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Human platelet detergent resistant membranes proteome

ABSTRACT: Here we characterized the proteins associated with detergent resistant membranes (DRMs) from resting and activated human platelets. We achieved a proteomic profile of 141 DRM proteins involved in many biological pathways including 'PAR-1-mediated thrombin signal events', 'Integrin family cell surface interactions', 'Platelet activation, signalling and aggregation' and 'Platelet degranulation'. Therefore, a high level of relevant platelet signalling and trafficking proteins were identified e.g., Rap 1b, Src, SNAP-23 and syntaxin-11, implicating platelet DRMs as concentrating platforms not only in the critical platelet function of activation but also in secretion/degranulation.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Patricia B. Maguire

PROVIDER: MSV000080708 | MassIVE | Tue Mar 28 22:37:00 BST 2017

SECONDARY ACCESSION(S): PXD002554

REPOSITORIES: MassIVE

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Analysis of the proteins associated with platelet detergent resistant membranes.

Szklanna Paulina B PB Foy Martina M Wynne Kieran K Byrne Dwayne D Maguire Patricia B PB

Proteomics 20160901 17

Proteomic studies have facilitated the identification of proteins associated with the detergent-resistant membrane (DRM) fraction in a variety of cell types. Here, we have undertaken label-free quantitative (LFQ) proteomic profiling of the proteins associated with detergent-resistant plasma and internal membranes from resting and activated platelets. One hundred forty-one proteins were identified and raw data is available via ProteomeXchange with identifier PXD002554. The proteins identified inc ...[more]

PMID: 27329341

Similar Datasets

Human platelet detergent resistant membranes proteome

Project description:Here we characterized the proteins associated with detergent resistant membranes (DRMs) from resting and activated human platelets. We achieved a proteomic profile of 141 DRM proteins involved in many biological pathways including 'PAR-1-mediated thrombin signal events', 'Integrin family cell surface interactions', 'Platelet activation, signalling and aggregation' and 'Platelet degranulation'. Therefore, a high level of relevant platelet signalling and trafficking proteins were identified e.g., Rap 1b, Src, SNAP-23 and syntaxin-11, implicating platelet DRMs as concentrating platforms not only in the critical platelet function of activation but also in secretion/degranulation.

2016-06-23 | PXD002554 | Pride

miRNA expression profiles of Platelet microparticles (PMPs)

Project description:To identify the miRNA expressing profiles of Platelet microparticlesï¼PMPs, we have employed the Agilent Human miRNA 8Ã60K (Design ID:046064) microarray. Platelet microparticles. The platelets were derived from citrated blood of healthy human donors under an Institutional Review Board-approved protocol. Platelets were isolated after centrifugation of blood (1200r for 30 min at 21â), then the supernatant (platelet-rich plasma) was centrifuged at 2000r for 30 min at 21â, and the pellet containing platelets was resuspended in RPMI-1640 medium (HyClone, Logan, UT). Platelets were counted (Clinical Laboratory, Shanghai First Maternity and Infant Hospital, Shanghai) and adjusted to a density of 150 Ã 106 cells/mL before supplement with 1.5% ACD(sigma ) and stimulated with thrombin (1.0 u/mL; Takeda Austria) for 1 h. PMPs were in the supernatant after centrifugation at 4000r for 10 min at 4â,then the supernatants were centrifuged at 50,000 Ã g for 60 min at 4 Â°C. The pellets containing MPs were resuspended in RPMI-1640 medium and quantified by BCA method. The gene expressions of three independent paired PMPs from platelets stimulated by thrombin or apoptosis.

2016-01-14 | E-GEOD-76789 | biostudies-arrayexpress

Platelet microRNA-mRNA Co-Expression Profiles Correlate with Platelet Reactivity

Project description:MicroRNAs (miRNAs) regulate cell physiology by altering protein expression, but the biology of platelet miRNAs is largely unexplored. We tested whether platelet miRNA levels were associated with platelet reactivity by genome-wide profiling using platelet RNA from 19 healthy subjects. We found that human platelets express 284 miRNAs. Unsupervised hierarchical clustering of miRNA profiles resulted in 2 groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed (DE) between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. Using whole genome mRNA expression data on these same subjects, we computationally generated a high-priority list of miRNA-mRNA pairs in which the DE platelet miRNAs had binding sites in 3'UTRs of DE mRNAs, and the levels were negatively correlated. Three miRNA-mRNA pairs (miR-200b:PRKAR2B, miR-495:KLHL5 and miR-107:CLOCK) were selected from this list and all 3 miRNAs knocked down protein expression from the target mRNA. Reduced activation from platelets lacking PRKAR2B supported these findings. In summary, (1) platelet miRNAs are able to repress expression of platelet proteins, (2) miRNA profiles are associated with and may predict platelet reactivity, and (3) bioinformatic approaches can successfully identify functional miRNAs in platelets. Total RNA from the platelets of 19 donors was harvested and labeled with Hy3. Reference RNA (a pool of all samples) was labeled with Hy5. This submission represents the miRNA expression component of the study.

2011-09-26 | E-GEOD-27917 | biostudies-arrayexpress

miRNA expression profiles of Platelet microparticles (PMPs)

Project description:To identify the miRNA expressing profiles of Platelet microparticles（PMPs, we have employed the Agilent Human miRNA 8×60K (Design ID:046064) microarray. Platelet microparticles. The platelets were derived from citrated blood of healthy human donors under an Institutional Review Board-approved protocol. Platelets were isolated after centrifugation of blood (1200r for 30 min at 21℃), then the supernatant (platelet-rich plasma) was centrifuged at 2000r for 30 min at 21℃, and the pellet containing platelets was resuspended in RPMI-1640 medium (HyClone, Logan, UT). Platelets were counted (Clinical Laboratory, Shanghai First Maternity and Infant Hospital, Shanghai) and adjusted to a density of 150 × 106 cells/mL before supplement with 1.5% ACD(sigma ) and stimulated with thrombin (1.0 u/mL; Takeda Austria) for 1 h. PMPs were in the supernatant after centrifugation at 4000r for 10 min at 4℃,then the supernatants were centrifuged at 50,000 × g for 60 min at 4 °C. The pellets containing MPs were resuspended in RPMI-1640 medium and quantified by BCA method.

2016-01-14 | GSE76789 | GEO

Platelet RNA-Seq

Project description:Platelet-rich plasma (PRP) has been obtained by centrifuging whole blood at 160 g for 15 min at room temperature. Platelets have been activated with 2 different physiologic agonists, namely collagen (60ug/mL) or Thrombin Receptor Activating Peptide (TRAP 25uM) under continuous stirring. An aliquot of platelets has been obtained at 120 minutes following collagen and TRAP and then processed to determine mRNA expression profiles. Time 0, indicating samples before any agonist treatment, was used as baseline. Total RNA was extracted using the mirVana PARIS kit (LifeTechnologies), according to manufacturers instruction. Indexed libraries were prepared using 500 ng of total RNA as starting material and sequenced on HiSeq 1500 (Illumina, USA) at a concentration of 8pM for 2x100 plus 7 additional cycles for indexes sequencing. Standard workflow for quality control through RTA and bcl2fastq conversion tool was applied.

2015-04-27 | E-MTAB-2355 | biostudies-arrayexpress

Platelet microRNA-mRNA Co-Expression Profiles Correlate with Platelet Reactivity

2011-09-27 | GSE27917 | GEO

Functionally-relevant splicing of resident pre-mRNAs in anucleate platelets upon activation by physiological stimuli

Project description:Platelet activation is the key event triggering thrombus formation in physiological and pathological conditions, such as acute coronary syndromes. Current therapies using antiaggregants still fail to prevent thrombotic coronary events in a significant number of patients, indicating that the mechanisms modulating platelet response during activation need to be clarified. The evidence that platelets are capable of de novo protein synthesis in response to stimuli raised the issue of how the activity of megakaryocyte-derived mRNAs is regulated in these anucleate cell fragments. We applied a combined multi-omics approach to investigate this phenomenon in platelets from healthy donors activated in vitro with Collagen or Thrombin Receptor Activating Peptide. Combining HiRIEF LC-MS to transcriptome analysis by RNA-Seq allowed platelet proteome characterization at deep coverage, revealing a significant effect of either stimulus on proteome composition. In silico intron retention analysis was then applied to search for splicing events induced by platelet activation, coupled to unbiased proteogenomics, to correlate intron retention in resting platelets to intron removal by RNA splicing during activation. This allowed identification of a set of transcripts, specifically involved in platelet shape changes, showing reduced intron retention and high peptide representation at exon-exon junctions in activated vs resting platelets. These results indicate that RNA splicing events takes place in platelets during activation and that pre-mRNA maturation of specific transcripts is part of the activation cascade and could therefore provide novel molecular markers of platelet activation status in acute coronary syndromes and other pathological conditions.

