TRANSCRIPTION FACTOR NF-E2 IN ALPHA IIB BETA 3 SIGNALING

血小板整合素AlphaIIbbeta3通过结合纤维蛋白原和触发调节细胞骨架的内部信号来响应细胞内信号。由于目前对AlphaIIbbeta3信号的了解有限，我们将研究原代小鼠巨核细胞中的AlphaIIbbeta3信号。巨核细胞是自然表达AlphaIIbbeta3的细胞，与血小板不同，它可以接受基因操作。我们发现，激动剂诱导的纤维蛋白原与AlphaIIbbeta3结合只发生在正常小鼠最大、最成熟的巨核细胞中。相比之下，转录因子NF-E2 P45亚基(NF-E2-/-)基因缺陷的小鼠的大巨核细胞无法结合可溶性纤维蛋白原，而且它们与固定的纤维蛋白原的粘附性很差，尽管AlphaIIbbeta3正常表达。这表明，由内向外的AlphaIIbbeta3信号需要一个或多个由NF-E2调节的基因。该项目的目标是识别和表征控制AlphaIIbbeta3功能的核因子-E2调节的基因。首先，将通过检测纤维蛋白原黏附细胞中的细胞骨架重组，通过将分析扩展到其他整合素，以及通过确定在NF-E2-/-巨核细胞中重组P45是否重构正常的AlphaIIbbeta3信号，来详细描述NF-E2-/-巨核细胞中的功能缺陷。重组p45将使用逆转录病毒和辛德比斯病毒系统表达。其次，将通过一系列互补的方法，通过检测野生型和NF-E2-/-巨核细胞中差异基因的表达来鉴定由NF-E2调节的基因，其中一个或多个可能重新调节AlphaIIbbeta3的功能。第三，通过差异表达确定的候选基因(S)将通过使用病毒载体在巨核细胞中表达它们或相关突变体来评估它们在AlphaIIbbeta3信号中的作用，并确定它们对AlphaIIbbeta3功能的影响。由核因子-E2-/-巨核细胞提供的AlphaIIbbeta3信号的独特窗口有望填补我们对双向整合素信号的理解的主要空白。与我们的合作者提出的关于凝血酶和AlphaIIbbeta3信号的补充工作相结合，这些机制信息可能有助于开发一类从血小板内抑制AlphaIIbbeta3功能的新型抗血栓药物。

Platelet integrin alphaIIbbeta3 responds to intracellular signals by binding fibrinogen and triggering inward signals that regulate the cytoskeleton. Since current understanding of alphaIIbbeta3 signaling is limited, we will study alphaIIbbeta3 signaling in primary murine megakaryocytes, cells that naturally express alphaIIbbeta3 and, unlike platelets, are amenable to genetic manipulation. We have discovered that agonist-induced fibrinogen binding to alphaIIbbeta3 occurs only in the largest, most mature megakaryocytes from normal mice. In contrast, large megakaryocytes from mice genetically deficient in the transcription factor NF-E2 p45 subunit (NF-E2-/-) fail to bind soluble fibrinogen and they adhere poorly to immobilized fibrinogen, despite normal expression of alphaIIbbeta3. This suggests that one or more NF-E2-regulated genes are required for inside-out alphaIIbbeta3 signaling. The goal of this project is to identify and characterize NF-E2-regulated genes that control alphaIIbbeta3 function. First, the functional defect in NF-E2-/- megakaryocytes will be characterized in detail by examining cytoskeletal reorganization in fibrinogen-adherent cells, by extending the analysis to other integrins, and by determining whether recombinant expression of p45 in NF-E2-/- megakaryocytes reconstitutes normal alphaIIbbeta3 signaling. Recombinant p45 will be expressed using retroviral and Sindbis virus systems. Second, NF-E2-regulated genes, one or more of which may re regulate alphaIIbbeta3 function, will be identified by examining differential gene expression in wild-type and NF-E2-/- megakaryocytes using a series of complementary approaches. Third, candidate genes identified by differential expression will be evaluated for their role(s) in alphaIIbbeta3 signaling by using viral vectors to express them, or relevant mutants, in megakaryocytes and determining their effects on alphaIIbbeta3 function. The unique window on alphaIIbbeta3 signaling provided by NF-E2-/- megakaryocytes promises to fill major gaps in our understanding of bidirectional integrin signaling. Taken together with the complementary work on thrombin and alphaIIbbeta3 signaling proposed by our collaborators, this mechanistic information may facilitate the development of a new class of antithrombotic drugs that inhibit alphaIIbbeta3 function from within the platelet.