscript. All authors have read the manuscript.


Supplementary Material



Publication History

Received: 04 December 2020

Accepted: 22 February 2021

Accepted Manuscript online:
26 February 2021

Article published online:
14 April 2021

© 2021. Thieme. All rights reserved.

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

 

  • References

  • 1 Moroi M, Jung SM. Platelet glycoprotein VI: its structure and function. Thromb Res 2004; 114 (04) 221-233
    CrossrefPubMedGoogle Scholar
  • 2 Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102 (02) 449-461
    CrossrefPubMedGoogle Scholar
  • 3 Bori-Sanz T, Inoue KS, Berndt MC, Watson SP, Tulasne D. Delineation of the region in the glycoprotein VI tail required for association with the Fc receptor gamma-chain. J Biol Chem 2003; 278 (38) 35914-35922
    CrossrefPubMedGoogle Scholar
  • 4 Berlanga O, Tulasne D, Bori T. et al. The Fc receptor gamma-chain is necessary and sufficient to initiate signalling through glycoprotein VI in transfected cells by the snake C-type lectin, convulxin. Eur J Biochem 2002; 269 (12) 2951-2960
    CrossrefPubMedGoogle Scholar
  • 5 Miura Y, Takahashi T, Jung SM, Moroi M. Analysis of the interaction of platelet collagen receptor glycoprotein VI (GPVI) with collagen. A dimeric form of GPVI, but not the monomeric form, shows affinity to fibrous collagen. J Biol Chem 2002; 277 (48) 46197-46204
    CrossrefPubMedGoogle Scholar
  • 6 Loyau S, Dumont B, Ollivier V. et al. Platelet glycoprotein VI dimerization, an active process inducing receptor competence, is an indicator of platelet reactivity. Arterioscler Thromb Vasc Biol 2012; 32 (03) 778-785
    CrossrefPubMedGoogle Scholar
  • 7 Jung SM, Tsuji K, Moroi M. Glycoprotein (GP) VI dimer as a major collagen-binding site of native platelets: direct evidence obtained with dimeric GPVI-specific Fabs. J Thromb Haemost 2009; 7 (08) 1347-1355
    CrossrefPubMedGoogle Scholar
  • 8 Jung SM, Moroi M, Soejima K. et al. Constitutive dimerization of glycoprotein VI (GPVI) in resting platelets is essential for binding to collagen and activation in flowing blood. J Biol Chem 2012; 287 (35) 30000-30013
    CrossrefPubMedGoogle Scholar
  • 9 Horii K, Kahn ML, Herr AB. Structural basis for platelet collagen responses by the immune-type receptor glycoprotein VI. Blood 2006; 108 (03) 936-942
    CrossrefPubMedGoogle Scholar
  • 10 Berlanga O, Bori-Sanz T, James JR. et al. Glycoprotein VI oligomerization in cell lines and platelets. J Thromb Haemost 2007; 5 (05) 1026-1033
    CrossrefPubMedGoogle Scholar
  • 11 Onselaer MB, Hardy AT, Wilson C. et al. Fibrin and D-dimer bind to monomeric GPVI. Blood Adv 2017; 1 (19) 1495-1504
    CrossrefPubMedGoogle Scholar
  • 12 Ebrahim M, Jamasbi J, Adler K. et al. Dimeric glycoprotein VI binds to collagen but not to fibrin. Thromb Haemost 2018; 118 (02) 351-361
    Article in Thieme ConnectPubMedGoogle Scholar
  • 13 Mangin PH, Onselaer MB, Receveur N. et al. Immobilized fibrinogen activates human platelets through glycoprotein VI. Haematologica 2018; 103 (05) 898-907
    CrossrefPubMedGoogle Scholar
  • 14 Xu R-G, Gauer JS, Baker SR. et al. GPVI (glycoprotein VI) interaction with fibrinogen is mediated by avidity and the fibrinogen αC-region. Arterioscler Thromb Vasc Biol 2021; 41 (03) 1092-1104
    CrossrefPubMedGoogle Scholar
  • 15 Slater A, Perrella G, Onselaer MB. et al. Does fibrin(ogen) bind to monomeric or dimeric GPVI, or not at all?. Platelets 2019; 30 (03) 281-289
    CrossrefPubMedGoogle Scholar
  • 16 Zhang D, Ebrahim M, Adler K. et al. Glycoprotein VI is not a functional platelet receptor for fibrin formed in plasma or blood. Thromb Haemost 2020; 120 (06) 977-993
    Article in Thieme ConnectPubMedGoogle Scholar
  • 17 Marsh RJ, Pfisterer K, Bennett P. et al. Artifact-free high-density localization microscopy analysis. Nat Methods 2018; 15 (09) 689-692
    CrossrefPubMedGoogle Scholar
  • 18 Hummert J, Yserentant K, Fink T, Euchner J, Herten D-P. Photobleaching step analysis for robust determination of protein complex stoichiometries. bioRxiv 2020; DOI: 10.1101/2020.08.26.268086.
    CrossrefPubMedGoogle Scholar
  • 19 Schindelin J, Rueden CT, Hiner MC, Eliceiri KW. The ImageJ ecosystem: an open platform for biomedical image analysis. Mol Reprod Dev 2015; 82 (7–8): 518-529
    CrossrefPubMedGoogle Scholar
  • 20 Ovesný M, Křížek P, Borkovec J, Svindrych Z, Hagen GM. ThunderSTORM: a comprehensive ImageJ plug-in for PALM and STORM data analysis and super-resolution imaging. Bioinformatics 2014; 30 (16) 2389-2390
