In this paper, human platelet 12-lipoxygenase [h12-LOX (ALOX12)], human reticulocyte 15-lipoxygenase-1 [h15-LOX-1 (ALOX15)], and human epithelial 15-lipoxygenase-2 [h15-LOX-2 (ALOX15B)] were observed to react with docosahexaenoic acid (DHA) and produce 17S-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17S-HpDHA). The k_cat/K_M values with DHA for h12-LOX, h15-LOX-1, and h15-LOX-2 were 12, 0.35, and 0.43 s^-1 μM^-1, respectively, which demonstrate h12-LOX as the most efficient of the three. These values are comparable to their counterpart k_cat/K_M values with arachidonic acid (AA), 14, 0.98, and 0.24 s^-1 μM^-1, respectively. Comparison of their product profiles with DHA demonstrates that the three LOX isozymes produce 11S-HpDHA, 14S-HpDHA, and 17S-HpDHA, to varying degrees, with 17S-HpDHA being the majority product only for the 15-LOX isozymes. The effective k_cat/K_M values (k_cat/K_M × percent product formation) for 17S-HpDHA of the three isozymes indicate that the in vitro value of h12-LOX was 2.8-fold greater than that of h15-LOX-1 and 1.3-fold greater than that of h15-LOX-2. 17S-HpDHA was an effective substrate for h12-LOX and h15-LOX-1, with four products being observed under reducing conditions: protectin DX (PDX), 16S,17S-epoxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid (16S,17S-epoxyDHA), the key intermediate in neuroprotection D1 biosynthesis [NPD1, also known as protectin D1 (PD1)], 11,17S-diHDHA, and 16,17S-diHDHA. However, h15-LOX-2 did not react with 17-HpDHA. With respect to their effective k_cat/K_M values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective k_cat/K_M in producing 16S,17S-epoxyDHA and a 27-fold lower effective k_cat/K_M in generating PDX. This is the first direct demonstration of h15-LOX-1 catalyzing this reaction and reveals an in vitro pathway for PDX and NPD1 intermediate biosynthesis. In addition, epoxide formation from 17S-HpDHA and h15-LOX-1 was negatively affected via allosteric regulation by 17S-HpDHA (K_d = 5.9 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (K_d = 2.5 μM), and 17S-hydroxy-13Z,15E,19Z-docosatrienoic acid (17S-HDTA) (K_d = 1.4 μM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC₅₀ values of approximately 1 and 3 μM, respectively. The in vitro results presented here may help advise in vivo PDX and NPD1 intermediate (i.e., 16S,17S-epoxyDHA) biosynthetic investigations and support the benefits of DHA rich diets.

Human platelet 12-lipoxygenase (h12-LOX) is responsible for the formation of oxylipin products that play an important role in platelet aggregation. Single nucleotide polymorphisms (SNPs) of h12-LOX have been implicated in several diseases. In this study, we investigate the structural, dynamical, and functional impact of a h12-LOX SNP that generates a tyrosine-to-cysteine mutation at a buried site (Y649C h12-LOX) and was previously ascribed with reduced levels of 12(S)-hydroxyeicosatetraenoic acid (12S-HETE) production in isolated platelets. Herein, in vitro Michaelis-Menten kinetics show reduced catalytic rates for Y649C compared to WT h12-LOX at physiological or lower temperatures. Both proteins exhibited similar melting temperatures, metal content, and oligomerization state. Liposome binding for both proteins was also dependent upon the presence of calcium, temperature, and liposome composition; however, the Y649C variant was found to have lowered binding capacity to liposomes compared to WT at physiological temperatures. Further, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments revealed a regional defined enhancement in the peptide mobility caused by the mutation. This increased instability for the mutation stemmed from a change in an interaction with an arched helix that lines the substrate binding site, located ≥15 Å from the mutation site. Finally, differential scanning calorimetry demonstrated a reduced protein (un)folding enthalpy, consistent with the HDX results. Taken together, these results demonstrate remarkable similarity between the mutant and WT h12-LOX, and yet, subtle changes in activity, membrane affinity and protein stability may be responsible for the significant physiological changes that the Y649C SNP manifests in platelet biology.

