Comparison of the release of microRNAs and extracellular vesicles from platelets in response to different agonists

<p>On activation platelets release microRNAs and extracellular vesicles (EV) into circulation. The release of EV from platelets has been shown to be dependent on the agonist; in this study, we investigated whether the microRNA profile or EV released from platelets was also agonist specific.</p> <p>Washed platelets from healthy subjects were maximally stimulated with agonists specific for the receptors for collagen (Glycoprotein VI (GPVI)), thrombin (PAR1/PAR4), or ADP (P2Y1/P2Y12) with/without inhibiting secondary mediators, using aspirin to block cyclooxygenase-1 and apyrase to remove ADP. The released microRNAs were profiled using TaqMan microRNA microarray cards. Platelet-derived EV (pdEV) were characterized by size (Nanoparticle Tracking Analysis, NTA), for procoagulant activity (Annexin-V binding and support of thrombin generation), and for the EV markers CD63 and HSP70.</p> <p>Platelet activation triggered the release of 57–79 different microRNAs, dependent upon agonist, with a core of 46 microRNAs observed with all agonists. There was a high level of correlation between agonists (<i>r</i>² > 0.98; <i>p</i> < 0.0001 for all), and with the microRNA content of the parent platelets (<i>r</i>² > 0.98; <i>p</i> < 0.0001). The 46 microRNAs seen in all samples are predicted to have significant effects on the translation of proteins involved in endocytosis, cell cycle control, and differentiation. MiR-223-3p was the most abundant in all samples and has previously been implicated in myeloid lineage development and demonstrated to have anti-inflammatory effects. Stimulation through GPVI produced a pdEV population with significantly more procoagulant activity than the other agonists. Apyrase significantly reduced microRNA and pdEV release, while aspirin had little effect.</p> <p>These data suggest that all tested agonists trigger the release of a similar microRNA profile while the procoagulant activity of the pdEV was agonist dependent. ADP was shown to play an important role in the release of both microRNAs and pdEV.</p

Size range of microvesicles as measured by nanoparticle tracking analysis.

<p>Data are shown as the mean±SD for haemodialysis (HD) patients, peritoneal dialysis (PD) patients and healthy controls (HC).</p

The effects of microvesicles in patients’ plasma on thrombin generation.

<p>(A) Representative thrombin generation curves for each subject group (B) Peak thrombin, (in nM.min^-1) (C) Endogenous thrombin potential (in nM thrombin. min^-1) and (D) Lag time (in mins), before (dark grey bars) and after (light grey bars) removal of MVs by filtration through a 0.22µM filter. Data are shown as the mean±SD for haemodialysis (HD) patients, peritoneal dialysis (PD) patients and healthy controls (HC).</p

Example electron micrographs demonstrating the presence of microvesicles (MVs) prepared from normal plasma.

<p>(A) Negatively stained transmission electron micrograph showing two MVs of approximately 400nm and 500nm and (B) an embedded and sectioned transmission electron micrograph of an MV showing a bilayer appearance lacking any intracellular structures.</p

Measurement of thrombin generation in restored microvesicle controls.

<p>Peak thrombin (A) and endogenous thrombin potential (B) were measured post-filtration (light grey bars) and in restored MV controls (dark grey bars), and are expressed as a percentage of pre-filtered levels (black bars). HD, haemodialysis; PD, peritoneal dialysis; HC, healthy control.</p

Representative Western blot results demonstrating the presence of both CD144+ ve endothelial (A) and CD42b+ ve platelet (B) derived MVs. LC, lysate control; HD, haemodialysis; PD, peritoneal dialysis; HC, healthy control.

<p>Representative Western blot results demonstrating the presence of both CD144+ ve endothelial (A) and CD42b+ ve platelet (B) derived MVs. LC, lysate control; HD, haemodialysis; PD, peritoneal dialysis; HC, healthy control.</p

Relative concentrations of endothelial microvesicles (A) and platelet microvesicles (B) in dialysis patients compared to healthy controls.

<p>Graphs show relative optical density measurements against a standardised endothelial or platelet lysate control in each of the three subject groups. Horizontal bars represent the mean (A) or median (B). HD, haemodialysis; PD, peritoneal dialysis; HC, healthy control.</p

Illustration of a region with a SNP from genome wide association studies (GWAS) which is associated with ASE of lncRNAs.

<p>The tracks are from top to bottom in each panel: Horizontal red bars represent lncRNA transcript windows (with genomic coordinates) used for determination of ASE levels; grey lines show p-values for the association of GWAS SNPs with ASE levels in the transcript window; a grey line overlayed with a red dotted line indicates that a <i>cis</i>-rSNP overlaps with the reported SNP in the GWAS catalog; red vertical lines are median ASE-levels for each SNP.</p

Comparison of total expression levels of regions annotated to intergenic lncRNAs, exons, introns, and intergenic regions.

<p>The horizontal axes in the panels show bins of fluorescence signals from the genotyping data, summed for both alleles to give a measure for total expression. The average expression levels of annotated transcripts were 4900 fluorescence units in exons, 2100 in introns, 590 in intergenic regions, compared to 3300 in the intergenic lncRNA regions that were used in the ASE analysis. The vertical axes show the number of observations in each bin.</p

SNPs associated with allele-specific expression of lncRNA windows with published trait- or disease-associations from genome-wide association studies.

1<p>The distance to the lncRNA region is 0 if the SNP is located within the region and the smallest distance otherwise,</p>2<p>Slope is given in absolute numbers,</p>3<p>Listed are all cis-rSNPs that are also found in the GWAS catalog together with the associated lncRNA,</p>4<p>The trait is taken from the GWAS catalog,</p>5<p>Within 2.5 kb,</p>6<p>rs6663565 as proxy,</p>7<p>rs2303393 as proxy,</p>8<p>rs6922111 as proxy,</p>9<p>rs7739434 as proxy,</p>10<p>rs13214831 as proxy,</p>11<p>rs1153862 as proxy,</p>12<p>rs12442557 as proxy.</p