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:Circulating tumor cell clusters/micro-emboli (CTM) possess greater metastatic capacity and survival advantage compared to individual circulating tumor cell (CTCs). However, the formation of CTM subtypes and their role in tumor metastasis remain unclear. In this study, we used a microfluidic Cluster-chip with easy operation and high efficiency to isolate CTM from peripheral blood, which confirmed their correlation with clinicopathological features and identified the critical role of CTC-platelet clusters in BC metastasis. The correlation between platelets and CTM function was further confirmed in a mouse model and RNA-seq of CTM identified high-expressed genes related to hypoxia stimulation and platelet activation which possibly suggested the correlation of hypoxia and CTC-platelet cluster formation. In conclusion, we successfully developed the Cluster-chip platform to realize the clinical capture of CTMs and analyze the biological properties of CTC-platelet clusters, which could benefit the design of potential treatment regimens to prevent CTM-mediated metastasis and tumor malignant progression.
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: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: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:Background and Aims: Recent studies have implicated platelets, particularly α-granules, in the development of steatohepatitis (NASH). However, the specific mechanisms involved have yet to be determined. Notably, thrombospondin 1 (TSP1) is a major component of the platelet α-granules released during platelet activation. The role of platelet-derived TSP1 in NASH remains unknown and was investigated in this study. Approach and Results: Platelet-specific TSP1 knockout mice (TSP1Δpf4) and their wild type littermates (TSP1F/F) were used. NASH was induced by feeding the mice a diet enriched in fat, sucrose, fructose, and cholesterol (AMLN diet). A human liver NASH organoid model was also employed. Although TSP1 deletion in platelets did not affect diet-induced steatosis, TSP1Δpf4 mice exhibited attenuated NASH and liver fibrosis, accompanied by improvements in plasma glucose and lipid homeostasis. Moreover, intrahepatic platelet accumulation, activation and chemokine production were reduced in TSP1Δpf4 mice, which was correlated with decreased immune cell infiltration into the liver. Consequently, this leads to diminished pro-inflammatory signaling in the liver and mitigated the progression of NAFLD. Moreover, in vitro data revealed that co-culturing TSP1-deficient platelets in a human liver NASH organoid model attenuated hepatic stellate cell activation and NASH progression. Additionally, TSP1 deficient platelets play a role in regulating brown fat endocrine function, specifically affecting Nrg4 production. Crosstalk between brown fat and the liver may also influence NAFLD progression. Conclusions: These data suggest that platelet α-granule-derived TSP1 is a significant contributor to diet-induced NASH and fibrosis, and may serve as a new therapeutic target for this severe liver disease.
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.
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.
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.