OC 10.5 - Protein Disulfide Isomerase Links Sulfenylation to a Prothrombotic Phenotype in Glutathione Peroxidase 3-Deficient Oxidative Stress Mice

Saturday, June 22, 2024

14:00 – 14:15 ICT

Room: 209 A-D

Presenting Author(s)

Moua Yang, PhD

Instructor
Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States

Co-Author(s)

AP

Anika Patel

Medical Student
Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
CS

Christina Scartelli

Research Assistant II
Beth Israel Deaconess Medical Center
Boston, Massachusetts, United States
KC

Kate S. Carroll

Professor
UF Scripps Biomedical Research
Jupiter, Florida, United States
DH

Diane E. Handy

Assistant Professor
Brigham and Women's Hospital
Boston, Massachusetts, United States
JL

Joseph Loscalzo

Hersey Distinguished Professor of the Theory and Practice of Physics
Brigham and Women's Hospital
Boston, Massachusetts, United States
Nicola Pozzi

Associate Professor
Saint Louis University School of Medicine
St. Louis, MO 63104, Missouri, United States
Robert Flaumenhaft, MD, PhD

Professor
BIDMC/Harvard Medical School
Boston, Massachusetts, United States

Background: Glutathione Peroxidase 3 (GPX3) is an extracellular anti-oxidative seleno-enzyme. Its deficiency or polymorphism in mice or human increases the risk of stroke through poorly understood mechanisms that include increased oxidant levels. Protein disulfide isomerase (PDI) is a prothrombotic redox-responsive enzyme, the oxidized form of which enhances thrombus formation. How oxidized PDI enhance thrombosis in oxidative stress is poorly understood.

Aims: We hypothesized that PDI senses the redox environment through specific oxidation events in glutathione peroxidase 3 deficiency to enhance thrombus formation.

Methods: Biochemical protein and enzymatic assays, morpholino knockdown (KD), and in vivo murine thrombosis studies were used.

Results: We previously found that the active site CGHC motif in the PDI a domain is sulfenylated during the oxidative stress of dyslipidemia, augmenting thrombus formation. Using site directed mutagenesis, we found that C56 is sensitive to oxidation. The proximal conserved T101, R120, and E47 nearby the CGHC motif is required for efficient sulfenylation of PDI by oxidants by stabilizing the thiolate anion (Figure 1A). Mutation of these amino acids still preserves oxidoreductase activity despite sulfenylation deficiency (Figure 1B). Using translational-blocking morpholino KD of PDI, thrombus formation was impaired in PDI-KD wildtype mice that could be rescued by the infusion of recombinant PDI. GPX3 deficiency is prothrombotic with enhanced platelet and fibrin accumulation compared to wildtype mice and KD of PDI prevents the augmented thrombogenic phenotype. Infusion of PDI in PDI KD GPX3 deficient mice restored thrombus formation. In contrast, infusion of a catalytically inactive PDI or sulfenylation-deficient R120D mutant failed to restore thrombosis (Figure 2A-B).

Conclusion(s): The prothrombotic phenotype observed in oxidative stress is sensed by PDI through specific structural elements (R120, T101, E47) by converting oxidants to sulfenic acids within the CGHC motif. Oxidation of PDI thus serves as the thrombogenic mechanism in antioxidant deficiency and could potentially be an anti-thrombotic target.