Effect of Genetic Factors, Age and Sex on Levels of Circulating Extracellular Vesicles and Platelets

Figures

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  Figure 1. Gating strategy to identify EVs and platelets. (a) Counting beads (red) were identified based on their high fluorescence for FITC and PE fluorochromes; (b) the phalloidin negative area (blue) contained both extracellular vesicles and platelets. The EV area (violet) was identified by intersecting the lipophilic cationic dye (LCD) positive EV area in (c) with the EV area morphologically assessed in (d). Platelets (green) were identified by intersecting platelets morphologically detected in (d) with those identified by CD31 and CD41a positivity in (e). (e) EVs double positive for CD31 and CD41a were considered of platelet origin and further divided into CD31dim and CD31hi; EVs positive for CD31 but negative for CD41a were considered of endothelial origin. EVs positive for CD45, CD171 and CD11b were considered leukocyte (f), neuronal (g), and glial (h) origins, respectively. In (f,g), we also gated CD45hi and CD171hi events, respectively, that could be either EVs expressing high levels of these specific markers or crystals. The comparison between the size of EVs (i) and of the Megamix beads (j) is shown.

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  Figure 2. Most relevant age–sex tendencies of EVs and platelets. Each scatter plot represented males in blue and females in pink, age in the x axis, and normalized trait in the y axis.

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  Figure 3. Regional association plots of the most significant identified signals. The significance of the association (−log10[p value]; left y axis) for each trait was plotted relative to the genomic positions on the hg19/GRCh37 genomic build (x axis). SNPs were colored to reflect their linkage disequilibrium with the top SNP (indicated with a purple dot and with its genomic position at the built GRCh37/hg19).