Vascular Thiol Isomerases in Thrombosis

项目概要/摘要 巯基异构酶在血栓形成中起关键作用，如在几个独立的研究中所证实的。 使用巯基异构酶、抑制性抗体和小分子的基因缺失的血栓形成模型 分子拮抗剂。靶向蛋白质二硫键异构酶（PDI）的治疗目前处于II/III期 研究，以评估其作为抗血栓的有效性和安全性。然而，尽管有令人信服的证据表明， 异构酶在血栓形成中起作用，关于这些酶如何促进血栓形成知之甚少。 血栓形成血管巯基异构酶- PDI、ERp 5和ERp 57-控制血管巯基的形成和切割。 变构二硫键和修饰蛋白质功能。修改的性质及其影响 蛋白质的功能在很大程度上尚未研究。利用血栓的分子、细胞和整体动物研究 形成，我们将解决关键的问题，巯基异构酶在血栓形成中的作用。在 目的1，我们将确定巯基异构酶如何逃脱滞留在内质网， 异构酶在血小板和内皮细胞中被组织和包装成颗粒， 调节这些细胞中巯基异构酶的胞吐作用的机制。在目标2中，我们将确定 将血管巯基异构酶与血小板血栓形成和纤维蛋白 一代该起始途径的蛋白质组分将使用基于机制的动力学方法鉴定。 捕获以鉴定来自血小板、血浆和内皮细胞的底物。在目标3中，我们将评估 通过一氧化氮调节硫醇异构酶并测试硫醇异构酶控制一氧化氮的能力 在血栓形成期间。我们将研究血小板受体如何被巯基异构酶介导的 去亚硝基化我们还将在活小鼠中对一氧化氮和活性氧进行成像，以评估 通过硫醇异构酶控制一氧化氮和氧化应激。该项目将提供新的基础 了解巯基异构酶在血管损伤后如何释放，它们如何修饰血管蛋白 基质，以及它们是如何被调节的。鉴于对细胞外巯基异构酶的了解很少- 介导的途径，这些研究将提供重要的信息，开始解密这一重要的， 但很大程度上未被探索的血栓形成层。

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.