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

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

• References

• 1 Moroi M, Jung SM. Platelet glycoprotein VI: its structure and function. Thromb Res 2004; 114 (04) 221-233
• 2 Nieswandt B, Watson SP. Platelet-collagen interaction: is GPVI the central receptor?. Blood 2003; 102 (02) 449-461
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 11 Onselaer MB, Hardy AT, Wilson C. et al. Fibrin and D-dimer bind to monomeric GPVI. Blood Adv 2017; 1 (19) 1495-1504
• 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
• 13 Mangin PH, Onselaer MB, Receveur N. et al. Immobilized fibrinogen activates human platelets through glycoprotein VI. Haematologica 2018; 103 (05) 898-907
• 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
• 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
• 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
• 17 Marsh RJ, Pfisterer K, Bennett P. et al. Artifact-free high-density localization microscopy analysis. Nat Methods 2018; 15 (09) 689-692
• 18 Hummert J, Yserentant K, Fink T, Euchner J, Herten D-P. Photobleaching step analysis for robust determination of protein complex stoichiometries. bioRxiv 2020;
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 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
• 31 Kim SA, Heinze KG, Schwille P. Fluorescence correlation spectroscopy in living cells. Nat Methods 2007; 4 (11) 963-973
• 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
• 33 Meseth U, Wohland T, Rigler R, Vogel H. Resolution of fluorescence correlation measurements. Biophys J 1999; 76 (03) 1619-1631
• 34 Chen Y, Müller JD, So PT, Gratton E. The photon counting histogram in fluorescence fluctuation spectroscopy. Biophys J 1999; 77 (01) 553-567
• 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
• 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
• 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
• 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
• 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
• 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;
• 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