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. However, whether PDI functions primarily as a reductase or an oxidase in the context of thrombus formation remains unknown. Here, we used pharmacological and genetic approaches along with PDI mutants to determine how the PDI redox state affects thrombus formation. LOC14, which inhibits PDI reductase activity and induces PDI oxidation, promoted thrombus formation in arteries exposed to ferric chloride and enhanced laser injury-induced platelet accumulation and fibrin formation in cremaster arterioles. Blocking antibodies targeting PDI reversed the prothrombotic effect of LOC14. Evaluation of sulfenylation-mediated PDI oxidation using the C53A, C56A, R120D, and T101A PDI mutants showed that the sulfenylation mechanism of PDI resembles that of hydrogen peroxide (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 morpholino-induced knockdown of PDI or in mice with platelet-specific knockout of PDI. In contrast, the sulfenylation-impaired R120D mutant PDI was unable to fully restore thrombus formation in the setting of oxidative stress induced in mice by genetic deletion of glutathione peroxidase 3 or by infusion of oxidized low-density lipoproteins. These studies show that PDI-catalyzed oxidation drives thrombosis and demonstrates a mechanism of peroxide-mediated oxidation of PDI that contributes to the prothrombotic response of oxidative stress.