Human Platelet Protein Ubiquitylation and Changes following GPVI Activation

Amanda J. Unsworth; Izabela Bombik; Adan Pinto-Fernandez; Joanna F. McGouran; Rebecca Konietzny; René P. Zahedi; Steve P. Watson; Benedikt M. Kessler; Catherine J. Pears

doi:10.1055/s-0038-1676344

Thrombosis and Haemostasis, Inhaltsverzeichnis

CC BY 4.0 · Thromb Haemost 2019; 119(01): 104-116
DOI: 10.1055/s-0038-1676344

Cellular Haemostasis and Platelets

Georg Thieme Verlag KG Stuttgart · New York

Human Platelet Protein Ubiquitylation and Changes following GPVI Activation

Authors

Amanda J. Unsworth^*

¹Department of Biochemistry, University of Oxford, Oxford, United Kingdom

²Institute for Cardiovascular and Metabolic Research, University of Reading, Reading, United Kingdom
Izabela Bombik^*

¹Department of Biochemistry, University of Oxford, Oxford, United Kingdom
Adan Pinto-Fernandez

³Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, United Kingdom
Joanna F. McGouran

³Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, United Kingdom
Rebecca Konietzny

³Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, United Kingdom
René P. Zahedi

⁴JGH Proteomics Centre, Lady Davis Institute, Jewish General Hospital, Montreal, Quebec, Canada
Steve P. Watson

⁵Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom

⁶Centre of Membrane Proteins and Receptors, Universities of Birmingham and Nottingham, The Midlands, United Kingdom
Benedikt M. Kessler^**

³Nuffield Department of Medicine, Target Discovery Institute, University of Oxford, Oxford, United Kingdom
Catherine J. Pears^**

¹Department of Biochemistry, University of Oxford, Oxford, United Kingdom

Abstract

Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibition of platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here, we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen-related peptide (CRP-XL). Using platelets from six individuals over three independent experiments, we identified 1,634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. Note that 925 of these peptides show an increase of more than twofold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on several proteins including Syk, filamin and integrin heterodimer sub-units. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.

Keywords

platelets - sites of ubiquitinylation - GPVI - deubiquitylation inhibition

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