Natural Human Tissue Factor: Structure, Function and Presentation

尽管组织因子（TF）在各种生理过程中起着重要作用，主要是在那些与TF相关的疾病中。 对于凝血，缺乏关于天然TF的结构和功能的信息 存在于体内的形式。大多数与TF相关的实验数据都是使用各种形式的 重组蛋白我们的初步数据表明，单核细胞TF存在于天然膜上， 150-400-比人工膜上存在的任何重组或天然TF活性高1倍。的 这项提案的主要目标是通过评估其结构来研究来自多种来源的天然人类TF 和功能特性，并通过鉴定负责高TF活性的细胞膜组分。 大量的胎盘、单核细胞和微粒TF蛋白将通过免疫亲和法纯化， 方法.纯化的TF物质的表征将利用免疫测定、结构、生物化学和生物化学方法。 功能测定以及结构-功能分析。TF蛋白的功能表征 纯化并呈现在天然膜上的酶将包括因子Vila驱动的一种酶-一种 底物系统（荧光检测，外源性因子Xase和因子IX激活），然后是更多 复杂的系统，如TF-启动合成凝血蛋白质组中的凝血酶生成， 血纯化的TF物质的翻译后修饰将使用 去糖基化、胰蛋白酶消化、质谱和测序。这些变化的影响 将对TF活性进行分析。来自胎盘的纯化的人TF将用作所有研究中的标准品。 功能和结构评价。三种不同的重组人TF将用于 也可以进行比较，即TF 12 ea（全长）、TF 1242（缺乏胞质结构域）和TF 121 s（可溶性; 仅细胞外结构域）。我们还将试图了解高单核细胞TF的机制， 通过纯化、分析和评价单核细胞膜、脂筏的组分， 和单核细胞衍生的微粒。这项研究积累的数据将扩大我们的知识 与天然人TF的结构和功能特性有关。新知识链接TF结构 并获得具有功能活性的环境。 相关性：该项目将使人们更好地了解调节组织活动的机制， 凝血因子，一种启动血液凝固的关键蛋白质，也是炎症和炎症之间的一种新兴联系。 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.