Investigating the catalytic efficiency of C22-Fatty acids with LOX human isozymes and the platelet response of the C22-oxylipin products

Polyunsaturated fatty acids (PUFAs) have been extensively studied for their health benefits because they can be oxidized by lipoxygenases to form bioactive oxylipins. In this study, we investigated the impact of double bond placement on the kinetic properties and product profiles of human platelet 12-lipoxygenase (h12-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), and human endothelial 15-lipoxygenase-2 (h15-LOX-2) by using 22-carbon (C22) fatty acid substrates with differing double bond content. With respect to kcat/KM values, the loss of Δ4 and Δ19 led to an 18-fold loss of kinetic activity for h12-LOX, no change in kinetic capability for h15-LOX-1, but a 24-fold loss for h15-LOX-2 for both C22-FAs. With respect to the product profiles, h12-LOX produced mainly 14-oxylipins. For h15-LOX-1, the 14-oxylipin production increased with the loss of either Δ4 and Δ19, however, the 17-oxylipin became the major species upon loss of both Δ4 and Δ19. h15-LOX-2 produced mostly the 17-oxylipin products throughout the fatty acid series. This study also investigated the effects of various 17-oxylipins on platelet activation. The results revealed that both 17(S)-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-DHA (17-HDHA) and 17-hydroxy-4Z,7Z,10Z,13Z,15E-DPAn6 (17-HDPAn6) demonstrated anti-aggregation properties with thrombin or collagen stimulation. 17-hydroxy-7Z,10Z,13Z,15E,19Z-DPAn3 (17-HDPAn3) exhibited agonistic properties, and 17-hydroxy-7Z,10Z,13Z,15E-DTA (17-HDTA) showed biphasic effects, inhibiting collagen-induced aggregation at lower concentrationsbut promoting aggregation at higher concentrations. Both 17-hydroxy-13Z,15E,19Z-DTrA (17-HDTrA), and 17-hydroxy-13Z,15E-DDiA (17-HDDiA) induced platelet aggregation. In summary, the number and placement of the double bonds affect platelet activation, with the general trend being that more double bonds generally inhibit aggregation, while less double bonds promote aggregation. These findings provide insights into the potential role of specific fatty acids and their metabolizing LOX isozymes with respect to cardiovascular health

Supplementation with omega‐3 or omega‐6 fatty acids attenuates platelet reactivity in postmenopausal women

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

DHA 12- LOX- derived oxylipins regulate platelet activation and thrombus formation through a PKA- dependent signaling pathway

BackgroundThe effects of docosahexaenoic acid (DHA) on cardiovascular disease are controversial and a mechanistic understanding of how this omega- 3 polyunsaturated fatty acid (Ï - 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 Ï - 3 PUFAs or DHA itself.ObjectivesTo determine the effects of DHA, 11- HDHA, and 14- HDHA on platelet function and thrombus formation, and to elucidate the mechanism by which these Ï - 3 PUFAs regulate platelet activation.Methods and resultsDHA, 11- HDHA, and 14- HDHA attenuated collagen- induced human platelet aggregation, but only the oxylipins inhibited - ºIIbβ3 activation and decreased - º- 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.ConclusionsThis 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.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/167023/1/jth15184-sup-0001-Supinfo.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167023/2/jth15184.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/167023/3/jth15184_am.pd