Project Summary Thiol isomerases (TIs) represent a previously unrecognized layer of regulation of thrombus formation. The role of protein disulfide isomerase (PDI) family TIs in thrombosis has now been shown in animal studies using genetically modified mice, antibodies that block TI activity, and small molecule antagonists of TIs. We have used a PDI inhibitor prophylactically in a phase II study of patients with advanced malignancy to reduce cancer- associated thrombosis. Yet despite the rapid progress in the therapeutic development of PDI antagonists, a comprehensive explanation of how TIs promote clot formation has yet to emerge. TIs are fundamentally endoplasmic reticulum (ER) redox sensors. Fueled by the oxidative environment of the ER, they use oxidants to generate disulfides in nascent proteins. The studies described in this application will evaluate whether this fundamental property of PDI family TIs powers their prothrombotic potential by translating vascular oxidants into a thrombotic response. We will evaluate the hypothesis that oxidants generated in disease states promote PDI sulfenylation and formation of oxidized PDI (oxPDI), which promotes thrombus formation through disulfide formation in extracellular substrates. How the redox environment induces oxidative cysteine modifications in PDI, which in turn control PDI function and enables PDI to oxidize thrombotic targets, will be assessed using novel probes of cysteine modifications and chemical-proteomic approaches (Aim 1). These experiments will define the cysteine modifications that PDI undergoes in the circulation and identify substrates oxidized by oxPDI during platelet activation. Approaches described in Aim 2 will use structure-function studies of PDI mutants and smFRET to define the molecular determinants of oxidant-induced disulfide formation in PDI. PDI mutants deficient in oxidant-induced disulfide formation will be used in murine models of thrombus formation to determine the role of oxidant-induced PDI disulfide formation in thrombosis. How oxPDI is released into the extracellular environment to interact with the thrombotic machinery will be determined by evaluating the role of the unfolded protein response (UPR) in promoting thrombus formation in an alveolus-on-chip model of pulmonary inflammation (Aim 3). These studies will define specific UPR and TI pathways that activate coagulation and determine how their activation causes a prothrombotic transformation of endothelium that results in microvascular occlusion. The premise that inhibition of UPR receptors such as IRE1a, PERK, and ATF6 impairs thrombus formation will be tested. Similarly, PDI and ERp46 will be evaluated as antithrombotic targets. This program will test a new model by which to conceptualize the function of TIs in thrombus formation based on understanding of determinants of redox environment and TI cysteine modifications. Validation of this model will lead to the identification of new biomarkers and therapeu...