Evidence that GPVI is expressed as a mixture of monomers and dimers, and that the D2 domain is not essential for GPVI activation: GPVI dimerisation is not critical for ligand binding

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@article{465fb71dae03487d9a60c25b73cd0c2b,
title = "Evidence that GPVI is expressed as a mixture of monomers and dimers, and that the D2 domain is not essential for GPVI activation: GPVI dimerisation is not critical for ligand binding",
abstract = "Collagen has been proposed to bind to a unique epitope in dimeric 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 dimerisation. 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 dimerisation 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 F{\"o}rster 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 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.",
author = "Joanne Clark and Neagoe, {Raluca Alexandra Iulia} and Malou Zuidscherwoude and Deirdre Kavanagh and Alexandre Slater and Eleyna Martin and Mark Soave and David Stegner and Bernhard Nieswandt and Natalie Poulter and Johan Hummert and Dirk-Peter Herten and Michael Tomlinson and Hill, {Stephen J} and Steve Watson",
year = "2021",
month = feb,
day = "26",
doi = "10.1055/a-1401-5014",
language = "English",
volume = "2021",
pages = "1--26",
journal = "Thrombosis and Haemostasis",
issn = "0340-6245",
publisher = "Schattauer",
number = "00",

}

RIS

TY - JOUR

T1 - Evidence that GPVI is expressed as a mixture of monomers and dimers, and that the D2 domain is not essential for GPVI activation

T2 - GPVI dimerisation is not critical for ligand binding

AU - Clark, Joanne

AU - Neagoe, Raluca Alexandra Iulia

AU - Zuidscherwoude, Malou

AU - Kavanagh, Deirdre

AU - Slater, Alexandre

AU - Martin, Eleyna

AU - Soave, Mark

AU - Stegner, David

AU - Nieswandt, Bernhard

AU - Poulter, Natalie

AU - Hummert, Johan

AU - Herten, Dirk-Peter

AU - Tomlinson, Michael

AU - Hill, Stephen J

AU - Watson, Steve

PY - 2021/2/26

Y1 - 2021/2/26

N2 - Collagen has been proposed to bind to a unique epitope in dimeric 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 dimerisation. 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 dimerisation 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 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.

AB - Collagen has been proposed to bind to a unique epitope in dimeric 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 dimerisation. 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 dimerisation 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 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.

U2 - 10.1055/a-1401-5014

DO - 10.1055/a-1401-5014

M3 - Article

VL - 2021

SP - 1

EP - 26

JO - Thrombosis and Haemostasis

JF - Thrombosis and Haemostasis

SN - 0340-6245

IS - 00

ER -