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The platelet transcriptome reveals changes in arginine metabolic pathways in patients with sickle cell disease

ABSTRACT: 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. Keywords: Platelets, genes, enzymes, metabolism, signal transduction, sickle cell disease

ORGANISM(S): Homo sapiens

PROVIDER: GSE11524 | GEO | 2008/05/22

SECONDARY ACCESSION(S): PRJNA106361

REPOSITORIES: GEO

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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.

Project description:Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologs of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in ?-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1? mutant of a guanidinobutyrase (EC.3.5.3.7), an enzyme not previously demonstrated in fungi. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. The goal of the present study was to characterize arginine catabolism in K. lactis. To this end, CAR1, CAR2 and PRO3 orthologs in K. lactis were identified and functionally analysed by deletion, expression in S. cerevisiae and enzyme activity assays. Since deletion of the arginase gene in K. lactis was found not to completely abolish growth on arginine as a sole nitrogen source, the alternative pathway for arginine catabolism operating in this yeast was studied by a combination of transcriptome analysis, 13C and 15N isotope-based flux analysis and enzyme activity assays in cell extracts. To investigate arginine metabolism in the arginase-negative K. lactis strain, strains GG1632 (Klku80? KlCAR1 reference strain) and IMS0367 (Klcar1? Arg+) were grown in aerobic bioreactor batch cultures on glucose chemically defined medium with arginine as sole nitrogen source. RNA sequencing of samples taken during the exponential phase of growth on glucose-arginine media of the reference strain G1631 and the arginase less strain IMS0367 were compared resulting in the characterization of a new function.

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The platelet transcriptome reveals changes in arginine metabolic pathways in patients with sickle cell disease

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