Abstract/Summary

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.