Dataset Information

Deciphering changes to human platelet proteome and degradome during storage before transfusion

ABSTRACT: Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, albeit the mechanism remains unknown. We used a dedicated N-terminomics technique, multiplex iTRAQ-TAILS (Terminal Amine Isotope Labeling of Substrates), to characterize the human platelet proteome, N-terminome, and their post-translational modifications throughout platelet storage under blood banking conditions. From the identified 2,938 proteins and 7,503unique peptides we characterized N-terminal methionine excision, co- and post-translational N-acetylation, maturation and proteolytic processing of proteins in human platelets. We also identified for the first time in platelets 10 proteins previously classified as “missing” in the human proteome. Most of identified N-termini (77%) were internal, of which 105 were novel potential alternative translation start sites, with 2,180 representing stable proteolytic products, thus highlighting a prominent previously uncharacterized role of proteolytic processing during platelet storage. Protease inhibitor studies revealed metalloproteinases as being primarily responsible for proteolytic processing (as opposed to degradation) during storage. System-wide identification of metallo- and other proteinase substrates and their respective cleavage sites offers novel potential mechanisms of their effect on protein activity and platelet function during storage.

INSTRUMENT(S): QSTAR

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Platelet, Platelet

DISEASE(S): Platelet Storage Pool Deficiency

SUBMITTER: Anna Prudova

LAB HEAD: Christopher Mark Overall

PROVIDER: PXD000906 | Pride | 2014-10-24

REPOSITORIES: Pride

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Publications

TAILS N-terminomics of human platelets reveals pervasive metalloproteinase-dependent proteolytic processing in storage.

Prudova Anna A Serrano Katherine K Eckhard Ulrich U Fortelny Nikolaus N Devine Dana V DV Overall Christopher M CM

Blood 20141020 26

Proteases, and specifically metalloproteinases, have been linked to the loss of platelet function during storage before transfusion, but the underlying mechanisms remain unknown. We used a dedicated N-terminomics technique, iTRAQ terminal amine isotopic labeling of substrates (TAILS), to characterize the human platelet N-terminome, proteome, and posttranslational modifications throughout platelet storage over 9 days under blood-banking conditions. From the identified 2938 proteins and 7503 uniqu ...[more]

PMID: 25331112

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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

Deciphering changes to human platelet proteome and degradome during storage before transfusion

Publications

TAILS N-terminomics of human platelets reveals pervasive metalloproteinase-dependent proteolytic processing in storage.

Similar Datasets

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