Functional Tissue Factor on Activated Endothelium

描述（来自申请人摘要的逐字）：扩增和传播 局部血栓形成的可能性取决于局部微环境。 由上游刺激的内皮细胞产生的活化因子Xa和IXa (EC)在主要的剪切应力条件下，以及任何血液传播的 组织因子（TF）和组织因子途径抑制物（TFPI）由这些细胞分泌 相同的EC可诱导血栓形成。调查人员计划检验这一假设 表达TF EC促进动脉和小动脉中的局部壁血栓， 并且剪切应力从基线水平的降低决定了 刺激EC成为功能性促凝剂。调查人员将 关注滋贺毒素对溶血性尿毒综合征血管壁的损伤 (HUS)作为一般内皮损伤的范例。具体目标1设计 为了显示对于同时暴露于剪切应力和TNF-α， 内皮细胞通过立即增加其功能性TF的表达， 它分泌TFPI，只有在几个小时后才能下调 TF基因相反，将剪切应力降低到接近停滞水平允许 这样的内皮变成促凝血剂。转录的重要性 Egr-1、Sp1、NFKb和AP-1因子在内皮细胞移植后上调TF mRNA中的作用 细胞暴露于剪切应力和TNF-α，单独和一起， 考察目的2是评估血小板对肾小球的粘附/聚集 内皮细胞被滋贺毒素损伤，并暴露于流动的血液，以及 确定内皮暴露后滋贺毒素的作用机制 TNF-α，进一步增加功能性TF，如先前通过 investigators.一种候选机制是对预先形成的TF进行解密。目标3是 为了证明在真实的时间和受控的血流条件下，TF 内皮和/或白细胞起源与生长的血小板共定位 聚集体，并存在于粘附于滋贺的血小板聚集体上 灵长类动物肾段毒素损伤的肾小球内皮细胞 HUS模型这些目标包括测量因子Xa和TFPI TF和TFPI抗原的免疫金标记、RT-PCR扩增 TF和TFPI mRNA的产生，以及核径流研究和电泳 迁移率变动分析。研究人员还将采用免疫胶体标记法 对于纤维蛋白原涂覆玻璃上血小板聚集体表面的纤维蛋白， 肾小球内皮血小板和TF共定位的实时成像 将通过双标记落射荧光视频显微镜实现。一 更清楚地了解剪切应力调节 激活的内皮表达TF对于血管内皮细胞的发展是重要的。 策略中断TF途径在HUS和概念上相关 动脉/小动脉血栓形成疾病。

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.