Abstract Body: Background: Oxidative stress is characteristic of systemic inflammatory conditions. Oxidants in oxidative stress promote thrombosis through poorly defined mechanisms. Protein disulfide isomerase (PDI) is an enzyme sensitive to oxidation. PDI supports thrombosis when released from blood and vascular cells by regulating disulfides. Yet the redox state of PDI mediating thrombus formation is unknown.

Hypothesis: We hypothesize that oxidized PDI promotes thrombosis in oxidative stress.

Method: We used enzymatic assays and mutagenesis approaches to understand the biochemistry of PDI oxidation. We employed intravital microscopy to assay the in vivo relevance of oxidation by imaging platelet and fibrin dynamics following laser- or chemical-injury to the arterial walls in mice. We used multi-complimentary models of oxidative stress along with PDI functional blocking antibody, morpholino PDI knockdown (KD), and platelet PDI deficient (KO) mice.

Results: Oxidizing PDI with peroxides increased sulfenic acid formation, an oxidative cysteine modification, by 2.5 fold on the catalytic cysteines and converted PDI to a disulfide-making oxidase. Sulfenylation of C56 requires positively charged R120 and T101 amino acids. This is akin to peroxiredoxin 5, a protein that uses sulfenic acids as part of the peroxide detoxifying mechanism. Structural comparison of peroxiredoxin with PDI revealed similar regulatory elements. Using the PDI selective oxidizing agent LOC14, platelet and fibrin accumulation were enhanced by 4.6 and 2.4-fold in wildtype mice after laser injury to the cremaster arteriole walls (n > 24 injuries). PDI blocking antibody prevented this prothrombotic phenotype compared to IgG control. Using the GPX3-/- oxidant stress mice, whole body PDI KD decreased enhance platelet and fibrin accumulation by 77% and 64%. In the ferric chloride carotid artery thrombosis model, oxLDL infusion decreased the time to vessel occlusion to 4.1 min in wildtype mice (n = 4 mice), representing accelerated thrombosis. OxLDL’s thrombogenic phenotype was delayed to 10.9 min in platelet PDI deficiency (n = 4). The infusion of recombinant wildtype PDI in both GPX3-/- PDI KD or oxLDL-infused platelet PDI KO mice restored the enhanced and accelerated thrombus formation. Infusion of catalytically inactive mutant or the sulfenylation-deficient R120D mutant failed to restore the prothrombotic phenotype.

Conclusion: Oxidized PDI promotes thrombus formation through specific cysteine modification.