Arterial thrombosis is the underlying cause for a number of cardiovascular-related events. Although dietary supplementation that includes polyunsaturated fatty acids (PUFAs) has been proposed to elicit cardiovascular protection, a mechanism for antithrombotic protection has not been well established. The current study sought to investigate whether an omega-6 essential fatty acid, docosapentaenoic acid (DPAn-6), and its oxidized lipid metabolites (oxylipins) provide direct cardiovascular protection through inhibition of platelet reactivity. Human and mouse blood and isolated platelets were treated with DPAn-6 and its 12-lipoxygenase (12-LOX)-derived oxylipins, 11-hydroxy-docosapentaenoic acid and 14-hydroxy-docosapentaenoic acid, to assess their ability to inhibit platelet activation. Pharmacological and genetic approaches were used to elucidate a role for DPA and its oxylipins in preventing platelet activation. DPAn-6 was found to be significantly increased in platelets following fatty acid supplementation, and it potently inhibited platelet activation through its 12-LOX-derived oxylipins. The inhibitory effects were selectively reversed through inhibition of the nuclear receptor peroxisome proliferator activator receptor-α (PPARα). PPARα binding was confirmed using a PPARα transcription reporter assay, as well as PPARα-/- mice. These approaches confirmed that selectivity of platelet inhibition was due to effects of DPA oxylipins acting through PPARα. Mice administered DPAn-6 or its oxylipins exhibited reduced thrombus formation following vessel injury, which was prevented in PPARα-/- mice. Hence, the current study demonstrates that DPAn-6 and its oxylipins potently and effectively inhibit platelet activation and thrombosis following a vascular injury. Platelet function is regulated, in part, through an oxylipin-induced PPARα-dependent manner, suggesting that targeting PPARα may represent an alternative strategy to treat thrombotic-related diseases.

Postmenopausal women are at increased risk for a cardiovascular event due to platelet hyperactivity. There is evidence suggesting that ω-3 polyunsaturated fatty acids (PUFAs) and ω-6 PUFAs have cardioprotective effects in these women. However, a mechanistic understanding of how these fatty acids regulate platelet function is unknown. In this study, we supplemented postmenopausal women with fish oil (ω-3 fatty acids) or evening primrose oil (ω-6 fatty acids) and investigated the effects on their platelet activity. The effects of fatty acid supplementation on platelet aggregation, dense granule secretion, and activation of integrin αIIbβ3 at basal levels and in response to agonist were tested in postmenopausal women following a supplementation and washout period. Supplementation with fish oil or primrose oil attenuated the thrombin receptor PAR4-induced platelet aggregation. Supplementation with ω-3 or ω-6 fatty acids decreased platelet dense granule secretion and attenuated basal levels of integrin αIIbβ3 activation. Interestingly, after the washout period following supplementation with primrose oil, platelet aggregation was similarly attenuated. Additionally, for either treatment, the observed protective effects post-supplementation on platelet dense granule secretion and basal levels of integrin activation were sustained after the washout period, suggesting a long-term shift in platelet reactivity due to fatty acid supplementation. These findings begin to elucidate the underlying mechanistic effects of ω-3 and ω-6 fatty acids on platelet reactivity in postmenopausal women. Hence, this study supports the beneficial effects of fish oil or primrose oil supplementation as a therapeutic intervention to reduce the risk of thrombotic events in postmenopausal women. https://clinicaltrials.gov/ct2/show/NCT02629497.

The oxylipins, 5S,12S-dihydroxy-6E,8Z,10E,14Z-eicosatetraenoic acid (5S,12S-diHETE) and 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5S,15S-diHETE), have been identified in cell exudates and have chemotactic activity toward eosinophils and neutrophils. Their biosynthesis has been proposed to occur by sequential oxidations of arachidonic acid (AA) by lipoxygenase enzymes, specifically through oxidation of AA by h5-LOX followed by h12-LOX, h15-LOX-1, or h15-LOX-2. In this work, h15-LOX-1 demonstrates altered positional specificity when reacting with 5S-HETE, producing 90% 5S,12S-diHETE, instead of 5S,15S-diHETE, with kinetics 5-fold greater than that of h12-LOX. This is consistent with previous work in which h15-LOX-1 reacts with 7S-HDHA, producing the noncanonical, DHA-derived, specialized pro-resolving mediator, 7S,14S-diHDHA. It is also determined that oxygenation of 5S-HETE by h15-LOX-2 produces 5S,15S-diHETE and its biosynthetic k_cat/K_M flux is 2-fold greater than that of h15-LOX-1, suggesting that h15-LOX-2 may have a greater role in lipoxin biosynthesis than previously thought. In addition, it is shown that oxygenation of 12S-HETE and 15S-HETE by h5-LOX is kinetically slow, suggesting that the first step in the in vitro biosynthesis of both 5S,12S-diHETE and 5S,15S-diHETE is the production of 5S-HETE.

