Tissue Factor Pathway Inhibitor Binding Proteins on Endo

描述(申请人提供)：组织因子途径抑制物(TFPI)快速抑制因子Xa和因子VIIa/组织因子催化复合体。因此，它被认为是最重要的凝血启动抑制剂。虽然它的结构表明它是一种可溶性蛋白质，但大多数TFPI与血管内皮细胞有关。本提案的目的是确定TFPI与生物表面联系的生化机制。肝素注射导致循环中TFPI浓度迅速增加2-10倍，因此，与糖胺多糖的相互作用被认为是细胞表面结合的主要模式。然而，流式细胞仪研究表明，超过95%的培养内皮细胞表面的TFPI以不被肝素改变的方式通过糖基化磷脂酰肌醇(GPI)锚点结合。我们以前已经证明，GPI锚定的蛋白多糖GLYPICA-3可以与TFPI结合，并可能解释TFPI与内皮的结合。最近，我们发现凝血酶敏感蛋白-1(TSP)是第二种TFPI结合蛋白，它可以在出血创面的血管外表面募集和定位TFPI。这份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作为组织因子引发的血液凝固的表面相关抑制物的功能。具体目标#1重点是TFPI的体外结构/功能研究。建议的项目包括：研究TFP I和TSP之间的相互作用在TFP I的细胞分解代谢中的作用；确定TFP I对内皮细胞增殖影响的机制；以及通过改变TFP I的形式进行酶动力学研究，以进一步确定TFP I的第三个kunitz结构域和C末端区域在TFP I抗凝活性中的作用。具体目标2涉及使用人胎盘和TSP基因敲除小鼠进行体内/体外研究，以确定体内血管床中发现的GPI锚定和肝素释放的TFP I的相对数量，并描述其结构。对TSP基因敲除小鼠的研究将确定TSP在TFPI与内皮表面联系中的作用。具体目标#3旨在描述TFPI是如何在细胞内被处理以产生可肝素释放和GPI锚定的池。缺少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构建物进行测试。此外，已经用HA或FLAG标签标记的TFPI构建体将被导入细胞，以便在流式细胞仪实验中分化内源性和重组表面TFPI。脉冲追逐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附着在GPI锚上的总TFP I的百分比。这些拟议的研究将提供TFP I如何与血管内皮细胞相关联的详细表征，并为不同血管床内TFP I的研究建立模型。这些研究具有很高的临床意义，因为TFPI是一种重要的组织因子引发的凝血抑制物，在中风、心脏病发作和许多其他血栓性疾病中会产生病理性血栓。

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.