Platelet extracellular vesicles and the transfer of microRNA in atherosclerosis
thesisposted on 15.08.2016, 14:12 by Ashley Robert Ambrose
On activation, platelets release two types of extracellular vesicle (EV); procoagulant microvesicles and exosomes. Platelets contain abundant microRNAs which can be packaged into EV and released into the blood, making a significant contribution to circulating microRNA. We aimed to characterise the EV released from platelets, profile their microRNA content and then identify and observe the effects of the EV microRNA on targets in monocytes. Washed platelets from healthy subjects were maximally stimulated with agonists specific for GPVI (CRP-XL), PAR1 (SFFLRN), PAR4 (AYGPKF), P2Y1/P2Y12 (ADP). Released EVs isolated by differential centrifugation were characterised by size (Nanosight), the exosome-specific markers CD63 & HSP70 (western blotting), and the procoagulant characteristics of microvesicles (Annexin-V binding and thrombin generation). RNA was isolated from EV populations, reverse transcribed, amplified, and the microRNA profiled on TaqMan microRNA microarrays. Targets for platelet-derived EV (pdEV) were then identified using a detailed bioinformatic approach, and the effect of the pdEV on the protein expression of these targets in monocytes was investigated. Stimulation through GPVI was the most potent generator of pdEV and produced a mixed population of exosomes and microvesicles. Stimulation at the PAR and P2Y receptors resulted in predominantly exosome production. Degradation of ADP with apyrase, significantly reduced both microvesicles and exosome production following stimulation with all agonists. The pdEVs contained between 57-79 different miRNA with a core of 45 miRNA observed throughout. From these 45 microRNA we identified 3592 predicted targets and 811 previously validated targets. Incorporation of platelet-monocyte interaction transcriptome datasets identified 34 targets in monocytes with strong evidence for regulation by pdEV microRNA. Addition of pdEV to whole blood revealed that they were preferentially targeted to monocytes and incubating them with primary monocytes and THP-1 cells demonstrated their uptake (flow cytometry and confocal microscopy) and the transfer of their microRNA cargo (RT-PCR). Critically, the delivery of microRNA via pdEV caused reduced protein expression of two selected targets; the haematopoietic transcription factor LMO2 and the scavenger receptor SCARB1 in culture. Together, these results indicate that platelets produce EV in an agonist-dependent manner which is reliant on secondary ADP signalling. The microRNA profile released is consistent regardless of agonist and comprises a group of microRNA with many potential interactions in monocytes. The pdEV are preferentially taken up by monocytes in the blood where they are able to alter protein expression of targets such as SCARB1, which has potential implications in the development of atherosclerosis.