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

描述（申请人提供）：在体内，血小板在活化内皮上的粘附/聚集可能是通过上调血小板粘附的内皮细胞（EC）分子，特别是1v23（玻连蛋白受体）、ICAM-1、P-选择素和超大血管性血友病因子（vWF）串来进行的，伴随并随后是组织因子（TF）驱动的动脉或小动脉壁血小板血栓形成。必要的 EC 激活可能通过多种途径产生，包括氧化的低密度脂蛋白、细菌毒素和/或细胞因子。目前的工作将检验这一假设和范式，即这些粘附分子的上调允许血小板在内皮上粘附/聚集的启动，然后该过程被 EC 相关 TF 和血源性 TF 放大。血源性 TF 以源自白细胞、血小板和 EC 的微粒形式存在。 EC 相关类型 TF 的上调可能通过多种机制发生，本文研究：a) TF mRNA 稳定性增加和/或 TFPI mRNA 稳定性降低，b) EC 膜外叶上磷脂酰丝氨酸增强，c) EC 相关 TFPI 调节能力降低。我们进一步假设，附壁血小板血栓反过来会充分减慢血流，使活化的内皮细胞呈现出更促凝血的表型。这种范例的一个典型例子是流行性儿童溶血性尿毒症综合征 (HUS)，其中食源性、产生志贺毒素 (STX) 的大肠杆菌会导致全身炎症和血栓形成状态，并产生包括肾功能衰竭、癫痫发作、昏迷和死亡等并发症。在 HUS 体外模型中，我们证明 STX 显着增强人肾小球 EC (HGEC) 单层上的功能性 TF，并显着增强血小板串（与超大 vWF 串相关）和这些单层上的小聚集体形成。具体目标 1 是确定将活化的 HGEC 暴露于 STX 时观察到的 TF 增强机制。具体目标 2 是证明 STX 通过上调上述粘附分子以及上调 TF 通路，增强活化 HGEC 上流动血液中的血小板粘附/聚集。这些研究应阐明针对 HUS 的治疗干预的潜力，旨在阻断粘附分子和阻断 EC 表面和/或血源性 TF。公共健康相关性：我们将检验以下假设：血管内皮（血管壁）上存在某些与血小板粘附的分子，是引发发炎血管壁上血小板血栓形成的原因。然后，这种血栓形成被称为组织因子的凝血活性放大，组织因子表达在同一发炎内皮的表面。我们认为，这一假设的主要应用是流行性儿童溶血性尿毒症综合征（HUS），其中食源性产毒素大肠杆菌会导致一种疾病状态，并伴有肾衰竭、癫痫发作、昏迷和死亡等并发症。我们的研究有望阐明针对 HUS 的治疗潜力，即通过阻断上述粘附分子和血管壁组织因子来治疗 HUS。

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.