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: 7237995
• 负责人: THOMAS Scott EDGINGTON
• 金额: $ 64.72万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7237995
• 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，血管内皮表面微域环境在组织因子 (TF) 与其底物因子 X 和 IX 的关联中的作用是什么？目前正在以一种新颖的方式解决这一问题，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进行体内观察。结合分子水平上的先进成像方法、转基因小鼠的使用，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（包括高人类 TF 表达小鼠）的背景，与项目 4 合作研究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，即将出现新水平的数据和见解。推进对关键相互作用蛋白表面位点的小受限肽基配体的初步发现，该位点涉及因子 X 与功能性 TF:VIIa 的关联，我们将首次能够选择性抑制凝血/血栓形成的外在肢体 途径不抑制因子 IX 激活并影响凝血的内在分支 止血途径。这需要与所有项目和核心进行协作才能执行。这组研究还将通过质谱法对真正的内皮表面因子 Xa 特异性受体进行正确的分子鉴定。目前正在进行中，潜在的候选分子已被识别，与当前文献的科学内容没有关系。我们将探讨这种受体 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。我们通过独立的搜索方法建立了一组对动脉粥样硬化斑块表面内皮具有选择性的 92 个肽基探针。我们已经完成了其中一个目标的识别，并正在推进其他三个目标的蛋白质识别。最初解决的靶标在体内动脉粥样硬化 ApoE 敲除小鼠中具有很高的选择性，在离体人髂动脉动脉粥样硬化斑块的表面上也有类似的表达。我们将评估这些标记物组的斑块表达与组织学、huTF 敲入小鼠/TF 敲除小鼠中的人类 TF 含量之间的联系，并探索泡沫细胞含量和其他 斑块血栓形成风险的可能标志物。这些数据和探针可以提供很多 缺乏对人体脉管系统进行体内成像来识别高风险的手段 “易感”斑块。