Purification and characterisation of the platelet-activating GPVI/FcRγ complex in SMALPs

Xueqing Wang, Alexandre Slater, Sarah C Lee, Neale Harrison, Naomi L Pollock, Saskia E Bakker, Stefano Navarro, Bernhard Nieswandt, Tim R Dafforn, Ángel García, Steve P Watson, Michael G Tomlinson*

*Corresponding author for this work

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Abstract

The collagen/fibrin(ogen) receptor, glycoprotein VI (GPVI), is a platelet activating receptor and a promising anti-thrombotic drug target. However, while agonist-induced GPVI clustering on platelet membranes has been shown to be essential for its activation, it is unknown if GPVI dimerisation represents a unique conformation for ligand binding. Current GPVI structures all contain only the two immunoglobulin superfamily (IgSF) domains in the GPVI extracellular region, so lacking the mucin-like stalk, transmembrane, cytoplasmic tail of GPVI and its associated Fc receptor γ (FcRγ) homodimer signalling chain, and provide contradictory insights into the mechanisms of GPVI dimerisation. Here, we utilised styrene maleic-acid lipid particles (SMALPs) to extract GPVI in complex with its two associated FcRγ chains from transfected HEK-293T cells, together with the adjacent lipid bilayer, then purified and characterised the GPVI/FcRγ-containing SMALPs, to enable structural insights into the full-length GPVI/FcRγ complex. Using size exclusion chromatography followed by a native polyacrylamide gel electrophoresis (PAGE) method, SMA-PAGE, we revealed multiple sizes of the purified GPVI/FcRγ SMALPs, suggesting the potential existence of GPVI oligomers. Importantly, GPVI/FcRγ SMALPs were functional as they could bind collagen. Mono-dispersed GPVI/FcRγ SMALPs could be observed under negative stain electron microscopy. These results pave the way for the future investigation of GPVI stoichiometry and structure, while also validating SMALPs as a promising tool for the investigation of human membrane protein interactions, stoichiometry and structure.

Original languageEnglish
Article number109944
JournalArchives of Biochemistry and Biophysics
Volume754
Early online date22 Feb 2024
DOIs
Publication statusPublished - 1 Apr 2024

Bibliographical note

Copyright © 2024 The Authors.

Acknowledgements
We are grateful to the Birmingham Platelet Group for helpful advice and comments, the Birmingham Advanced Mass Spectrometry Facility for mass spectrometry analyses, and Dr. Stephanie Nestorow for sending our samples to the Advanced Bioimaging RTP facility at the University of Warwick. X.W. and this project was supported by the European Union's Horizon 2020 research and innovation programme under a Marie Skłodowska-Curie grant agreement (766118). A.S. was supported by an Accelerator Award from the British Heart Foundation [AA/18/2/34218] during this project. N.H. was funded by BBSRC grant BB/P00783X/1. S.P.W. is a British Heart Foundation Professor (CH 03/003).

Keywords

  • GPVI
  • SMALPs
  • Platelets
  • Collagen receptor
  • SMA-PAGE

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