Functional Tissue Factor on Activated Endothelium

描述(逐字摘自申请者摘要)：放大和传播 局部血栓的形成可能取决于局部微环境。 上游刺激内皮细胞产生的激活因子Xa和IXa (EC)在现行剪应力条件下，以及任何血液传播的情况下 它们分泌的组织因子(TF)和组织因子途径抑制物(TFPI) 同样的内皮细胞可能会导致血栓形成。研究人员种植植物来检验这一假说 表达转铁蛋白的EC促进动脉和小动脉局部壁血栓形成， 剪切力从基线水平的降低决定了 刺激的EC成为功能性促凝剂。调查人员将 溶血性尿毒症综合征中志贺毒素对血管壁损伤的研究 (HUS)作为全身内皮损伤的范例。具体目标1是设计 为了表明同时暴露在剪切力和肿瘤坏死因子-a下， 内皮通过即刻增加来限制其功能性转铁蛋白的表达 其分泌的TFPI，只有在几个小时后才能下调 转铁蛋白基因。相反，剪切力降低到接近停滞的水平 这样的内皮细胞会变成促凝剂。抄写的重要性 Egr-1、Sp1、NFkB和AP-1在血管内皮细胞后上调TF mRNA中的作用 细胞单独和联合暴露于剪切力和肿瘤坏死因子-a将是 检查过了。目标2是评估血小板对肾小球的黏附/聚集 志贺毒素损伤内皮细胞，暴露于流动的血液中，并 确定志贺毒素在内皮暴露后的作用机制 对于肿瘤坏死因子-a，进一步增强了功能转铁蛋白，如前面的 调查人员。候选机制是对预先形成的TF进行解密。目标3是 在血流受控的情况下实时演示转铁蛋白 内皮细胞和/或白细胞起源与生长的血小板共定位 聚集，并存在于附着在志贺的血小板聚集物上 灵长类动物肾节段毒素损伤的肾小球内皮 HUS的模型。这些目标包括Xa因子和TFPI的测量 Tf和TFPI免疫金标记物的制备、RT-PCR扩增 Tf和TFPI mRNAs的产生、核径流研究和电泳法 迁移率变化分析。调查人员还将使用免疫胶体标记法 对于纤维蛋白原涂层玻璃上的血小板聚集物表面的纤维蛋白和 肾小球内皮细胞。血小板与转铁蛋白共定位的实时成像 将通过双标记荧光视频显微镜来实现。一个 更清楚地理解剪切力调节 活化的内皮细胞表达转铁蛋白在血管病变的发生发展中起重要作用 HUS中阻断转铁蛋白途径的策略及其概念上的相关 动脉/小动脉血栓形成障碍。

DESCRIPTION (Verbatim from Applicant's Abstract): Amplification and propagation of local thrombosis is likely to be dependent upon the local microenvironment. Activated factors Xa and IXa produced by upstream, stimulated endothelial cells (EC) under prevailing shear stress conditions, as well as any blood-borne tissue factor (TF) and tissue factor pathway inhibitor (TFPI) secreted by these same ECs may induce thrombosis. The investigators plant to test the hypothesis that TF-expressing EC promote local mural thrombi in arteries and arterioles, and that reductions in shear stress from baseline levels determine the extent to which stimulated EC become functionally procoagulant. The investigators will focus on Shiga toxin injury to the vessel wall in the hemolytic uremic syndrome (HUS) as a paradigm for general endothelial injury. Specific Aim 1 is designed to show that for simultaneous exposure to shear stress and TNF-a, the endothelium limits its expression of functional TF by immediately increasing its secretion of TFPI, only after several hours being able to downregulate the gene for TF. Conversely, reduction of shear stress to near-stasis levels allows such endothelium to become procoagulant. The importance of the transcription factors Egr-1, Sp1, NFKb, and AP-1 in upregulating TF mRNA after endothelial cell exposure to shear stress and TNF-a, individually and together, will be examined. Aim 2 is to assess platelet adhesion / aggregation to glomerular endothelial cells injured by Shiga toxin, and exposed to flowing blood, and to determine the mechanism by which Shiga toxin, following exposure of endothelium to TNF-a, further augments functional TF, as demonstrated previously by the investigators. A candidate mechanism is de-encryption of preformed TF. Aim 3 is to demonstrate in real time and under controlled blood flow conditions that TF on endothelial and/or leukocyte origin co-localizes with growing platelet aggregates, and is present on platelet aggregates adherent to Shiga toxin-injured glomerular endothelium in kidney segments derived from a primate model of HUS. These aims incorporate measurement of factor Xa and TFPI production, immunogold labeling to TF and TFPI antigen, RT-PCR amplification production of TF and TFPI mRNAs, and nuclear runoff studies and electrophoretic mobility shift assays. The investigators will also employ immunoglold lableling for fibrin on the surface of platelet aggregates on fibrinogen-coated glass and glomerular endothelium. Real-time imaging of platelet and TF co-localization will be achieved by means of dual-label epiflourescence videomicroscopy. A clearer understanding of the manner in which shear stress regulates the expression of TF by activated endothelium is important to the development of strategies to interrupt the TF pathway in HUS and conceptually related disorders of arterial / arteriolar thrombosis.