Tissue Factor Pathway Inhibitor Binding Proteins on Endo

描述（由申请人提供）：组织因子途径抑制剂（TFPI）可快速抑制Xa因子和VIIa因子/组织因子催化复合物。因此，它被认为是开始凝血的最重要的抑制剂。虽然其结构表明它是一种可溶性蛋白，但大多数TFPI与血管内皮有关。本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超过95%的TFPI通过糖酰磷脂酰肌醇（GPI）锚点结合，其结合方式不受肝素的改变。我们之前已经表明，glypican-3，一种gpi锚定的蛋白聚糖，与TFPI结合，并可能解释TFPI与内皮的结合。最近，我们发现血栓反应蛋白- 1 （TSP）是第二个TFPI结合蛋白，可能在出血伤口的血管外表面招募和定位TFPI。修订后的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作为组织因子启动血液凝固的表面相关抑制剂的功能。Specific Aim #1专注于TFPI的体外结构/功能研究。拟开展的研究项目包括：TFPI与TSP相互作用在TFPI细胞分解代谢中的作用研究；明确TFPI对内皮细胞增殖作用的机制；以及改变TFPI形式的酶动力学研究，以进一步确定第三Kunitz结构域和c端区域在TFPI抗凝血活性中的作用。具体目标#2涉及使用人胎盘和TSP敲除小鼠进行体内/离体研究，以确定体内血管床中gpi锚定和肝素释放的TFPI的相对数量，并表征其结构。对TSP敲除小鼠的研究将确定TSP在TFPI与内皮表面关联中的作用。特异性Aim #3旨在描述TFPI如何在细胞内加工以产生肝素释放和gpi锚定池。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，前两个外显子是细胞内靶向gpi锚定结合蛋白所必需的，或者gpi锚定结合位点是饱和的。这些假设将使用包含前两个外显子的TFPI结构来验证。此外，用HA或FLAG标记的TFPI构建物将被转染到细胞中，以便在流式细胞术实验中分化内源性和重组表面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以及总TFPI与gpi锚点相连的百分比。这些拟议的研究将详细描述TFPI与血管内皮的关系，并为不同血管床中TFPI的研究建立模型。这些研究具有很高的临床相关性，因为TFPI是组织因子引发的血液凝固的重要抑制剂，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。

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.