Regulation of Endothelial Cell Procoagulant Properties

组织因子是凝血级联反应的中心激活剂，已成为一种信号受体 G蛋白偶联和整合素信号通路的十字路口。项目3是基于我们最近的发现 Tf胞外区关键二硫键的还原/氧化调节血栓前状态 活动。为了阐明这种新的调控机制是如何分别控制信号和血栓前状态的 工作组的活动是该项目的中心主题。具体地说，申请人建议将 蛋白质结构决定因素，使转铁蛋白在酶和 底物相互作用，膜组成对临界二硫键稳定性的作用，以及 降低的转铁蛋白相对细胞氧化途径的定位，包括蛋白质二硫键异构酶 与组织因子的氧化激活有关。Tf可以替代地变成S-亚硝化的发现是 实验的基础，用于表征将一氧化氮转移到转铁蛋白的细胞途径，并产生分析 在血栓形成的背景下检测这种翻译后修饰的工具。申请人提出建议 为了验证这样的假设，即通过TF与之相关的信号通路相互控制血栓形成 关联的。具体地说，开发了一些策略来证明整合素将转铁蛋白靶向细胞外 以及整合素激活对局部氧化还原电位的影响对基质的贡献 通过氧化转铁蛋白而致血栓。突变被用来明确地建立信号。 非凝血型转铁蛋白的性质。Tf的血栓形成和信号转导作用在 在易发生动脉粥样硬化的患者中使用湍流诱导病变发展的体内模型 动物。这些研究的成功完成将为新的监管机构提供有价值的信息 Tf依赖的血栓形成途径与血小板活化动力学特别相关 细胞外基质。

Tissue Factor is the central activator of the coagulation cascade and has emerged as a signaling receptor at the crossroads of G-protein coupled and integrin signaling pathways. Project 3 is based on our recent finding that reduction/oxidation of a critical disulfide bond in the extracellular domain of TF regulates prothrombotic activity. To elucidate how this novel regulatory mechanism separately controls signaling and prothrombotic activity of TF is the central theme of this project. Specifically, the applicant proposes to characterize the protein structural determinants that render TF susceptible to reduction in the context of enzyme and substrate interaction, the role of membrane composition on the stability of the critical disulfide bond, and the localization of reduced TF relative to cellular oxidative pathways, including protein disulfide isomerase implicated in oxidative activation of TF. The finding that TF can alternatively become S-nitrosylated is the basis for experiments to characterize cellular pathways that transfer NO to TF and to generate analytical tools to detect this posttranslation modification in the context of thrombus formation. The applicant proposes to test the hypothesis that thrombogenicity is under reciprocal control by signaling pathways with which TF is associated. Specifically, strategies are developed to demonstrate that integrins target TF to extracellular matrices and that the effects of integrin activation on local redox potential contribute to matrix thrombogenicty by oxidating TF. Mutagenesis is employed to unambiguously establish the signaling properties of the non-coagulant form of TF. Thrombogenic and signaling roles of TF are evaluated in atherosclerosis using an in vivo model of turbulent flow induced lesion development in atherosclerosis prone animals. The successful completion of these studies will provide valuable information on a novel regulatory pathway of TF-dependent thrombogenicity of particular relevance for the dynamics of platelet activation on extracellular matrices.