    CrossrefPubMedGoogle Scholar
  • 21 Clark JC, Kavanagh DM, Watson S. et al. Adenosine and Forskolin inhibit platelet aggregation by collagen but not the proximal signalling events. Thromb Haemost 2019; 119 (07) 1124-1137
    Article in Thieme ConnectPubMedGoogle Scholar
  • 22 Bender M, Stritt S, Nurden P. et al. Megakaryocyte-specific Profilin1-deficiency alters microtubule stability and causes a Wiskott-Aldrich syndrome-like platelet defect. Nat Commun 2014; 5: 4746
    CrossrefPubMedGoogle Scholar
  • 23 Epps TW, Singleton KJ. An omnibus test for the two-sample problem using the empirical characteristic function. J Stat Comput Simul 1986; 26 (3–4): 177-203
    CrossrefPubMedGoogle Scholar
  • 24 Virtanen P, Gommers R, Oliphant TE. et al; SciPy 1.0 Contributors. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat Methods 2020; 17 (03) 261-272
    CrossrefPubMedGoogle Scholar
  • 25 Machleidt T, Woodroofe CC, Schwinn MK. et al. NanoBRET: a novel BRET platform for the analysis of protein-protein interactions. ACS Chem Biol 2015; 10 (08) 1797-1804
    CrossrefPubMedGoogle Scholar
  • 26 Kilpatrick LE, Alcobia DC, White CW. et al. Complex formation between VEGFR2 and the beta2-Adrenoceptor. Cell Chem Biol 2019; 26 (06) 830-841.e9
    CrossrefPubMedGoogle Scholar
  • 27 Evans EJ, Esnouf RM, Manso-Sancho R. et al. Crystal structure of a soluble CD28-Fab complex. Nat Immunol 2005; 6 (03) 271-279
    CrossrefPubMedGoogle Scholar
  • 28 Zhang X, Schwartz JC, Almo SC, Nathenson SG. Crystal structure of the receptor-binding domain of human B7-2: insights into organization and signaling. Proc Natl Acad Sci U S A 2003; 100 (05) 2586-2591
    CrossrefPubMedGoogle Scholar
  • 29 Battin C, De Sousa Linhares A, Paster W. et al. Neuropilin-1 acts as a receptor for complement split products. Front Immunol 2019; 10 (2209): 2209
    CrossrefPubMedGoogle Scholar
  • 30 Peach CJ, Kilpatrick LE, Friedman-Ohana R. et al. Real-time ligand binding of fluorescent VEGF-a isoforms that discriminate between VEGFR2 and NRP1 in living cells. Cell Chem Biol 2018; 25 (10) 1208-1218.e5
    CrossrefPubMedGoogle Scholar
  • 31 Kim SA, Heinze KG, Schwille P. Fluorescence correlation spectroscopy in living cells. Nat Methods 2007; 4 (11) 963-973
    CrossrefPubMedGoogle Scholar
  • 32 Kavanagh DM, Smyth AM, Martin KJ. et al. A molecular toggle after exocytosis sequesters the presynaptic syntaxin1a molecules involved in prior vesicle fusion. Nat Commun 2014; 5 (01) 5774
    CrossrefPubMedGoogle Scholar
  • 33 Meseth U, Wohland T, Rigler R, Vogel H. Resolution of fluorescence correlation measurements. Biophys J 1999; 76 (03) 1619-1631
    CrossrefPubMedGoogle Scholar
  • 34 Chen Y, Müller JD, So PT, Gratton E. The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 1999; 77 (01) 553-567
    CrossrefPubMedGoogle Scholar
  • 35 Herrick-Davis K, Grinde E, Cowan A, Mazurkiewicz JE. Fluorescence correlation spectroscopy analysis of serotonin, adrenergic, muscarinic, and dopamine receptor dimerization: the oligomer number puzzle. Mol Pharmacol 2013; 84 (04) 630-642
    CrossrefPubMedGoogle Scholar
  • 36 Wong K, Briddon SJ, Holliday ND, Kerr ID. Plasma membrane dynamics and tetrameric organisation of ABCG2 transporters in mammalian cells revealed by single particle imaging techniques. Biochim Biophys Acta 2016; 1863 (01) 19-29
    CrossrefPubMedGoogle Scholar
  • 37 McGuire H, Aurousseau MR, Bowie D, Blunck R. Automating single subunit counting of membrane proteins in mammalian cells. J Biol Chem 2012; 287 (43) 35912-35921
    CrossrefPubMedGoogle Scholar
  • 38 Sekar RB, Periasamy A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations. J Cell Biol 2003; 160 (05) 629-633
    CrossrefPubMedGoogle Scholar
  • 39 Tomlinson MG, Calaminus SD, Berlanga O. et al. Collagen promotes sustained glycoprotein VI signaling in platelets and cell lines. J Thromb Haemost 2007; 5 (11) 2274-2283
    CrossrefPubMedGoogle Scholar
  • 40 Slater A, Di Y, Clark JC. et al. Structural characterisation of a novel GPVI nanobody-complex reveals a biologically active domain-swapped GPVI dimer. Blood 2021; DOI: 10.1182/blood.2020009440.
    CrossrefPubMedGoogle Scholar
  • 41 Pallini C, Pike JA, O'Shea C. et al. Immobilized collagen prevents shedding and induces sustained GPVI clustering and signaling in platelets. Platelets 2021; 32 (01) 59-73
    CrossrefPubMedGoogle Scholar