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Specific proteome changes in platelets from individuals with GATA1-, GFI1B- and RUNX1-linked bleeding disorders

ABSTRACT: Mutations in the transcription factors GATA1, GFI1B and RUNX1 (GATA Binding Factor 1, Growth Factor Independence 1B, Runt-related transcription factor 1) cause familial platelet and bleeding disorders. Mutant platelets exhibit common abnormalities including an α-granule reduction. This suggests that similar pathways are deregulated by different transcription factor mutations. To identify common factors, full platelet proteomes from individuals with mutant GATA1R216Q, GFI1BQ287*, RUNX1TD2-6, or RUNX1Q154Rfs, and healthy controls were examined by label free quantitative mass spectrometry. Unsupervised clustering analysis of 2823 reliably quantified proteins revealed profound differences between cases and controls. Among cases, 82 of 174 significantly downregulated proteins were assigned to platelet function, hemostasis and granule biology, in line with platelet dysfunction and bleedings. Remarkably, only two of these proteins were diminished in all affected cases. This indicates that transcription factor mutations alter platelet proteomes in distinct largely non-overlapping manners. In contrast to the two common diminished proteins, 46 proteins were overrepresented in all affected cases compared to controls. These proteins converged to mitochondrial, metabolic and other biological processes. This work provides the quantitative landscape of proteins that affect platelet function when deregulated by mutated transcription factors in inherited bleeding disorders.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Platelet, Platelet

DISEASE(S): Bleeding

SUBMITTER: Floris van Alphen

LAB HEAD: Bert A. van der Reijden

PROVIDER: PXD020343 | Pride | 2022-02-22

REPOSITORIES: Pride

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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.

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Specific proteome changes in platelets from individuals with GATA1-, GFI1B- and RUNX1-linked bleeding disorders

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