12-HETrE regulation of platelets

描述（由申请人提供）：已知由加氧酶形成的生物活性脂质在循环中具有保护和促血栓作用。12-脂氧合酶（12-LOX）及其氧化产物在血小板功能调控中起着重要但尚未明确的作用。脂肪酸二homo-?的12-LOX氧化-亚麻酸（DGLA）产生新的生物活性代谢物12-羟基二碳四烯酸（12-HETrE）。初步数据表明，12- HETrE在血小板中起保护作用，以限制活化。本研究探讨了DGLA是否通过产生12-HETrE抑制血小板活化。由于DGLA在细胞膜磷脂中高度表达，并且12-LOX代谢物可以以¿M浓度释放到循环中，描绘这种以前未知的代谢物调节细胞活性的机制对于开始了解DGLA的12-LOX氧化如何可能导致许多生理过程的调节至关重要，包括血栓形成，炎症，免疫和肿瘤生长的调节。为了解决这些问题，本应用程序提出：1)描述12-HETrE在血小板中的作用机制。初步数据表明，12-HETrE可改变cAMP水平，并支持受体介导的血小板功能调节。此外，我们最近的工作表明，Rap1活性受到DGLA和12- HETrE的负调控，初步数据表明，Rap1活性受到cAMP的减弱。12-HETrE调节细胞活性的作用模式将通过确定其是否a)以gpcr依赖的方式起作用，b)可以直接抑制血小板中的凝血酶和胶原受体信号传导，以及c)通过抑制Rap1介导其在血小板中的作用，以调节血小板中的生理终点（颗粒分泌、整合素激活、细胞粘附和血小板聚集）来确定。的重要性

DESCRIPTION (provided by applicant): 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).