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.