Human Platelet Protein Ubiquitylation and Changes following GPVI Activation

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.

Structure and function of the ubiquitin‐proteasome system in platelets

“…Ubiquitination can be reversed by the action of deubiquitinating enzymes, which cleave ubiquitin from the substrate protein or edit ubiquitin chains by the removal of single ubiquitin moieties. Deubiquitinating enzymes have also been detected in platelets and pre‐treatment of platelets with deubiquitinase inhibitors results in diminished thrombin‐stimulated platelet adhesion and reduced ADP‐, collagen‐, and thrombin‐induced platelet aggregation …”

“…In ANKRD26 thrombocytopenia the function of the ubiquitin‐proteasome system in platelets is impaired as evidenced by the accumulation of polyubiquitinated proteins . In addition, it has been observed that even platelet activation itself results in an accumulation of ubiquitinated proteins . This might be contradictory to the fact that platelets release their granule proteins upon activation, resulting in a significant loss in intracellular proteins.…”

“…Deubiquitinating enzymes have also been detected in platelets and pre-treatment of platelets with deubiquitinase inhibitors results in diminished thrombin-stimulated platelet adhesion and reduced ADP-, collagen-, and thrombin-induced platelet aggregation. 61,64 The biological relevance of a functional ubiquitin-proteasome system in platelets is demonstrated by patients with ANKRD26 thrombocytopenia, a rare form of dominantly inherited thrombocytopenia. In ANKRD26 thrombocytopenia the function of the ubiquitin-proteasome system in platelets is impaired as evidenced by the accumulation of polyubiquitinated proteins.…”

“…65 In addition, it has been observed that even platelet activation itself results in an accumulation of ubiquitinated proteins. 61 This might be contradictory to the fact that platelets release their granule proteins upon activation, resulting in a significant loss in intracellular proteins. One possible explanation could be the fact that various proteins required for cytoskeletal rearrangement, eg, Filamin A or Talin-1, are ubiquitinated but not released upon activation.…”

Deep analysis of the USP18-dependent ISGylome and proteome unveils important roles for USP18 in tumour cell antigenicity and radiosensitivity

“…HAP1 lysates were used for GlyGly immunoprecipitation using PTMScan Ubiquitin Remnant Motif Kit (Cell Signaling), according to manufacturer's protocol 45,46 . Briefly, 20 mg of extracts were solubilized and denatured in 10 mL lysis buffer (20 mM HEPES, pH 8.0, 9 M urea, 1 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate), reduced using dithiothreitol (4.5 mM final) for 30 minutes at 55 °C.…”