Megakaryocyte and erythroblast DNA in plasma and platelets
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PROVIDER: PRJNA852251 | ENA |
REPOSITORIES: ENA
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Project description:Circulating nucleic acids are present in plasma and are derived from a range of cell types, mainly cells of hematopoietic origin, enabling their use as biomarkers for diseases involving these cell types. Using cell type-specific methylation markers, we determined that megakaryocytes are major contributors to cfDNA (~26%), while erythroblasts contribute 1-4% of cfDNA in healthy individuals. Additionally, we identified megakaryocyte-derived DNA within platelets, providing non-invasive access to the megakaryocyte genome and epigenome. Analysis of cfDNA in women who have received male platelet transfusions indicates that megakaryocyte-derived cfDNA does not originate from platelets but is rather released directly from megakaryocytes. The concentration of megakaryocyte-derived cfDNA is elevated in individuals with pathologies involving increased platelet production (Essential Thrombocythemia, Idiopathic Thrombocytopenic Purpura) and decreased by bone marrow suppression (due to chemotherapy), while erythrocyte progenitor cfDNA is elevated in patients with Thalassemia. Megakaryocyte- and erythroblast-specific DNA methylation patterns may serve as novel biomarkers for pathologies involving increased or decreased thrombopoiesis and erythropoiesis.
2022-10-12 | GSE206818 | GEO
Project description:The TAR DNA Binding Protein (TDP-43) has been implicated in the pathogenesis of human neurodegenerative diseases and exhibits hallmark neuropathology in amyotrophic lateral sclerosis (ALS). Here, we explore its tractability as a plasma biomarker of disease and describe its localization and possible functions in the cytosol of platelets. Novel TDP-43 immunoassays were developed on three different technical platforms and qualified for specificity, signal-noise ratio, detection range, variation, spike recovery and dilution linearity in human plasma samples. Fractionation studies revealed that >95% of plasma TDP-43 protein [RL1] was located within the platelet cytosol, together with numerous RNAs. Platelet-derived TDP-43 exhibits TDP-43 proteoforms detected in neurodegenerative diseases, TARDBP RNA splice variants and TDP-43 RNA targets found in the central nervous system (CNS). We propose that TDP-43 serves similar functional roles in platelets and synapses, suggesting that the study of platelet TDP-43 might provide a window into TDP-43 proteinopathies within the CNS. The restricted compartmentalization of plasma TDP-43 in platelets provides a highly concentrated substrate for further biochemical analyses. Moreover, our results suggest that current plasma biobanking protocols are subject to considerable heterogeneity in platelet recovery and measurements of TDP-43 in plasma.
2024-09-25 | GSE245303 | GEO
Project description:We profiled gene expression of pelleted material containing platelets from frozen plasma of healthy controls and ME/CFS cases for bulk RNA-seq, collected before and 24h after participants conducted a cardiopulmonary exercise test (CPET).
2024-01-16 | GSE236402 | GEO
Project description:Dnm2fl/fl Pf4-Cre (Dnm2Plt-/-) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt-/- platelets. Additionally, Dnm2Plt-/- platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt-/- mice was due to JAK2 constitutive activation or elevated circulating TPO levels. In unstimulated Dnm2Plt-/- platelets, STAT3 and to a lower extent STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt-/- mice in the Mpl-/- background to generate Mpl-/- Dnm2Plt-/- mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl-/- Dnm2Plt-/- platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl-/- Dnm2Plt-/- mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl-/- Dnm2Plt-/- mice had severe erythroblast maturation defects, decreased expression of hemoglobin and heme homeostasis genes, increased expression of ribosome biogenesis and protein translation genes, and developed anemia with grossly elevated plasma erythropoietin levels, leading to early fatality by postnatal day 25. Mpl-/- Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.
2022-08-25 | GSE206343 | GEO
Project description:We developed a novel differentiation system that directionally induces hESCs into megakaryocytes and functional platelet in vitro by highly mimicking the in vivo developmental process of megakaryocytes and platelets. We then performed gene expression profiling analysis using data obtained by RNA-seq at different stages of differentiation during the differentiation of hESCs into megakaryocyte lineages.
