Rac inhibition causes impaired GPVI signalling in human platelets through GPVI shedding and reduction of PLCγ2 phosphorylation

Raluca Alexandra Iulia Neagoe, Elizabeth E Gardiner, David Stegner, Bernhard Nieswandt, Steve Watson, Natalie Poulter

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Abstract

Rac1 is a small Rho GTPase that is activated in platelets upon stimulation with various ligands, including collagen and thrombin, which are ligands for the glycoprotein VI (GPVI) receptor and the protease-activated receptors, respectively. Rac1-deficient murine platelets have impaired lamellipodia formation, aggregation, and reduced PLCγ2 activation, but not phosphorylation. The objective of our study is to investigate the role of Rac1 in GPVI-dependent human platelet activation and downstream signalling. Therefore, we used human platelets stimulated using GPVI agonists (collagen and collagen-related peptide) in the presence of the Rac1-specific inhibitor EHT1864 and analysed platelet activation, aggregation, spreading, protein phosphorylation, and GPVI clustering and shedding. We observed that in human platelets, the inhibition of Rac1 by EHT1864 had no significant effect on GPVI clustering on collagen fibres but decreased the ability of platelets to spread or aggregate in response to GPVI agonists. Additionally, in contrast to what was observed in murine Rac1-deficient platelets, EHT1864 enhanced GPVI shedding in platelets and reduced the phosphorylation levels of PLCγ2 following GPVI activation. In conclusion, Rac1 activity is required for both human and murine platelet activation in response to GPVI-ligands, but Rac1’s mode of action differs between the two species.
Original languageEnglish
Article number3746
JournalInternational Journal of Molecular Sciences
Volume23
Issue number7
DOIs
Publication statusPublished - 29 Mar 2022

Bibliographical note

Funding Information:
R.A.I.N. is supported by the European Union?s Horizon 2020 research and innovation program under the Marie Slodowska-Curie grant, agreement no. 766118 TAPAS, and is enrolled in a joint PhD program at the Universities of Birmingham (United Kingdom) and W?rzburg (Germany). S.P.W holds a BHF Chair (CH03/003). This work was also supported by the Centre of Membrane Proteins and Receptors (COMPARE), UK. The APC was funded by the University of Birmingham (United Kingdom).

Publisher Copyright:
© 2022 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

  • platelets
  • Rac1
  • glycoprotein VI
  • EHT1864
  • GPVI shedding
  • phospholipase C gamma 2

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