title: Effects of fish oil-derived N-3 polyunsaturated Fatty acids on the generation and functions of platelet-derived extracellular vesicles
Creators: name: Bozbas, Esra
Publisher: name: University of Reading
Year: 2022

description: Background: Extracellular vesicles (EVs) are membrane-enclosed vesicles derived from a 
wide range of cell types and these structures are suggested to have roles in the development 
of cardiovascular diseases (CVDs), thereby being considered as emerging markers of CVD.
Platelet-derived EVs (PDEVs) comprise the major EV population in the circulation and are an 
important contributor to the pathophysiology of CVDs through their procoagulant activities. N-3 polyunsaturated fatty acids (PUFAs) are abundant in oily fish and fish oil and reported to 
reduce CVD risk, but there has been little research to date examining the effects of n-3 PUFA 
on the generation and function of EVs, including those produced in vitro from platelets. 
Objective: The aim of this project was to investigate the effects of fish oil-derived n-3 PUFA
supplementation on conventional, thrombogenic and the ‘emerging’ cardiovascular risk
markers, extracellular vesicles (EVs), in subjects with moderate risk of CVDs. The generation, 
composition and procoagulant activities of EVs derived from platelets were of particular 
interest in this thesis, as well as their relationship with conventional and thrombogenic risk 
markers. 
Design: A total of 40 subjects aged 40-70 years with moderate risk for CVDs were recruited 
to a randomised, double-blind, placebo-controlled crossover intervention study. Subjects were 
supplemented with capsules containing either fish oil (1.8 g/d n-3 PUFA) or control oil (high-oleic safflower oil) for a period of 12 weeks. Effects of fish oil supplementation on 
thrombogenic risk markers, including coagulation, thrombin generation, platelet aggregation 
and fibrinolysis were investigated. Nanoparticle Tracking Analysis (NTA) and fluorescence 
flow cytometry were employed to analyse numbers of PDEVs. Total lipid fatty acid composition 
of PDEVs was analysed by gas chromatography (GS) and their coagulatory behaviour was 
assessed using a range of functional assays, including fibrin clot formation, thrombin 
generation, fibrinolysis and ex vivo thrombus formation.
Results: There was a strong relationship between numbers of circulating EVs and 
thrombogenic risk markers of CVDs. Supplementation with n-3 PUFAs resulted in significant 
modification of a range of CVD risk markers (both conventional and thrombogenic), modulation
of the fatty acid composition of in vitro-generated PDEVs from unstimulated and stimulated 
platelets, alteration of the surface expression of PS in the case of PDEVs derived from 
unstimulated platelets, a reduction in the ability of PDEVs generated in vitro from unstimulated 
and stimulated platelets to support fibrin clot formation and thrombin generation and an
accompanying improvement in fibrinolysis.
Conclusion: Circulating EVs are associated with thrombogenic activity, implicating them in 
increasing risk of CVDs. Dietary n-3 PUFA supplementation altered the fatty acid composition
of, and the surface expression of PS by, in vitro-generated PDEVs and these PDEVs were
less procoagulant, which indicates potential anti-coagulatory properties of n-3 PUFAs 
mediated through modification of PDEVs.