2024-08-14 | GSE216593 | GEO
Project description:Post-transcriptional and translational controls mediated by microRNAs (miRNA) regulate diverse biological processes and disease. We systematically dissected regulatory effects of miRNAs relevant to megakaryocytopoiesis and platelet biology by analyzing expression patterns in 79 subjects with thrombocytosis and healthy controls, and integrated these data with transcriptomic and proteomic platforms. We identified and validated a unique 21-miRNA genetic fingerprint associated with thrombocytosis, and demonstrated that a discrete 3-member subset effectively defines ET phenotypes. The genetic signature includes functional guide and passenger strands of the previously-uncharacterized miR 490 (5p and 3p), both of which displayed restricted, low-level expression in megakaryocytes/platelets (compared to leukocytes), and aberrant expression during thrombocytosis, most profound in essential thrombocythemia (ET). Overexpression of miR 490 in a bilineage differentiation model of megakaryocyte/erythroid progenitor formation was insufficient for hematopoietic stem cell colony differentiation and/or lineage specification. Systematic integration of transcriptomic and mass spectrometric datasets with functional reporter assays identified dishevelled associated activator of morphogenesis 1 (DAAM1) as a unique miR 490 5p protein target demonstrating decreased expression in ET platelets, putatively modulated by translational control (and not by mRNA target degradation). Our data define a dysregulated miRNA fingerprint in thrombocytosis, and collectively support a developmentally-restricted function of miR 490 (and its putative DAAM1 target) to conditions associated with exaggerated megakaryocytopoiesis and/or proplatelet formation. Human platelets from a total of 79 samples in 3 phenotypic groups: essential thrombocythemia (ET, N =27), reactive thrombocytosis (RT, N=22) and healthy controls (NO, N=30) were measured on array platform
2012-11-06 | E-GEOD-39046 | biostudies-arrayexpress
Project description:Platelets are anucleate cytoplasmic fragments that lack genomic DNA, but continue to synthesize protein using a pool of mRNAs, ribosomes, and regulatory small RNAs inherited from the precursor megakaryocyte (MK). The regulatory processes that shape the platelet transcriptome and the full scope of platelet translation have remained elusive. Using RNA-Seq and ribosome profiling of primary human platelets, we show the platelet transcriptome encompasses a subset of transcripts detected by RNA-Seq analysis of in vitro derived MK cells and these platelet-enriched transcripts are broadly occupied by ribosomes. We use RNA sequencing of synchronized populations of in vitro derived platelet-like particles (PLPs) to show that mRNA decay strongly shapes the nascent platelet transcriptome. Our data suggests that the decay of platelet mRNAs is slowed by the natural loss of the mRNA surveillance and ribosome rescue factor Pelota (PELO).
2017-02-02 | GSE94384 | GEO
Project description:Mutations of SMAD family member 4 (Smad4) gene caused Hereditary Hemorrhagic Telangiectasia (HHT). It was believed that bleeding disorders were caused by arteriovenous malformation in this syndrome. Although several studies indicated dysfunction of platelets from HHT patient, the role(s) of smad4 in platelet function has not been examined. In this study, using megakaryocyte/platelet-specific Smad4-deficient mice, we investigated the physiological function of Smad4 in platelet activation and the underlying mechanism. Microarray data demonstrated that the level of mRNA for multiple genes changed in Smad4 deficient platelet. For microarray analysis, total mRNA was extracted from washed platelets from Smad4f/f or Smad4M-bM-^HM-^R/M-bM-^HM-^R mice (for each group, n=6). mRNA was labeled and hybridized to Affymetrix Mouse Genome 430 2.0 chips according to manufacturer's instructions (Affymetrix).
2013-01-29 | E-GEOD-43814 | biostudies-arrayexpress
Project description:Thrombopoietin (TPO) acting via its receptor Mpl is the major cytokine regulator of platelet number. To precisely define the role of specific hematopoietic cells in TPO dependent hematopoiesis, we generated mice that express the Mpl receptor normally on stem/progenitor cells but lack expression on megakaryocytes and platelets (MplPF4cre/PF4cre). MplPF4cre/PF4cre mice displayed profound megakaryocytosis and thrombocytosis with a remarkable expansion of megakaryocyte-committed and multipotential progenitor cells, the latter displaying biological responses and a gene expression signature indicative of chronic TPO over-stimulation as the underlying causative mechanism, despite a normal circulating TPO level. Thus, TPO signaling in megakaryocytes is dispensable for platelet production; its key role in control of platelet number is via generation and stimulation of the bipotential megakaryocyte precursors. Nevertheless, Mpl expression on megakaryocytes and platelets is essential to prevent megakaryocytosis and myeloproliferation by restricting the amount of TPO available to stimulate the production of megakaryocytes from the progenitor cell pool.
2014-06-10 | E-MTAB-2389 | biostudies-arrayexpress
Project description: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.
2017-07-25 | PXD000282 | Pride