Abstract Number: OC 38.5
Meeting: ISTH 2022 Congress
Background: Oxidative stress contributes to thrombosis in atherosclerosis, inflammation, infection and malignancy. How oxidants induce thrombus formation, however, is poorly understood. Protein disulfide isomerase (PDI) is a prothrombotic redox-responsive enzyme. Yet the redox form of PDI responsible for thrombus formation is not known.
Aims: We hypothesized that oxidized PDI (oxPDI) transduces oxidative stress into a prothrombotic response.
Methods: Biochemical protein and enzymatic assays, platelet aggregometry, and in vivo murine thrombosis studies were used.
Results: Exposure of PDI to oxidants such as H2O2 and oxLDL resulted in loss of free thiols in PDI. Oxidants promoted generation of sulfenylated PDI, which spontaneously converted to disulfided PDI capable of transferring disulfides to substrate proteins. Evaluation of mutant PDIs and PDI fragments showed that sulfenylation required both PDI catalytic and substrate binding domains. While PDI alone failed to stimulate platelet aggregation, it augmented platelet aggregation induced by oxLDL, but not other agonists. The evaluation of oxPDI in vivo is complicated by the fact that infused oxPDI is quickly modified by the plasma redox environment. To circumvent this issue, we used LOC14, a compound that selectively binds PDI and forces it into an oxidized state. Evaluation by smFRET confirmed the oxidized conformation of PDI following LOC14 incubation and differential alkylation demonstrated the disulfided state. LOC14 enhanced platelet aggregation and this augmentation was inhibited by anti-PDI antibodies. When infused into mice, LOC14 stimulated both platelet accumulation (4.6-fold) and fibrin formation (2.4-fold) following laser injury. OxLDL also markedly promoted both platelet accumulation and fibrin formation in this model and inhibition of PDI blocked oxLDL-mediated thrombosis.
Conclusion(s): Structural elements within different PDI domains enable efficient conversion of oxygen free radicals to disulfides and transfer of these disulfides to substrate proteins. oxPDI is the prothrombotic form of the enzyme and serves as a link between oxidant stress and thrombus formation.
To cite this abstract in AMA style:Yang M, Khan S, Chinnaraj M, Scartelli C, Patel S, Carroll K, Pozzi N, Smith B, Flaumenhaft R. Oxidized Protein Disulfide Isomerase Links Oxidative Stress to Thrombus Formation [abstract]. https://abstracts.isth.org/abstract/oxidized-protein-disulfide-isomerase-links-oxidative-stress-to-thrombus-formation/. Accessed March 4, 2024.
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