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: 6619738
• 负责人: Alan E Mast
• 金额: $ 23.2万
• 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/6619738
• 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（由申请方提供）：组织因子途径抑制剂（TFPI）可快速抑制因子Xa和因子VIIa/组织因子催化复合物。因此，它被认为是血液凝固起始的最重要的抑制剂。虽然它的结构表明，它是一种可溶性蛋白质，大多数TFPI是与血管endothelial. Objective的建议是确定负责协会TFPI与生物表面的生化机制。肝素输注导致循环TFPI浓度迅速增加2- 10倍，因此，认为与糖胺聚糖的相互作用是细胞表面结合的主要模式。然而，流式细胞术研究表明，培养的内皮细胞表面上超过95%的TFPI通过糖基磷脂酰肌醇（GPI）-锚以不被肝素改变的方式结合。我们以前已经表明，磷脂酰肌醇蛋白聚糖-3，GPI锚定的蛋白聚糖，结合TFPI，并可能占TFPI结合内皮。最近，我们确定了血小板反应蛋白-1（TSP）作为第二个TFPI结合蛋白，它可以在出血伤口内募集和定位TFPI到血管外表面。本修订提案分为三个具体目标，反映了我们实验室正在采取的三种长期方法，以研究TFPI作为组织因子引发凝血的表面相关抑制剂的功能。具体目标#1侧重于TFPI的体外结构/功能研究。拟议的项目包括：研究TFPI和TSP之间的相互作用在TFPI的细胞增殖中的作用，确定TFPI对内皮细胞增殖作用的机制;和用改变形式的TFPI进行的酶动力学研究，以进一步确定第三Kunitz结构域和C-末端区域在TFPI的抗凝活性中的作用。使用人胎盘和TSP敲除小鼠的离体研究，以确定体内血管床内发现的GPI锚定的和肝素可释放的TFPI的相对量，并表征其结构。用TSP敲除小鼠进行的研究将确定TSP在TFPI与内皮表面结合中的作用。具体目标#3旨在表征TFPI如何在细胞内加工以产生肝素可释放和GPI锚定的库。缺乏前两个非翻译外显子的重组TFPI似乎完全分泌到培养基中。对这一观察结果的可能解释包括，前两个外显子是适当的细胞内靶向GPI锚定的结合蛋白所必需的，或者GPI锚定的结合位点是饱和的。将使用含有前两个外显子的TFPI构建体来检验这些假设。此外，将已经用HA或FLAG标签标记的TFPI构建体转染到细胞中，以允许在流式细胞术实验中区分内源性和重组表面TFPI。脉冲追踪免疫沉淀研究将使用Trition-X-114进行细胞裂解。这些实验将确定合成后多长时间和总TFPI的百分比，成为连接到GPI-anchor.These拟议的研究将提供一个详细的表征TFPI如何与血管内皮细胞和建立模型的研究TFPI在不同的血管床。这些研究是高度临床相关的，因为TFPI是组织因子引发的血液凝固的重要抑制剂，其在中风、心脏病发作和许多其他血栓性疾病中产生病理性血栓。