Dataset Information

EPAC1 knockout mouse effects in platelets and plasma

ABSTRACT: ABSTRACT FOR BOTH PLASMA AND PLATELET-LYSATE In this article we describe the involvement of exchange factor activated by cAMP 1 (Epac1) in hemostasis and platelet activation. We have used plasma and platelet-lysate from EPAC1 knockout mice and wild-type control mice in label-free proteomics with an Orbitrap Velos Pro. Blood was obtained from wild-type and Epac1-/- mice euthanized with CO2. Approximately 1000 ul was drawn from the left ventricle into a 2 ml syringe, containing 100 ul ACD and 200 ul modified Tyrode`s buffer. The blood was centrifuged at 200g for 5 minutes at room temperature, and the resulting platelet-rich plasma (PRP) centrifuged at 700g for another 10 minutes in the presence of 10 ul ACD. The resulting platelet-poor plasma (PPP) was transferred to Eppendorf tubes and the pelleted platelets resuspended in modified Tyrode`s buffer and adjusted to 2.5 x 108 platelets/ml. Platelets were allowed to rest at room temperature before experimentation. For proteomics analysis, platelets were further purified by size-exclusion through Sepharose CL-2B gel (Pharmacia Biotec, Sweden) as previously described by Jensen et al. in Blood 2004; 104. Plasma was crude or depleted for Albumin prior to trypsination and LCMS with quantification using Progenesis LCMS. The platelet-lysates were fractioned on SDS-PAGE prior to trypsination and LC-MS analysis with MaxQuant quantification. Label-free protein quantification for plasma The software Progenesis LC-MS® Ver 2.7 (Nonlinear Dynamics Ltd, Newcastle, UK) was used for label-free quantification and comparison of LC-MS proteomics data based on the volume, m/z and retention time of the MS1 features (peptides). In Progenesis, the LC-MS runs were automatically aligned, and only features with charges between +2 to +7 and containing associated MSMS spectra were accepted for export as an mgf file for identification. The mgf file was search against the human SwissProt Mus musculus database (version September 2012) using SearchGUI Ver 1.8.9. The search criteria were: trypsin as the protease with no miss-cleavages accepted, fixed carbamidomethylation on cystein, variable oxidation on methionine, precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.7 and OMSSA as the search engine. The search result and associated spectra were combined and assigned to proteins in Peptide Shaker Ver 0.17.3 (http://peptideshaker.googlecode.com) at 1% FDR. The results were exported from PeptideShaker as validated PSMs in a Phenyx format, and imported back into Progenesis. The protein abundances reported from Progenesis were based on the sum of the normalized abundance of the unique identified petides. The proteomics raw files and PRIDE XML identification files were uploaded to PRIDE using ProteomeXchange ver 1.0.4, and the projects are public available. Two PRIDE XML files were generated using PeptideShaker, one for crude plasma and one for albumin-depleted plasma.

INSTRUMENT(S): LTQ Orbitrap, instrument model

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Blood Platelet, Platelet, Blood Plasma

SUBMITTER: Eystein Oveland

LAB HEAD: Professor PhD Frode Selheim

PROVIDER: PXD000282 | Pride | 2017-07-25

REPOSITORIES: Pride

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Publications

Epac1-deficient mice have bleeding phenotype and thrombocytes with decreased GPIbβ expression.

Nygaard Gyrid G Herfindal Lars L Asrud Kathrine S KS Bjørnstad Ronja R Kopperud Reidun K RK Oveland Eystein E Berven Frode S FS Myhren Lene L Hoivik Erling A EA Lunde Turid Helen Felli THF Bakke Marit M Døskeland Stein O SO Selheim Frode F

Scientific reports 20170818 1

Epac1 (Exchange protein directly activated by cAMP 1) limits fluid loss from the circulation by tightening the endothelial barrier. We show here that Epac1-/- mice, but not Epac2-/- mice, have prolonged bleeding time, suggesting that Epac1 may limit fluid loss also by restraining bleeding. The Epac1-/- mice had deficient in vitro secondary hemostasis. Quantitative comprehensive proteomics analysis revealed that Epac1-/- mouse platelets (thrombocytes) h ...[more]

PMID: 28821815

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Project description:In sickle cell disease, ischemia-reperfusion injury and intravascular hemolysis produce endothelial dysfunction and vasculopathy characterized by reduced nitric oxide (NO) and arginine bioavailability. Recent functional studies of platelets in patients with sickle cell disease reveal a basally activated state, suggesting that pathological platelet activation may contribute to sickle cell disease vasculopathy. Studies were therefore undertaken to examine transcriptional signaling pathways in platelets that may be dysregulated in sickle cell disease. We demonstrate and validate here the feasibility of comparative platelet transcriptome studies on clinical samples from single donors, by the application of RNA amplification followed by microarray-based analysis of 54,000 probe sets. Data mining an existing microarray database, we identified 220 highly abundant genes in platelets and a subset of 72 relatively platelet-specific genes, defined by more than 10-fold increased expression compared to the median of other cell types in the database with amplified transcripts. The highly abundant platelet transcripts found in the current study included 82% or 70% of platelet abundant genes identified in two previous gene expression studies on non-amplified mRNA from pooled or apheresis samples, respectively. On comparing the platelet gene expression profiles in 18 patients with sickle cell disease in steady state to 12 African American controls, at a 3-fold cut-off and 5% false discovery rate, we identified ~100 differentially expressed genes, including multiple genes involved in arginine metabolism and redox homeostasis. Further characterization of these pathways using real time PCR and biochemical assays revealed increased arginase II expression and activity and decreased platelet polyamine levels. These studies suggest a potential pathogenic role for platelet arginase and altered arginine and polyamine metabolism in sickle cell disease and provide a novel framework for the study of disease-specific platelet biology. Experiment Overall Design: There are 18 sickle cell samples and 12 control samples from healthy African American volunteers.

Dataset Information

EPAC1 knockout mouse effects in platelets and plasma

Publications

Epac1-deficient mice have bleeding phenotype and thrombocytes with decreased GPIbβ expression.

Similar Datasets

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