Tissue Factor, Flow, and Platelet Adhesion/Aggregation on Activated Endothelium

描述（由申请人提供）：在体内，活化的内皮细胞上的血小板粘附/聚集可能通过粘附血小板的内皮细胞（EC）分子的上调进行，特别是1v23（玻璃体粘连蛋白受体）、ICAM-1、p -选择素和超大型血管性血液病因子（vWF）链，伴随着并跟随组织因子（TF）驱动的动脉或小动脉壁上血小板血栓形成。必要的EC激活可能通过几种途径出现，包括氧化低密度脂蛋白、细菌毒素和/或细胞因子。目前的工作将测试这些粘附分子的上调允许血小板在内皮上粘附/聚集的假设和范式，这一过程随后被ec相关的TF和血源性TF放大。血源性TF以来自白细胞、血小板和内皮细胞的微粒的形式存在。EC相关型TF的上调可能通过多种机制发生，本文研究了：a) TF mRNA稳定性增加和/或TFPI mRNA稳定性降低，b) EC膜外小叶磷脂酰丝氨酸增强，c) EC相关TFPI调节能力降低。我们进一步假设，壁血小板血栓反过来会减慢血流，从而使活化的内皮细胞具有更强的促凝表型。这种模式的一个主要例子是流行的儿童溶血性尿毒症综合征（HUS），其中食源性志贺毒素（STX）产生的大肠杆菌导致全身性炎症和血栓形成前状态，并伴有并发症，包括肾衰竭、癫痫发作、昏迷和死亡。在溶血性尿毒综合征的体外模型中，我们已经证明STX显著增加了人肾小球内皮细胞（HGECs）单层上的功能性TF，并显著增强了血小板串（与超大vWF串相关）和这些单层上小聚集体的形成。具体目的1是确定激活的hgec暴露于STX时观察到的TF增强的机制。具体目的2是证明STX通过上调上述粘附分子，也通过上调TF途径，增强活化的hgcs在流动血液中的血小板粘附/聚集。这些研究应该阐明针对溶血性尿毒症的治疗干预的潜力，这些干预旨在阻断粘附分子和阻断EC表面和/或血源性TF。公共卫生相关性：我们将验证以下假设：血管内皮（血管壁）上粘附血小板的某些分子是炎症血管壁上血小板血栓形成的原因。这种血栓的形成随后被称为组织因子的凝血活性放大，组织因子在同一发炎的内皮细胞表面表达。我们认为，这一假说的主要应用是流行的儿童溶血性尿毒症综合征（HUS），其中食源性产生毒素的大肠杆菌导致疾病状态，并发症包括肾衰竭，癫痫发作，昏迷和死亡。我们的研究有望阐明针对阻断上述粘附分子和血管壁组织因子的溶血性尿毒症治疗的潜力。

DESCRIPTION (provided by applicant): In vivo, platelet adhesion/aggregation on activated endothelium may proceed via upregulation of endothelial cell (EC) molecules adhesive for platelets, especially 1v23 (the vitronectin receptor), ICAM-1, P-selectin, and ultra large von Willebrand factor (vWF) strings, accompanied and followed by tissue factor (TF)-driven mural platelet thrombosis in arteries or arterioles. The necessary EC activation may arise via several routes, including oxidized low density lipoproteins, bacterial toxins, and/or cytokines. The present work will test the hypothesis and paradigm that upregulation of these adhesion molecules permits the initiation of platelet adhesion/aggregation on endothelium, a process which is then amplified by both EC-associated TF and blood- borne TF. Blood-borne TF exists in the form of microparticles derived from leukocytes, platelets, and ECs. Upregulation of the EC-associated type of TF may take place by a variety of mechanisms, studied here: a) increases in TF mRNA stability and/or decreases in TFPI mRNA stability, b) enhancement of phosphatidylserine on the EC membrane outer leaflet, and c) reduction in the regulatory ability of EC-associated TFPI. We further hypothesize that mural platelet thrombi, in turn, slow blood flow sufficiently to allow activated endothelium to assume an even more procoagulant phenotype. A prime example of this paradigm is the epidemic childhood hemolytic uremic syndrome (HUS), in which food-borne, Shiga toxin (STX)-producing E. coli lead to a systemic inflammatory and prothrombotic state, with complications which can include renal failure, seizures, coma, and death. In an in vitro model of HUS we have demonstrated that STX significantly augments functional TF on monolayers of human glomerular ECs (HGECs), and markedly enhances platelet strings (associated with ultra large vWF strings) and small aggregate formation on these monolayers. Specific Aim 1 is to determine the mechanism(s) of TF augmentation observed with exposure of activated HGECs to STX. Specific Aim 2 is to demonstrate that STX augments platelet adhesion/aggregation in flowing blood on activated HGECs via upregulation of the above adhesion molecules, but also via upregulation of the TF pathway. These studies should elucidate the potential of therapeutic interventions in HUS directed at blocking adhesion molecules and blocking EC surface and/or blood-borne TF. PUBLIC HEALTH RELEVANCE: We will test the hypothesis that the presence of certain molecules on vascular endothelium (the blood vessel wall) which are adhesive for blood platelets are what initiates platelet thrombus formation on the inflamed blood vessel wall. This thrombus formation is then amplified by the clotting activity known as tissue factor, expressed on the surface of this same inflamed endothelium. A prime application of this hypothesis, we believe, is the epidemic childhood hemolytic uremic syndrome (HUS), in which food- borne toxin-producing E. coli leads to a disease state with complications including kidney failure, seizures, coma, and death. Our studies promise to clarify the potential of treatments for HUS directed at blocking the above adhesive molecules and vessel wall tissue factor.