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The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

ObjectiveDietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis.Approach and resultsDGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets.ConclusionsThis study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner.