Tissue Factor the Vascular Proteome and Thrombosis

组织因子、血管蛋白质组和血栓形成

• 批准号: 6859745
• 负责人: THOMAS Scott EDGINGTON
• 金额: $ 63.81万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6859745
• 关键词: X ray crystallography; blood coagulation; clotting factor; coagulation factor IX; coagulation factor VII; coagulation factor X; confocal scanning microscopy; enzyme activity; enzyme structure; intravital microscopy; laboratory mouse; laboratory rat; membrane structure; nuclear magnetic resonance spectroscopy; nucleic acid sequence; polymerase chain reaction; protease inhibitor; proteasome; protein structure function; proteomics; site directed mutagenesis; structural biology; thromboplastin; thrombosis; tissue /cell culture; vascular endothelium

This project addresses issues in the thrombogenic pathways that have yet to be resolved. First, is the role of vascular endothelial surface microdomain context in the association of Tissue Factor (TF) with its substrates factors X and IX? This is being addressed in a novel manner with creation of various Selective Vascular Thrombogens that dock to different endothelial surface context and can be observed in vivo by real-time intravital microscopy as well as in in vitro cell assays and animal models of TF driven thrombosis and disseminated intravascular coagulation. Coupled with advanced imaging methods at the molecular level, use of genetically modified mice, and the collaboration with Project 2 to explore the differences in cell signaling, with Project 3 to explore the context in various genetically modified mice including high human TF expressing mice, and with Project 4 to approach the full three dimensional protein interactions coupled with the predicted atomic level interactions, a new level of data and insight will be forthcoming. Advancing initial discovery of a small constrained peptidyl ligand for a key interactive protein surface locus involved in association of the factor X with the functional TF:VIIa we will be able for the first time to selectively inhibit the extrinsic limb of the coagulation/thrombogenic pathway without inhibiting factor IX activation and effecting the intrinsic limb of the coagulation hemostatic pathways. This will require collaboration with all projects and cores for its execution. This set of studies will also address the correct molecular identification by mass spectrometry of the real endothelial surface factor Xa specific receptor. This is currently in process and potential candidate molecules have been identified and bear no relationship to the current scientific content of the literature. How important this receptor, FXaR, may be in thrombogenic and hemostatic pathways will be explored as well as its structure and basis of function. We have established by independent search methods a set of 92 peptidyl probes selective for the arterial atherosclerotic plaque surface endothelium. We have completed identification of one of the targets and are advancing three others to protein identification. The initially solved target that is very selective in vivo in atherosclerotic ApoE knockout mice is also similarly expressed on the surface of human iliac artery atherosclerotic plaques ex vivo. We will assess the linkage between plaque expression of these panels of markers and the histology, human TF content in the huTF knockin mouse/TF knockout mice as well as explore foam cell content and other possible markers of thrombotic risk of plaques. Such data and the probes may provide the much lacking means of in vivo imaging of the vasculature of humans for identification of the high risk 'susceptible' plaques.

该项目解决了血栓形成途径中尚未解决的问题。首先，血管内皮细胞表面微区在组织因子（TF）与其底物因子X和IX的结合中的作用？这一问题正在以一种新的方式得到解决，即创建各种选择性血管血栓原，这些血栓原与不同的内皮表面环境对接，并且可以通过实时活体显微镜以及体外细胞测定和TF驱动的血栓形成和弥散性血管内凝血的动物模型在体内观察。再加上分子水平上的先进成像方法，使用转基因小鼠，以及与项目2合作探索细胞信号传导的差异，与项目3合作探索各种转基因小鼠（包括高表达人TF的小鼠）的背景，与项目4合作接近完整的三维蛋白质相互作用以及预测的原子水平相互作用，数据和洞察力将达到新的水平。推进对参与因子X与功能性TF：VIIa关联的关键相互作用蛋白表面位点的小的受约束肽基配体的初步发现，我们将首次能够选择性地抑制凝血/血栓形成的外源性分支。 不抑制因子IX活化和影响凝血的内在分支的途径 止血通路。这将需要与所有项目和核心合作执行。这组研究还将通过质谱法对真实的内皮表面因子Xa特异性受体进行正确的分子鉴定。目前正在进行中，已经确定了潜在的候选分子，与文献的当前科学内容无关。该受体FXaR在血栓形成和止血途径中的重要性及其结构和功能基础将被探讨。我们已经建立了一套92肽探针选择性动脉粥样硬化斑块表面内皮细胞的独立搜索方法。我们已经完成了其中一个目标的鉴定，并正在推进其他三个目标的蛋白质鉴定。在动脉粥样硬化ApoE敲除小鼠体内非常有选择性的最初解决的靶标也在离体人髂动脉动脉粥样硬化斑块的表面上类似地表达。我们将评估huTF敲入小鼠/TF敲除小鼠中这些标志物组的斑块表达与组织学、人TF含量之间的联系，以及探索泡沫细胞含量和其他相关因素。 斑块血栓形成风险的可能标志物。这些数据和探测器可以提供更多的信息， 缺乏用于识别高风险的人体脉管系统的体内成像手段 “敏感”斑块。