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

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.

Publication History

Received: 04 December 2020

Accepted: 22 February 2021

Accepted Manuscript online:
26 February 2021

Article published online:
14 April 2021

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