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.
Highlights
Platelets play a primary role in haemostasis following vascular damage
Ubiquitylation of Platelet Proteins Western blot using an antibody (FK2) that recognizes both mono- and poly-ubiquitin revealed the ubiquitylation of multiple proteins in resting human platelets (►Fig. 1A)
PR619 inhibits CRP-XL-induced activation of the fibrinogen binding by the major platelet integrin αIIbβ[3] (►Fig. 1E), consistent with inhibition of the GPVI signalling pathway upstream of integrin activation
Summary
Platelets play a primary role in haemostasis following vascular damage. The major mediators of platelet responsesà These authors contributed to this study. Platelets play a primary role in haemostasis following vascular damage. à These authors contributed to this study. Ãà B.M.K. and C.J.P. are joint corresponding authors. Interact with receptors which converge on activation of the serine/threonine kinase protein kinase C and increases in intracellular Ca2þ levels. Soluble mediators such as thrombin signal through receptors coupled to heterotrimeric G proteins, whereas the glycoproteins (GPs) GPVI and CLEC-2 signal through Src family kinases (SFKs) and Syk.[1,2,3] All pathways lead to covalent modification of target proteins received June 4, 2018 accepted after revision October 10, 2018.
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