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: 7105004
• 负责人: THOMAS Scott EDGINGTON
• 金额: $ 63.57万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7105004
• 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域环境是否在组织因子(TF)与其底物因子X和IX的关联中起作用？这一问题正在以一种新颖的方式得到解决，各种选择性血管凝血素的产生对接到不同的内皮表面环境，并可以通过实时活体显微镜以及在体外细胞分析和TF驱动的血栓形成和弥散性血管内凝血的动物模型中进行观察。结合分子水平的先进成像方法，使用转基因小鼠，并与项目2合作探索细胞信号的差异，项目3探索各种转基因小鼠(包括高表达人TF的小鼠)的背景，并与项目4探讨全三维蛋白质相互作用以及预测的原子水平相互作用，将出现一个新的数据和洞察力水平。我们将首次发现与因子X和功能性Tf：VIIa相关的关键相互作用蛋白表面位点的一个受限制的小肽配体，我们将首次能够选择性地抑制凝血/血栓形成的外在肢体。 不抑制凝血因子IX激活和影响凝血的固有肢体的途径 止血通路。这将需要与执行其执行的所有项目和核心合作。这组研究还将解决通过质谱学对真正的内皮细胞表面因子Xa特异性受体进行正确的分子鉴定。这项研究目前正在进行中，潜在的候选分子已经被确定，与目前文献的科学内容没有关系。这种受体FXaR在血栓形成和止血途径中可能有多重要，以及它的结构和功能基础将被探索。我们通过独立的搜索方法建立了一套92个肽基探针，用于选择性地检测动脉粥样硬化斑块表面的内皮细胞。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。在动脉粥样硬化性ApoE基因敲除小鼠中，最初解决的靶点在体内是非常有选择性的，在体外也同样表达于人髂动脉粥样硬化斑块的表面。我们将在hutf基因敲除小鼠/tf基因敲除小鼠中评估这些标记物的斑块表达与组织学、人类tf含量之间的联系，并探索泡沫细胞含量和其他 斑块血栓风险的可能标记物。这样的数据和探测器可以提供更多 缺乏对人类血管系统进行活体成像以识别高危的手段 “易感”斑块。