Thiol isomerase ERp46 catalyzes the disulfide formation of coagulation factor XII enhancing its activity

Protein disulfide isomerases (PDIs) are a family of thiol oxidoreductases that catalyze the oxidation, reduction, and isomerization of disulfide bonds. While some PDIs enhance arterial thrombosis, their roles in coagulation system remain largely unknown.

The effect of a thiol blocker N-ethylmaleimide and a reducing agent dithiothreitol on factor XII activity was measured by chromogenic assay, activated partial thromboplastin time and thrombin generation assay. Redox states of FXII disulfides were determined by thiol labeling and mass spectrometry. Functional disulfides were evaluated through cysteine mutagenesis of FXII and predicting structural function via molecular dynamics simulations. Thiol modification and kinetic trapping were used to identify ERp46 substrates. The inferior vena cava stenosis model assessed the roles of ERp46 and FXII in venous thrombosis.

N-ethylmaleimide significantly prolonged activated partial thromboplastin time and inhibited FXIIa chromogenic activity, while dithiothreitol inhibited clotting, thrombin generation, and substrate HK cleavage. Of the PDIs tested, only oxidized ERp46 enhanced FXII activity. Screening identified Cys513-Cys529 and Cys540-Cys571 as crucial disulfides for FXII function. Notably, half of Cys540-Cys571 were in partially disulfide-bonded form. ERp46 oxidized Cys540-Cys571 and increased FXII activity. In vivo, ERp46 deficiency reduced venous thrombus growth, with no additive effect observed in mice with combined ERp46 and FXII deficiency. Only wild-type FXII protein, not FXII/C540S-C571S mutant, restored venous thrombus growth in FXII-deficient mice.

These findings reveal a novel redox-regulatory mechanism for FXII activity and identify the critical role of ERp46 in oxidization of Cys540-Cys571 disulfide, facilitating the activation of FXII and intrinsic coagulation pathway.