Amplified Expression Profiling of Platelet Transcriptome Reveals Global Activation of Arginine Metabolic Pathways in Patients with Sickle Cell Disease.

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 globally 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 for platelet specific or abundant genes identified 118 genes that showed more than a 100-fold increase in transcript expression level compared to all other cells in the database. Most of these genes were clearly annotated as platelet-specific and 84% of these transcripts overlapped with the platelet abundant genes identified in previous gene expression studies. 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/NO metabolism and redox homeostasis. Further functional characterization of these pathways using Gene Set Enrichment Analysis, real time PCR, arginase enzymatic assay, and polyamine quantification revealed arginase II-mediated catabolism and diversion of arginine from NO signaling and polyamine synthesis to proline formation. These studies suggest a potential pathogenic role for platelet arginase and ornithine decarboxylase antizyme and provide a novel framework for the study of disease-specific platelet biology.