Vascular Thiol Isomerases in Thrombosis

项目摘要/摘要 硫醇异构酶在血栓形成中起关键作用，正如在几个独立的 利用硫醇异构酶、抑制性抗体和Small基因缺失的血栓形成模型 分子拮抗剂。一种针对蛋白质二硫键异构酶(PDI)的治疗目前处于II/III期 评价其作为抗血栓药的有效性和安全性的研究。然而，尽管有令人信服的证据表明硫醇 异构酶在血栓形成中发挥作用，但人们对这些酶如何参与血栓形成知之甚少。 血栓形成。血管硫醇异构酶-PDI、ERp5和ERp57-控制着 变构二硫键和修饰蛋白质的功能。修改的性质及其影响 蛋白质的功能在很大程度上还没有被研究过。血栓的分子、细胞和整体动物研究 在血栓形成过程中，我们将解决与硫醇异构酶在血栓形成中的作用相关的关键问题。在……里面 目的1，我们将确定硫醇异构酶如何逃脱内质网中的滞留，硫醇如何 异构酶在血小板和内皮细胞中被组织和包装成颗粒，而 调节这些细胞中硫醇异构酶的胞吐作用的机制。在目标2中，我们将确定 血管硫醇异构酶与血小板血栓形成和纤维蛋白联系的途径和机制 一代。这一起始途径的蛋白质组分将使用基于机制的动力学方法进行鉴定 捕获以识别来自血小板、血浆和内皮细胞的底物。在目标3中，我们将评估 一氧化氮对硫醇异构酶的调控及硫醇异构酶控制一氧化氮能力的检测 在血栓形成过程中。我们将研究硫醇异构酶是如何激活血小板受体的。 脱亚硝化。我们还将成像活体小鼠体内的一氧化氮和活性氧物种，以评估 硫醇异构酶对一氧化氮和氧化应激的调控。这个项目将提供新的基础 关于硫醇异构酶在血管损伤后如何释放，它们如何修饰血管蛋白的知识 底物，以及它们是如何被监管的。鉴于对胞外硫醇异构酶的了解很少- 中介途径，这些研究将为开始解密这一关键的， 但在很大程度上还未被探索，形成了血栓层。

Project Summary/Abstract Thiol isomerases serve a critical role in thrombus formation, as demonstrated in several independent models of thrombus formation using genetic deletion of thiol isomerases, inhibitory antibodies, and small molecule antagonists. A therapy targeting protein disulfide isomerase (PDI) is currently in phase II/III studies to evaluate its efficacy and safety as an antithrombotic. Yet despite compelling evidence that thiol isomerases function in thrombus formation, little is known about how these enzymes contribute to thrombosis. Vascular thiol isomerases - PDI, ERp5, and ERp57 - control the formation and cleavage of allosteric disulfide bonds and modify protein function. The nature of the modifications and how they impact protein function are largely unstudied. Using molecular, cellular and whole animal studies of thrombus formation, we will address critical questions related to the role of thiol isomerases in thrombus formation. In Aim 1, we will determine how thiol isomerases escape retention in the endoplasmic reticulum, how thiol isomerases are organized and packaged into granules in platelets and endothelial cells, and the mechanisms that regulate the exocytosis of thiol isomerases in these cells. In Aim 2, we will identify the pathways and mechanisms that link vascular thiol isomerases to platelet thrombus formation and fibrin generation. Protein components of this initiation pathway will be identified using mechanism-based kinetic trapping to identify substrates from platelets, plasma, and endothelial cells. In Aim 3, we will evaluate the regulation of thiol isomerases by nitric oxide and test the ability of thiol isomerases to control nitric oxide during thrombus formation. We will study how platelet receptors are activated by thiol isomerase-mediated denitrosylation. We will also image nitric oxide and reactive oxygen species in live mice to assess the control of nitric oxide and oxidative stress by thiol isomerases. This project will provide new fundamental knowledge of how thiol isomerases are released following vascular injury, how they modify vascular protein substrates, and how they are regulated. Given the paucity of knowledge of extracellular thiol isomerase- mediated pathways, these studies will provide essential information for beginning to decrypt this essential, yet largely unexplored, layer of thrombus formation.