Formation of bioactive lipids by oxygenases is known to play both a protective and pro-thrombotic role in circulation. 12-lipoxygenase (12-LOX) and its oxidized products play an important but unresolved role in regulation of platelet function. 12-LOX oxidation of the fatty acid, dihomo-?-linolenic acid (DGLA), produces the novel bioactive metabolite 12-hydroxyeicosatetrienoic acid (12-HETrE). Preliminary data suggests that 12- HETrE acts in a protective manner in platelets to limit activation. This proposal investigates if DGLA inhibits platelet activation through the production of 12-HETrE. Since DGLA is highly expressed in the phospholipids of the cell membrane and 12-LOX metabolites can be released into circulation in ?M concentrations, delineating the mechanism by which this previously unknown metabolite regulates cellular activity is essential to begin to understand how 12-LOX oxidation of DGLA can potentially lead to regulation of a number of physiological processes including thrombosis, inflammation, immunity and regulation of tumor growth. To address these questions, this application proposes to 1) delineate the mechanisms(s) of action of 12-HETrE in platelets. Preliminary data suggests 12-HETrE alters cAMP levels and is supportive of receptor-mediated regulation of platelet function. Additionally, our recent work showed Rap1 activity is negatively regulated by DGLA and 12- HETrE and preliminary data suggests Rap1 activity is attenuated by cAMP. The mode of action for 12-HETrE regulation of cellular activity will be determined by identifying if it a) functions in a GPCR-dependent manner, b) can directly inhibit thrombin and collagen receptor signaling in the platelet, and c) mediates its actions in the platelet via inhibition of Rap1 in order to regulate physiological endpoints (granule secretion, integrin activation, cell adhesion, and platelet aggregation) in the platelet. 2) The importance of DGLA and 12-HETrE in regulation of hemostasis and thrombosis in vivo will also be assessed in wild-type mice and 12-LOX-/- mice who are unable to produce 12-HETrE. Preliminary data supports an important role for 12-LOX in production of 12- HETrE from DGLA and dietary studies suggesting that increasing DGLA intake results in deficits in platelet function. The abilit of DGLA supplementation to protect vessels from occlusive thrombus formation will be assessed in these mice. Finally, 3) the role of 12-LOX in DGLA product formation and its importance to DGLA- mediated effects in the cell will be determined by characterizing DGLA effects in the presence of a potent 12- LOX inhibitor and mutant 12-LOX enzymes as well as determining the physiological effect of 12-HETrE in the release of cells partially or fully deficiet in functional 12-LOX. Understanding the mechanism by which 12- HETrE elicits its effects on cellular activation will give insight into the potential role of ?-6 fatty acid regulation of the vssel. Hence, these studies will fill a significant gap in understanding how fatty acids such as DGLA can impinge upon the regulation of a number of physiological and pathophysiological processes through its 12-LOX oxidized metabolite(s).

Public Health Relevance

12(S)-HETrE is a novel metabolite produced from 12-lipoxygenase oxidation of the fatty acid DGLA. Once formed, 12(S)-HETrE negatively regulates platelet activity. Elucidating the mechanism by which 12(S)-HETrE regulates cell activity will increase our understanding of how DGLA protects the vessel in vivo.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Dunsmore, Sarah
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Thomas Jefferson University
Internal Medicine/Medicine
Schools of Medicine
United States
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Tourdot, Benjamin E; Ahmed, Intekhab; Holinstat, Michael (2014) The emerging role of oxylipins in thrombosis and diabetes. Front Pharmacol 4:176
Luci, Diane K; Jameson 2nd, J Brian; Yasgar, Adam et al. (2014) Synthesis and structure-activity relationship studies of 4-((2-hydroxy-3-methoxybenzyl)amino)benzenesulfonamide derivatives as potent and selective inhibitors of 12-lipoxygenase. J Med Chem 57:495-506
Yeung, Jennifer; Tourdot, Benjamin E; Fernandez-Perez, Pilar et al. (2014) Platelet 12-LOX is essential for Fc?RIIa-mediated platelet activation. Blood 124:2271-9