TISSUE FACTOR IN THE ARTERIAL WALL

动脉壁中的组织因子

• 批准号: 6866584
• 负责人: Yale R Nemerson
• 金额: $ 17.94万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-03-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6866584
• 关键词: atherosclerotic plaque; binding proteins; blood coagulation; cell cell interaction; coagulation factor X; flow cytometry; human subject; mathematical model; molecular biology information system; monoclonal antibody; platelet aggregation; protein purification; protein sequence; protein structure function; thromboembolism; thromboplastin; thrombosis; tissue /cell culture

DESCRIPTION (Applicant's list of Aims) Specific Aim 1: To elaborate the mechanism by which TF is transferred to the platelet surface. We have documented that during ex-vivo thrombus formation platelets become TF-positive as judged by light-and electron- microscopy. Because platelets with bound TF would be an extremely thrombogenic species, we plan to study the detailed mechanism by which this transfer takes place. Initial studies will focus on the interaction of platelets and leukocytes in order to define the molecules required for this transfer. The transfer of TF will be evaluated morphometrically following shearing of leukocyte derived microparticles with fresh human platelets. We also have utilized FACS analysis for quantitative assessment of the transfer. Both techniques indicate that the CD-15 p-selectin ligand-receptor pair is involved as is TF,itself. Because we and others have identified TF- positive neutrophils (PMNs) in thrombi and in tissues, we propose to investigate the ability of these cells to synthesize TF. Specific Aim 2: Purification and characterization of a Tissue Factor Binding Protein from platelets: Our hypothesis is that the interaction of leukocyte-derived TF with platelets is an essential step in ex-vivo thrombus formation and these experiments are designed to provide the tools to test this hypothesis. Because we have shown that a monoclonal antibody to TF blocks this transfer, we believe that there likely is a tissue factor binding protein on platelets. We have initiated the purification of this protein using a combination of classical and affinity steps. As judged from ligand blots of proteins electro-tranferred from SDS gels, we have identified a candidate protein with an apparent mol wt of approximately 200,000, unreduced, and approximately 80,000 when reduced. We propose to purify and characterize this protein. We will identify the protein from amino acid sequence data performed on eluates from SDS gels. If the protein is present in the Brookhaven or other national databases, we will search for any known functions. If the cDNA is available, we propose to express it in an appropriate cell line. If not, we propose to clone the cDNA coding for it and then express it. Further, the extra-cellular domain of TF (sTF, TF1-218) binds to platelets as judged by enzyme kinetics utilizing sTF and factor VIIa and estimating the velocity of factor X activation. These kinetic experiments indicate that sTF and platelets interact to form an active catalytic complex. We propose to evaluate this hypothesis by directly measuring the binding by using fluorescence techniques. Knowing the equilibrium binding constant for this interaction will enable us to interpret the kinetic results and to test our hypothesis relating to the activity of TF bound to platelets. Specific aim 3: To Describe ex-vivo thrombus growth in detail. The order in which platelets, fibrin and TF are deposited is largely unknown and our hypothesis regarding the incorporation of circulating TF in thrombi can be tested by the experiments described in this aim in which we address the microscopic details of thrombus growth. Specifically, we propose to develop a system in which we can visualize in real time the deposition of fibrin, platelets and TF as ex vivo thrombi develop. We also propose to examine the permeability of thrombi to proteins. And, lastly, we plan to develop simulations of the diffusion of proteins generated at the vessel wall, e.g., factor Xa, into an overlying platelet mass by real-time observation of the deposition of these species. Three monoclonal antibodies or their fragments, which do not inhibit thrombus growth, will be labeled with different fluorophores. Thrombi will be formed by perfusion of blood over a collagen-coated glass slide in a parallel plate chamber. Our existing equipment will enable us to visualize three fluorophores each five seconds. (To be done in collaboration with Dr. Eric Grabowski, Harvard Medical School and Dr. Scott Diamond, Institute for Engineering and Medicine, U. Penn).

具体目的1：阐明TF转移至血小板表面的机制。我们已经证明，在离体血栓形成过程中，血小板通过光镜和电子显微镜判断为TF阳性。由于结合TF的血小板是一种极易形成血栓的物质，我们计划研究这种转移发生的详细机制。最初的研究将集中在血小板和白细胞的相互作用，以确定这种转移所需的分子。在用新鲜人血小板剪切白细胞衍生微粒后，将对TF转移进行形态测定评价。我们还利用流式细胞仪分析的定量评估的转移。两种技术均表明CD-15 p-选择素配体-受体对与TF本身一样参与。因为我们和其他人已经在血栓和组织中鉴定出TF阳性中性粒细胞（PMNs），我们建议研究这些细胞合成TF的能力。具体目标二：血小板中组织因子结合蛋白的纯化和表征：我们的假设是白细胞衍生的TF与血小板的相互作用是体外血栓形成的重要步骤，这些实验旨在提供检验这一假设的工具。因为我们已经证明了TF的单克隆抗体阻断了这种转移，我们认为血小板上可能存在组织因子结合蛋白。我们已经开始使用经典和亲和步骤的组合纯化这种蛋白质。根据SDS凝胶电转移蛋白质的配体印迹判断，我们已经鉴定出一种表观摩尔重量约为200，000（未还原）和约为80，000（还原）的候选蛋白质。我们建议纯化和表征这种蛋白质。我们将根据SDS凝胶洗脱液的氨基酸序列数据鉴定蛋白质。如果该蛋白存在于Brookhaven或其他国家数据库中，我们将搜索任何已知的功能。如果cDNA是可用的，我们建议在适当的细胞系中表达它。此外，TF的细胞外结构域（sTF，TF 1 -218）与血小板结合，如利用sTF和因子VIIa的酶动力学和估计因子X活化的速度所判断的。这些动力学实验表明sTF和血小板相互作用形成活性催化复合物。我们建议通过使用荧光技术直接测量结合来评估这一假设。了解这种相互作用的平衡结合常数将使我们能够解释动力学结果，并测试我们的假设有关的活动TF结合血小板。具体目标3：详细描述离体血栓生长。血小板、纤维蛋白和TF沉积的顺序在很大程度上是未知的，我们关于血栓中循环TF掺入的假设可以通过本目标中描述的实验进行测试，其中我们讨论了血栓生长的微观细节。具体地说，我们建议开发一种系统，在该系统中，我们可以在真实的时间的纤维蛋白，血小板和TF作为离体血栓发展的沉积可视化。我们还建议检查血栓对蛋白质的渗透性。最后，我们计划开发血管壁产生的蛋白质扩散的模拟，例如，通过实时观察这些物质的沉积，将因子Xa沉积到覆盖的血小板团中。将用不同的荧光团标记不抑制血栓生长的三种单克隆抗体或其片段。在平行板室中，通过将血液灌注到胶原包被的载玻片上形成血栓。我们现有的设备将使我们能够每五秒看到三个荧光团。(To这项研究是与哈佛医学院的埃里克·格拉博夫斯基博士和美国工程与医学研究所的斯科特·戴蒙德博士合作完成的。Penn）。