Natural Human Tissue Factor: Structure, Function and Presentation

尽管组织因子(Tf)在不同的生理过程中起着突出的作用，主要是在与 在凝血方面，天然转铁蛋白的结构和功能缺乏相关信息。 存在于活体中的形式。大多数与转移因子相关的实验数据都是使用不同形式的 重组蛋白。我们的初步数据表明，天然膜上存在的单核细胞转铁蛋白是 比人造膜上的任何重组或天然转移因子的活性高150-400倍。这个 该方案的主要目标是通过评估天然人转铁蛋白的结构，从多个来源研究它们 和功能特性，并通过鉴定负责高转铁蛋白活性的细胞膜成分(S)。 大量的胎盘、单核细胞和线粒体转铁蛋白将通过免疫亲和纯化。 方法：研究方法。对纯化的转铁蛋白种类的鉴定将利用免疫分析、结构、生化和 功能分析和结构-功能分析。Tf蛋白的功能特性研究 纯化并在天然膜上呈现将在一个酶-One中包括因子Vila驱动的反应 底物系统(荧光分析、外源性因子Xase和因子IX激活)以及更多 合成凝血蛋白质组和整体凝血酶生成等复杂系统 血。纯化的转铁蛋白的翻译后修饰将用 去糖基化、胰酶消化、质谱学和测序。这些修改的影响 对转铁蛋白活性进行了分析。从胎盘中提纯的人转铁蛋白将作为所有 评价、职能评价和结构评价。三种不同种类的重组人转铁蛋白将用于 Tf、Ea(全长)、TFI_242(缺少细胞质结构域)和Tfi.2is(可溶性； 仅限细胞外域)。我们还将试图了解高单核细胞转铁蛋白的机制 纯化、分析和评价单核细胞膜脂筏组分(S)的原位活性 以及单核细胞衍生的微粒。在这次研究中积累的数据将扩大我们的知识 与天然人转铁蛋白的结构和功能特性有关。新的知识链接TF结构 并获得具有功能活性的环境。 相关性：该项目将有助于更好地理解组织活动的调节机制 因子是启动血液凝结的关键蛋白，也是炎症和炎症之间的新纽带 血栓形成。

Despite the prominent role for tissue factor (TF)in various physiological processes, primarily in those related to blood coagulation, there is a lack of information with respect to the structure and function of natural TF forms existing in vivo. Most experimental data related to TF have been derived using various forms of recombinant proteins. Our preliminary data indicate that monocytic TF present on the native membrane is 150-400-fold more active than any TF, recombinant or natural, presented on an artificial membrane. The primary goal of this proposal is to study natural human TF from multiple sources by evaluating their structural and functional properties and by identifying the cell membrane component(s) responsible for high TF activity. Substantial amounts of placental, monocyte and mtcroparticle TF proteins will be purified by immunoaffinity methods. Characterization of purified TF species will utilize immunoassays, structural, biochemical and functional assays as well as structure-function analyses. Functional characterization of TF proteins both purified and presented on native membranes will include factor Vila-driven reactions in one enzyme-one substrate systems (fluorogenic assays, extrinsic factor Xase and factor IX activation) followed by more complex systems, such as TF-initiated thrombin generation in synthetic coagulation proteome and whole blood. The posttranslational modifications of the purified TF species will be characterized using deglycosylation, tryptic digestion, mass-spectroscopy and sequencing. The influence of these modifications on TF activity will be analyzed. Purified human TF from placenta will be used as a standard in all evaluations, functional and structural. Three different species of recombinant human TF will be used for comparison as well, i.e. TF^ea (full-length), TFi_242 (lacking the cytoplasmic domain), and TFi.2is (soluble; extracellular domain only). We will also attempt to understand the mechanism underlying high monocyte TF in situ activity by purifying, analyzing and evaluating the component(s) of the monocyte membrane, lipid rafts and monocyte-derived microparticles. The data accumulated during this study will expand our knowledge related to the structural and functional properties of natural human TF. New knowledge linking TF structure and environment with functional activity will be obtained. Relevance: This project will lead to a better understanding of the mechanisms regulating the activity of tissue factor, a key protein in the initiation of blood clotting and an emerging link between inflammation and thrombosis.