12-HETrE regulation of platelets

说明(申请人提供)：已知加氧酶形成生物活性脂质在循环中起保护和促进血栓形成的作用。12-脂氧合酶(12-LOX)及其氧化产物在调节血小板功能中发挥着重要但尚未解决的作用。12-LOX氧化脂肪酸--亚麻酸(DGLA)，生成新的生物活性代谢物12-羟基二十碳三烯酸(12-HETrE)。初步数据表明，12-HETrE在血小板中起保护作用，以限制激活。这项建议调查DGLA是否通过产生12-HETrE来抑制血小板活化。由于DGLA在细胞膜的磷脂中高度表达，12-LOX代谢物可以在�M浓度下释放到循环中，因此阐明这种先前未知的代谢物调节细胞活动的机制对于开始了解DGLA的12-LOX氧化如何潜在地导致包括血栓形成、炎症、免疫和调节肿瘤生长在内的许多生理过程是至关重要的。为了解决这些问题，本申请建议1)描绘12-HETrE在血小板中的作用机制(S)。初步数据显示，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的负性调节，初步数据表明，cAMP减弱了RAP1活性。12-HETrE调节细胞活性的作用模式将通过确定它是否a)以GPCR依赖的方式发挥作用，b)直接抑制血小板中的凝血酶和胶原受体信号，以及c)通过抑制Rap1介导其在血小板中的作用，从而调节血小板中的生理终点(颗粒分泌、整合素激活、细胞黏附和血小板聚集)来确定。2)重要的是 DGLA和12-HETrE对体内止血和血栓形成的调节作用也将在野生型小鼠和无法产生12-HETrE的12-LOX-/-小鼠身上进行评估。初步数据支持12-LOX在DGLA生产12-HETrE中的重要作用，饮食研究表明增加DGLA摄入量会导致血小板功能缺陷。补充DGLA保护血管免受闭塞性血栓形成的能力将在这些小鼠中进行评估。最后，12-LOX在DGLA产物形成中的作用及其在DGLA介导的细胞效应中的重要性将通过确定DGLA在有效的12-LOX抑制剂和突变的12-LOX酶存在下的DGLA效应以及12-HETrE在释放部分或完全缺乏功能性12-LOX的细胞中的生理作用来确定。了解12-HETrE诱导其对细胞激活的影响的机制将有助于深入了解？-6脂肪酸调节VSSEL的潜在作用。因此，这些研究将填补在理解脂肪酸如何通过其12-LOX氧化代谢产物(S)影响一些生理和病理生理过程的方面的重大空白(S)。

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).