Platelet Derived Exosomes Are Enriched for Specific microRNAs Which Regulate WNT Signalling in Endothelial Cells
Authors: Dempsey Eugene , Dervin Feidhlim , Maguire Patricia B. 
Published: November 2019

Abstract
Activated platelets release exosomal vesicles which contain a wide range of proteins and nucleic acids including microRNAs (miRNAs) and crucially these can be delivered to other cells1. The expression of approximately 50% of genes are regulated by miRNAs, therefore, they serve as powerful modulators of signalling networks within cells. In this study, we assess the miRNAs which platelets have the potential to deliver into target cells via secreted exosomes and we particularly focus on how this influences signalling through the WNT pathway in endothelial cells.
Exosomes were isolated by differential ultracentrifugation from thrombin-activated platelet releasate and were assessed for microparticle vesicle contamination using western blotting, transmission electron microscopy and nanoparticle tracking analysis. miRNAs (372) were quantified using low density arrays using real-time PCR (Qiagen miScript miRNA PCR Arrays) from both whole platelets and isolated platelet exosomes. Identification of putative miRNA target genes was performed using multiple prediction algorithms (TargetScan, miRDB, DIANA-MicroT). Pathway analysis of the target genes was performed using ClueGO/Clupedia within Cytoscape (http://www.cytoscape.org/; version 3.01). Luciferase reporter assay and miRNA transfections in EA.hy926 endothelial cells were performed using Attractene (Qiagen). Exosome uptake by target cells was monitored using a lipophilic fluorescent stain (Dil) followed by fluorescent imaging.
We successfully quantified 370 miRNAs in platelets and 277 miRNAs specifically in platelet-derived exosomes and identified a 32 miRNA signature which was preferentially enriched (>2 fold, p<0.05) in secreted exosomes when compared to whole platelets. Some of the most highly expressed miRNAs in platelet exosomes included miR-223 and miR-328, which have both been linked to numerous human pathologies. Bioinformatically, we predicted 1453 target genes of this 32 miRNA signature, which highlighted that platelet exosomes are enriched for miRNAs which regulate key signalling networks including the WNT, TGFβ, and Hippo pathways. By transfecting miRs into endothelial cells we demonstrate that these platelet exosomal miRNAs can regulate WNT signalling in endothelial cells. In brief, they specifically modulated the cytosolic and nuclear expression of beta-catenin affecting tight junction formation and cell motility.
In conclusion, we have now established the miRNome of exosomes derived from human platelets. From our comparative analysis of whole platelets and exosomal miRNA we have determined that platelets contain both miRNAs which may specifically be required to regulate platelet function and other miRNAs which are preferentially enriched in exosomes and secreted upon activation to influence the behaviour of targeted cells. We believe this study is a key step in gaining a greater understanding of how platelets can contribute to the development of many human diseases including atherosclerosis and cancer by influencing the WNT signalling network and altering endothelial cell permeability and migration.
1. Laffont B, Corduan A, Ple H, et al. Activated platelets can deliver mRNA regulatory Ago2*microRNA complexes to endothelial cells via microparticles. Blood. 2013;122(2):253-261.

Disclosures
No relevant conflicts of interest to declare.