Oxidized PDI promotes thrombus formation in oxidative stress

Protein disulfide isomerase (PDI) functions in thrombus formation in vivo and represents a viable target for antithrombotic therapy. PDI is a redox sensor that can either reduce or oxidize substrates depending on the redox environment. Yet whether PDI functions primarily as a reductase or an oxidase in the context of thrombus formation is unknown. We have used pharmacological approaches and PDI mutants to determine how the redox state of PDI affects thrombus formation. LOC14, which inhibits PDI reductase activity and induces PDI oxidation, promoted thrombus formation in arteries exposed to FeCl3 and enhanced injury-induced platelet accumulation and fibrin formation in cremaster arterioles. Substitution of a single sulfur atom with oxygen in LOC14 reversed these prothrombotic effects. Blocking antibodies targeting PDI also reversed the effect of LOC14. Evaluation of sulfenylation-mediated PDI oxidation using C53A, C56A, R120D and T101A PDI mutants showed that the sulfenylation mechanism of PDI resembles that of H2O2 reduction by peroxiredoxins. These studies identified PDI mutants that failed to undergo H2O2-mediated oxidation, but showed normal reductase activity. When tested in vivo, either wild-type PDI or the R120D mutant fully restored normal thrombus formation following morphilino-induced knockdown of PDI. In contrast, the R120D mutant PDI was unable to fully restore thrombus formation in the setting of oxidative stress induced in mice with genetic deletion of glutathione peroxidase 3 null (GPx3-/-). These studies show that PDI-catalyzed oxidization drives thrombus formation in vivo and demonstrate a mechanism of peroxide-mediated oxidation of PDI that contributes to the prothrombotic response of oxidative stress.