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向信号来响应细胞内信号。由于目前对alphaIIbbeta3信号传导的理解有限，我们将研究小鼠原代巨核细胞中的alphaIIbbeta3信号传导，这些细胞自然表达alphaIIbbeta3，与血小板不同，它们易于基因操纵。我们已经发现，激动剂诱导的纤维蛋白原与alphaIIbbeta3的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，来自基因缺乏转录因子NF-E2 p45亚基（NF-E2-/-）的小鼠巨核细胞不能结合可溶性纤维蛋白原，并且它们对固定纤维蛋白原的粘附性很差，尽管alphaIIbbeta3正常表达。这表明一个或多个nf - e2调控基因是由内而外的alphaIIbbeta3信号传导所必需的。该项目的目标是鉴定和表征控制alphaIIbbeta3功能的nf - e2调节基因。首先，NF-E2-/-巨核细胞的功能缺陷将通过检查纤维蛋白原粘附细胞中的细胞骨架重组，将分析扩展到其他整合素，并确定NF-E2-/-巨核细胞中p45的重组表达是否重建正常的alphaIIbbeta3信号传导来详细表征。重组p45将使用逆转录病毒和Sindbis病毒系统表达。其次，NF-E2调节基因，其中一个或多个可能重新调节alphaIIbbeta3功能，将通过使用一系列互补方法检测野生型和NF-E2-/-巨核细胞中的差异基因表达来鉴定。第三，通过差异表达鉴定的候选基因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，并确定它们对alphaIIbbeta3功能的影响，来评估它们在alphaIIbbeta3信号传导中的作用。NF-E2-/-巨核细胞提供的关于alphaIIbbeta3信号传导的独特窗口有望填补我们对双向整合素信号传导理解的主要空白。结合我们的合作者提出的关于凝血酶和alphaIIbbeta3信号传导的补充工作，这一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.