12-Hepe Regulates the Antiplatelet Effects of Epa, the ω-3 Fatty Acid

Fish oil supplements containing omega-3 polyunsaturated fatty acids (PUFAs) are popular over-the-counter supplements due to their ability to reduce the risk for cardiovascular disease. Fish oil supplements are enriched with the omega-3 fatty acid, eicosapentaenoic acid (EPA). Supplementation with EPA alone provides cardiovascular protection, and the recent REDUCE-IT trial demonstrated the effectiveness of icosapent ethyl, a modified form of EPA, at reducing the risk of major cardiovascular events in at risk patients. Despite the wide use of dietary supplements containing high levels of EPA, the mechanisms regulating the cardiovascular protective effects of EPA remain unclear. The previous hypothesis was that high levels of EPA reduced the production of arachidonic acid (AA) metabolites, the most abundant proplatelet fatty acid, by competing at the oxygenase enzymes, however our group has shown the antiplatelet effects of several PUFAs are regulated through their 12-lipoxygease (12-LOX) metabolites. Therefore, we set out to determine whether the cardiovascular protective effects observed in individuals taking dietary supplements with EPA are realized by altering platelet function, and if these effects are mediated through the 12-lipoxygenase derived metabolite, 12-hydroxyeicosapentaenoic acid (12-HEPE). To determine if the presence of EPA alters the production of 12-LOX and cyxlooxygnease-1 (COX-1) metabolites, the lipid releasate from platelets treated with EPA and stimulated with collagen was analyzed via mass spectrometry. Production of 12-HEPE, the 12-LOX metabolite of EPA, is significantly increased in the presence of EPA, while COX-1 derived metabolites of EPA remain undetected. The presence of EPA does not alter levels of the 12-LOX and COX-1 derived metabolites of arachidonic acid. To assess the ability of EPA and 12-HEPE to alter platelet activation, isolated platelets from healthy human donors were treated with EPA or 12-HEPE and stimulated with various agonists targeting different steps of the hemostatic response to vascular injury. Both EPA and 12-HEPE dose-dependently inhibit collagen and thrombin-induced platelet aggregation with 12-HEPE having increased potency compared to EPA. Furthermore, 12-HEPE is a more potent inhibitor of surface expression of platelet integrin αIIbβ3 activation and surface exposure of P-selectin analyzed via flow cytometry in comparison to EPA. Additionally, EPA fully inhibits thrombus formation in whole blood under arterial shear via Total Thrombus formation Analysis System (T-TAS), while 12-HEPE only partially inhibits thrombus formation. Similarly, only EPA attenuates platelet adhesion to collagen in whole blood under arterial shear, while 12-HEPE has no effect. Our findings show for the first time 12-HEPE, the 12-LOX metabolite of EPA, is the most abundant metabolite produced by platelets when activated in the presence of EPA, suggesting the effects of EPA on platelets are regulated by 12-HEPE and are not by reducing the production of proplatelet arachidonic acid derived metabolites. We also show both EPA and 12-HEPE directly inhibit platelet activation, and 12-HEPE has more potent antiplatelet effects in isolated platelets. Interestingly, in whole blood, 12-HEPE has a reduced effect compared to EPA, suggesting 12-HEPE may be unstable or bound by circulating proteins in whole blood, making the local production of 12-HEPE within the platelet critical to its antiplatelet effect. These findings provide further insight into the mechanisms underlying the cardioprotective effects of EPA. A better understanding of current PUFA supplements containing EPA can inform treatment and prevention of cardiovascular diseases.