Objective Dietary supplementation with polyunsaturated fatty acids (PUFAs) has been widely used for primary and secondary prevention of CVD in individuals at risk; however, the cardioprotective benefits of PUFAs remain controversial due to lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 PUFA, 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)-HETrE, inhibiting platelet activation and thrombosis. Approach and Results DGLA 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) following 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, two hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis following 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked GPCR pathway in human platelets. Conclusions This study provides the first direct evidence that an omega-6 PUFA, 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 Gαs-linked GPCR-dependent manner.

The reaction of 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 LXA 4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB 4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are k cat = 0.17 s-1 and k cat /K M = 0.011 μM-1s-1 (106-fold and 1600-fold lower than for 12-LOX oxygenation of AA, respectively). On the other hand, for 15-LOX-1 the equivalent parameters are k cat = 4.6 s −1 and k cat /K M = 0.21 μM-1s-1 (3-fold higher and similar to that 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 (Biochemistry 55, 2832-2840, 2016). Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB 4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM, however, LXB 4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB 4 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. LXA 4 is the primary product with 5-LOX, but only if 15-HpETE is the substrate. Approximately equal proportions of LXA 4 and LXB 4 are produced by 12-LOX, but only if LTA 4 is the substrate, as described previously (Biochimica et Biophysica Acta 1133, 223-234, 1992).

Lipoxins are an important class of lipid mediators that induce the resolution of inflammation and arise from transcellular exchange of arachidonic acid (AA)-derived lipoxygenase products. Human epithelial 15-lipoxygenase-2 (h15-LOX-2), the major lipoxygenase in macrophages, has exhibited strict regiospecificity, catalyzing only the hydroperoxidation of carbon 15 of AA. To determine the catalytic potential of h15-LOX-2 in transcellular synthesis events, we reacted it with the three lipoxygenase-derived monohydroperoxy-eicosatetraenoic acids (HPETE) in humans: 5-HPETE, 12-HPETE, and 15-HPETE. Only 5-HPETE was a substrate for h15-LOX-2, and the steady-state catalytic efficiency (kcat/Km) of this reaction was 31% of the kcat/Km of AA. The only major product of h15-LOX-2’s reaction with 5-HPETE was the proposed lipoxin intermediate, 5,15-dihydroperoxy-eicosatetraenoic acid (5,15-diHPETE). However, h15-LOX-2 did not react further with 5,15-diHPETE to produce lipoxins. This result is consistent with the specificity of h15-LOX-2 despite the increased reactivity of 5,15-diHPETE. Density functional theory calculations determined that the radical, after abstracting the C10 hydrogen atom from 5,15-diHPETE, had an energy 5.4 kJ/mol lower than that of the same radical generated from AA, demonstrating the facility of 5,15-diHPETE to form lipoxins. Interestingly, h15-LOX-2 does react with 5S,6R-diHETE, forming LipoxinA4, indicating the gemdiol does not prohibit h15-LOX-2 reactivity. Taken together, these results demonstrate the strict regiospecificity of h15-LOX-2 that circumscribes its role in transcellular synthesis.