2018-01-15 | E-MTAB-6148 | biostudies-arrayexpress

Platelet microRNA expression profiles of acute coronary syndrome patients with high and low platelet reactivity

Project description:To explore the diverse platelet microRNA (miRNA) expression between high platelet reactivity (HPR) and low platelet reactivity (LPR) patients with acute coronary syndromes (ACS), we enrolled a cohort of ACS patients and performed miRNA expression profiling of platelets from four HPR and four LPR patients using human miRNA microarray system. VerifyNow P2Y12 assay was applied to indentify HPR and LPR. Venous blood was drawn from the patients and was centrifuged to prepare platelets. Among the candidate differentially expressed miRNAs, miR-15b expression was further confirmed to be lower in platelets of 22 HPR patients than 17 LPR by quantitative reverse-transcription polymerase chain reaction (RT-qPCR). We enrolled a consecutive cohort of 290 ACS patients and assessed the platelet reactivity using VerifyNow P2Y12 assay. In this study, HPR was defined as M-bM-^IM-%300 platelet reactivity unit (PRU) while LPR <170 PRU. miRNA microarray analysis was performed in platelets of four HPR and four LPR patients with ACS.

2014-07-17 | E-GEOD-59488 | biostudies-arrayexpress

Thrombin-reduced miR-27b attenuates platelet angiogenic activities via enhancing platelet de novo synthesis of anti-angiogenic thrombospondin-1

Project description:To identify if platelet activation would result in altered platelet miRNA profile, not only in total RNA sample, also in AGO2-immunoprecipitation(AGO2-IP) products. To determinate if altered platelet miRNAs could regulate de novo protein synthesis of angiogenic factors in activated platelets, and even more importantly, if miRNA-regulated platelet angiogenic factor synthesis could result in changes of platelet angiogenic activities.

2017-03-14 | GSE96533 | GEO

NanoString- Transcriptional profiling of LPS-primed and inflammasome-activated untouched, or platelet-depleted human CD14+ monocytes

Project description:The goal of this experiment was to explore how human platelets affect the transcriptional responses of primary human CD14+ blood monocytes to lipopolysaccharide (LPS), and NLRP3 activation with Nigericin. For this purpose, we analyzed the mRNA expression of 770 myeloid-specific transcripts using the nCounter® Nanostring Human Myeloid Innate Immunity Panel v2. We isolated classical monocytes (CD14+CD16−) from the peripheral blood of healthy volunteers. Monocytes were derived from PBMCs using negative selection with the EasySep™ Human Monocyte Isolation Kit from STEMCELLTM Technologies. We modified this process by either including or excluding a platelet-depleting cocktail, creating two groups: \\"Standard Monocytes (StdMo)\\" and \\"Platelet-depleted Monocytes (PdMo).\\" To examine the impact of platelets further, we supplemented PdMos with fresh autologous platelets at a ratio of 50 platelets per monocyte, resulting in a third group, \\"PdMo + Plts.\\" StdMos were prepared according to the standard protocol provided by the EasySep™ Kit. For the PdMos, a Platelet Removal Component (50 µl ml−1) from the kit was used during isolation. We also reconstituted platelet-depleted monocytes with platelets and analyzed platelets alone to determine their specific mRNA contributions. The groups—StdMo, PdMo, PdMo + Plts, and platelets alone—were then exposed to 2 ng/ml of Ultrapure LPS (from E. coli O111:B4) for 4.5 hours, or stimulated with LPS for 3 hours followed by inflammasome activation with Nigericin (10 µM) for 90 min before extracting RNA for analysis.

2024-05-31 | E-MTAB-14072 | biostudies-arrayexpress

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