Identification of the phosphotyrosine proteome from thrombin activated platelets

Abstract

Signalling cascades are regulated both positively and negatively by tyrosine phosphorylation. Integrin mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signalling complexes to be assembled. In resting platelets, a small number of phosphorylated proteins are evident with molecular mass of 50-62 kDa and 120-130 kDa. In thrombin activated human platelets, however, there is a large increase in the number of tyrosine phosphorylated signalling proteins detected. These proteins include pCas (130 kDa), FAK (125 kDa), PI(3)k (85 kDa) and src (85 kDa). However, it is unlikely that this list of proteins represents all the dynamic changes that occur after platelet activation and it is understood that more proteins remain unidentified. In this study, we propose a method for the isolation of the phosphotyrosine proteome from both resting and thrombin activated human platelets. All the dynamic phosphotyrosine events that occur in the platelet after thrombin activation were isolated by immunoprecipitation, using the monoclonal antibody 4G10, allowing us to obtain higher concentrations of relatively low abundant proteins. The resulting phosphotyrosine proteomes were separated by two-dimensional gel electrophoresis. Sixty-seven proteins were reproducibly found to be unique in the thrombin activated platelet proteome when compared to resting platelets. We have positively identified ten of these proteins by Western blotting and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and these include FAK, Syk, ALK-4, P2X6 and MAPK kinase kinase. This proteomics approach to understanding the signalling events following platelet activation may elucidate potential drug targets for the treatment of coronary thrombosis.

Canonical Wnt signaling negatively regulates platelet function

“…Fzd6Ϫ/Ϫ mice and Fzd6ϩ/Ϫ littermate controls were kindly provided by Dr. Jeremy Nathans and Dr. Yanshu Wang (The Johns Hopkins University) (32). Isolation of human and murine platelets was performed as described (33,34). Platelet releasate was prepared as before (35).…”

What Can Proteomics Tell Us About Platelets?

“…In contrast, state-of-the-art procedures use quantitative phosphoproteomics to monitor changes in phosphorylation levels of hundreds to thousands of peptides from relatively low sample amounts (≈0.1-1 mg of protein per condition), thus providing valuable insights into important biochemical processes. To date, mostly gel-based approaches in conjunction with phosphotyrosine immunoprecipitation have been used to study platelet signaling processes (eg, mediated by activation of thrombin receptors, 93,106,107,137 collagen receptors, 82,112 C-type lectin-like receptor 2, 138 and integrin α IIb β 3 outside-in signaling). 139 Because of the limitation in sensitivity, these approaches often did not provide MS-based identification of phosphorylation sites but identified new phosphoproteins and signaling pathways, hence expanding the knowledge about platelet regulation.…”

Proteomic analysis of membrane microdomain-associated proteins in the dorsolateral prefrontal cortex in schizophrenia and bipolar disorder reveals alterations in LAMP, STXBP1 and BASP1 protein expression

“…Determination of flotillin-1 and transferrin receptor distribution The methods for SDS-PAGE and western blotting were undertaken as described previously with the following modifications. 33 For western blot analysis, gels were transferred to nitrocellulose membranes and probed with specific antibodies as described previously. 34 Mouse monoclonal antibodies to flotillin-1 (clone 18, 1:500 dilution) and transferrin receptor (anti-CD71, 1:200 dilution) were purchased from BD Biosciences (Oxford, UK) and Chemicon (Hampshire, UK) respectively.…”