Tissue Factor Pathway Inhibitor Binding Proteins on Endo

Endo 上的组织因子途径抑制剂结合蛋白

• 批准号: 6833861
• 负责人: Alan E Mast
• 金额: $ 8.58万
• 依托单位: VERSITI WISCONSIN, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6833861
• 关键词: binding proteins; binding sites; blocking antibody; blood coagulation; cell membrane; chemical association; clinical research; coagulation factor X; fibroblast growth factor; flow cytometry; gene targeting; genetically modified animals; glycosylphosphatidylinositols; heparin; human tissue; immunocytochemistry; immunoprecipitation; inhibitor /antagonist; laboratory mouse; lipid metabolism; protein metabolism; protein structure function; proteoglycan; thromboplastin; tissue /cell culture; vascular endothelial growth factors; vascular endothelium

DESCRIPTION (provided by applicant): Tissue factor pathway inhibitor (TFPI) rapidly inhibits both factor Xa and the factor VIIa/tissue factor catalytic complex. Thus, it is thought to be the most important inhibitor of the initiation of blood coagulation. Although its structure suggests that it is a soluble protein, most TFPI is associated with the vascular endothelium.The objective of this proposal is to define the biochemical mechanisms responsible for the association of TFPI with biological surfaces. Heparin infusion results in a prompt 2-to 10-fold increase in circulating TFPI concentration, therefore, interactions with glycosaminoglycans are considered a primary mode of cell surface association. However, flow cytometry studies demonstrate that over 95 percent of the TFPI on the surface of cultured endothelial cells is bound through a glycosyiphosphatidylinositol (GPI)-anchor in a manner that is not altered by heparin. We have previously shown that glypican-3, a GPI-anchored proteoglycan, binds to TFPI and may account for TFPI binding to the endothelium. Recently, we identified thrombospondin - 1 (TSP) as a second TFPI binding protein that may act to recruit and localize TFPI to extravascular surfaces within a bleeding wound. This revised proposal is organized into three specific aims that reflect the three long term approaches our laboratory is taking to investigate the function of TFPI as a surface associated inhibitor of tissue factor initiated blood coagulation.Specific Aim #1 is focused on in vitro structure/function studies of TFPI. Proposed projects include: the investigation of the role of the interaction between TFPI and TSP in the cellular catabolism of TFPI; defining the mechanisms responsible for the proliferative effect of TFPI on endothelial cells; and enzyme kinetic studies with altered forms of TFPI to further define the role of the third Kunitz domain and C-terminal region in the anticoagulant activity of TFPI.Specific Aim #2 involves in vivo/ex vivo studies using human placenta and TSP knock-out mice to define the relative amounts of GPI-anchored and heparin-releasable TFPI found within vascular beds in vivo and to characterize their structures. The studies with the TSP knock-out mice will define the role of TSP in the association of TFPI with the endothelial surface.Specific Aim #3 is designed to characterize how TFPI is processed within the cell to produce the heparin-releasable and GPI-anchored pools. Recombinant TFPI lacking the first two, non-translated, exons appears to be entirely secreted into the culture medium. Possible explanations for this observation include that the first two exons are required for proper intracellular targeting to a GPI-anchored binding protein or that the GPI-anchored binding sites are saturated. These hypotheses will be tested using TFPI constructs containing the first two exons. Additionally, TFPI constructs that have been tagged either with the HA or the FLAG tag will be transfected into cells to allow differentiation of endogenous and recombinant surface TFPI in flow cytometry experiments. Pulse-chase immunoprecipitation studies will be performed using Trition-X-1 14 for cell lysis. These experiments will define how long after synthesis and the percentage of the total TFPI that becomes attached to a GPI-anchor.These proposed studies will provide a detailed characterization of how TFPI associates with the vascular endothelium and establish models for the study of TFPI within different vascular beds. These studies are highly clinically relevant because TFPI is a an important inhibitor of tissue factor initiated blood coagulation that produces the pathological thrombi in stroke, heart attack and many other thrombotic diseases.

描述（由申请人提供）：组织因子途径抑制剂（TFPI）迅速抑制因子XA和VIIA/组织因子因子催化复合物。因此，被认为是血液凝血开始的最重要的抑制剂。尽管它的结构表明它是一种可溶性蛋白，但大多数TFPI都与血管内皮相关。该提案的目的是定义负责TFPI与生物表面关联的生化机制。肝素输注导致循环TFPI浓度迅速增加2至10倍，因此，与糖胺聚糖的相互作用被认为是细胞表面缔合的主要模式。然而，流式细胞仪研究表明，培养的内皮细胞表面上有超过95％的TFPI通过乙二醇糖酰肌醇（GPI）锚固结合，并以肝素不改变的方式结合。我们以前已经表明，GPI锚定的蛋白聚糖Glypican-3与TFPI结合，可能解释了TFPI与内皮的结合。最近，我们将血小板传播-1（TSP）鉴定为第二种TFPI结合蛋白，可以起作用并将TFPI定位于出血伤口内的血管外表面。这项修订后的提案分为三个特定目标，反映了我们的实验室采取的三种长期方法来研究TFPI作为组织因子的表面抑制剂引发的血液凝结的功能。特异性目标＃1集中于TFPI的体外结构/功能研究。拟议的项目包括：研究TFPI和TSP之间相互作用在TFPI细胞分解代谢中的作用；定义负责TFPI对内皮细胞增殖作用的机制；和酶动力学研究，随着TFPI的改变形式，进一步定义了第三个Kunitz结构域和C末端区域在TFPI的抗凝活性中的作用。特定的目标＃2涉及体内/外胎盘研究中使用人类胎盘和TSP敲除小鼠在体内研究中的研究，以确定gpi-casti的相对量，并在gpi-castior中识别了gpi-castiorcount titt t t t t t t t t t t t t。体内并表征其结构。对TSP敲除小鼠的研究将定义TSP在TFPI与内皮表面的关联中的作用。特定的AIM＃3旨在表征细胞内TFPI的处理方式，以生成可透明的肝素可释放和GPI锚定的池。重组TFPI缺乏前两个未翻译的外显子似乎完全分泌到培养基中。该观察结果的可能解释包括前两个外显子是对GPI锚定蛋白的适当细胞内靶向所必需的，或者GPI锚定的结合位点已饱和。这些假设将使用包含前两个外显子的TFPI构建体进行测试。此外，已将其标记为HA或FLAG标签标记的TFPI构建体将被转染到细胞中，以在流式细胞仪实验中允许内源性和重组表面TFPI分化。将使用Trition-X-1 14进行细胞裂解，进行脉冲培养基免疫沉淀研究。这些实验将定义合成后多长时间以及与GP-thor相连的总TFPI的百分比。这些拟议的研究将提供有关TFPI如何与血管内皮相关联的详细表征，并为不同血管内TFPI的研究建立模型。这些研究在临床上具有很高的相关性，因为TFPI是组织因子的重要抑制剂，引发了血液凝结，可导致中风，心脏病发作和许多其他血栓性疾病的病理血栓。