Project description:A platelet proteomic approach to reveal the regulatory network by which AEE inhibits platelet activation, thereby preventing thrombosis.
Project description:Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibits platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen related peptide (CRP-XL). We identified 1634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. 925 of these peptides show an increase of more than 2-fold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on a number of proteins including Syk, filamin and integrins. Adhesion and spreading on fibrinogen mediated by the major platelet integrin IIb3 is blocked by inhibition of deubiquitylases. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.
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.
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.
Project description:Genome-wide platelet transcriptomics is increasingly being used to uncover new aspects of platelet biology and as a diagnostic and prognostic tool. Nevertheless, platelet isolation methods for transcriptomic studies are not standardized, introducing challenges for cross-study comparisons, data integration, and replication. In this prospective multicenter study, we assessed how three of the most commonly-used platelet isolation protocols influence metrics from next-generation bulk RNA sequencing and functional assays. Compared with washing alone, more stringent removal of leukocytes by anti-CD45+ beads or PALL filters resulted in sufficient quantity of RNA for next-generation sequencing and similar quality of RNA sequencing metrics. The more stringent removal of leukocytes resulted in lower relative expression of known leukocyte-specific genes and higher relative expression of known platelet-specific genes. The results were consistent across enrolling sites, suggesting that the techniques are transferrable and reproducible. The use of anti-CD45+ beads reduced integrin aIIbb3 activation to PAR-1 activating peptide (SFLLRN-TRAP) but not ADP, compared to washing alone, while the isolation method had no influence on basal platelet reactivity. In conclusion, genome-wide transcriptional and functional assays in platelets are influenced by isolation technique. These results should help the research community make informed choices about platelet isolation techniques in their own platelet studies.
Project description:Here we suggest that NM1 regulation of oxidative phosphorylation in mitochondria has an effect on hematopoietic progenitor stem cells during their differentiation to terminal blood and bone marrow stromal cells. Deletion of NM1 in the bone marrow tissue leads to differential gene expression associated with platelet activation and blood coagulation; immune system response and osteoclast differentiation. The platelet activation gene programs which are dependent on glycolysis are upregulated in NM1 KO tissue, while lymphocyte and osteoclast differentiation which is dependent on oxidative phosphorylation is suppressed in NM1 KO bone marrow.
Project description:Pulmonary hypertension (PH) patients typically present with a diminished platelet count, but the role of platelets in the development and progression of PH remains unclear.Transcriptomic analysis revealed that platelets from PH patients exhibited an upregulation of genes associated with cellular adhesion, platelet activation, and adhesion. Notably, the hub genes, glycoprotein IIb/IIIa (GP IIb/IIIa), were implicated in mediating platelet-endothelium adhesion through their interaction with intercellular adhesion molecule-1 (ICAM-1) on pulmonary artery endothelial cells, triggering platelet activation and the subsequent release of platelet-derived growth factor BB (PDGF-BB).
Project description:Renowned for their role in hemostasis and thrombosis, platelets are increasingly recognized for their involvement in innate immunity, immunothrombosis and inflammatory diseases. Platelets, heterogeneous in size and molecular content, express a wide range of receptors enabling versatile activation endpoints relevant for immune functions. Upon activation, platelet release extracellular vesicles (PEVs), whose formation and molecular cargo exhibit remarkable tunability based on receptor-mediated activation or environmental cues. This study aimed to compare the immune modulatory roles of PEVs released through activation by specific platelet receptors, GPVI, CLEC-2, and thrombin-collagen (TC). Functional assays in vivo in zebrafish and using an in vitro human macrophage models highlighted distinct immune-inflammatory responses triggered by PEVs. Surprisingly, despite extensive characterization using omics analyses and basic EV particle characterization, the differences in the protein and miRNA cargo and the EV physicochemical properties between the PEV types were subtle and insufficient to precisely define their mechanism of action to distinguish a distinct functional profile. Moreover, the constitutively formed PEVs used as controls displayed a disparate activation profile from the receptor-induced PEVs.
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.