Background

The effects of docosahexaenoic acid (DHA) on cardiovascular disease are controversial and a mechanistic understanding of how this omega-3 polyunsaturated fatty acid (ω-3 PUFA) regulates platelet reactivity and the subsequent risk of a thrombotic event is warranted. In platelets, DHA is oxidized by 12-lipoxygenase (12-LOX) producing the oxidized lipids (oxylipins) 11-HDHA and 14-HDHA. We hypothesized that 12-LOX DHA-oxylipins may be involved in the beneficial effects observed in dietary supplemental treatment with ω-3 PUFAs or DHA itself.

Objectives

To determine the effects of DHA, 11-HDHA, and 14-HDHA on platelet function and thrombus formation, and to elucidate the mechanism by which these ω-3 PUFAs regulate platelet activation.

Methods and results

DHA, 11-HDHA, and 14-HDHA attenuated collagen-induced human platelet aggregation, but only the oxylipins inhibited ⍺IIbβ3 activation and decreased ⍺-granule secretion. Furthermore, treatment of whole blood with DHA and its oxylipins impaired platelet adhesion and accumulation to a collagen-coated surface. Interestingly, thrombus formation was only diminished in mice treated with 11-HDHA or 14-HDHA, and mouse platelet activation was inhibited following acute treatment with these oxylipins or chronic treatment with DHA, suggesting that under physiologic conditions, the effects of DHA are mediated through its oxylipins. Finally, the protective mechanism of DHA oxylipins was shown to be mediated via activation of protein kinase A.

Conclusions

This study provides the first mechanistic evidence of how DHA and its 12-LOX oxylipins inhibit platelet activity and thrombus formation. These findings support the beneficial effects of DHA as therapeutic intervention in atherothrombotic diseases.

The antiplatelet effect of polyunsaturated fatty acids is primarily attributed to its metabolism to bioactive metabolites by oxygenases, such as lipoxygenases (LOX). Platelets have demonstrated the ability to generate 15-LOX-derived metabolites (15-oxylipins); however, whether 15-LOX is in the platelet or is required for the formation of 15-oxylipins remains unclear. This study seeks to elucidate whether 15-LOX is required for the formation of 15-oxylipins in the platelet and determine their mechanistic effects on platelet reactivity. In this study, 15-HETrE, 15-HETE, and 15-HEPE attenuated collagen-induced platelet aggregation, and 15-HETrE inhibited platelet aggregation induced by different agonists. The observed anti-aggregatory effect was due to the inhibition of intracellular signaling including αIIbβ3 and protein kinase C activities, calcium mobilization, and granule secretion. While 15-HETrE inhibited platelets partially through activation of peroxisome proliferator-activated receptor β (PPARβ), 15-HETE also inhibited platelets partially through activation of PPARα. 15-HETrE, 15-HETE, or 15-HEPE inhibited 12-LOX in vitro, with arachidonic acid as the substrate. Additionally, a 15-oxylipin-dependent attenuation of 12-HETE level was observed in platelets following ex vivo treatment with 15-HETrE, 15-HETE, or 15-HEPE. Platelets treated with DGLA formed 15-HETrE and collagen-induced platelet aggregation was attenuated only in the presence of ML355 or aspirin, but not in the presence of 15-LOX-1 or 15-LOX-2 inhibitors. Expression of 15-LOX-1, but not 15-LOX-2, was decreased in leukocyte-depleted platelets compared to non-depleted platelets. Taken together, these findings suggest that 15-oxylipins regulate platelet reactivity; however, platelet expression of 15-LOX-1 is low, suggesting that 15-oxylipins may be formed in the platelet through a 15-LOX-independent pathway.