OC 25.1 - Thiol isomerase ERp46 catalyzes the disulfide formation of coagulation factor XII enhancing its activation

Background: Redox regulation of disulfide bonds by oxidoreductases has emerged as an important contributor to thrombosis and hemostasis. Protein disulfide isomerases (PDI) family, a subset of oxidoreductases catalyzing thiol-disulfide exchange, has been demonstrated to play a critical role in thrombus formation, however their involvement in coagulation activation needs further investigation.

Aims: To determine whether and how PDIs play a role in the activation of factor XII (FXII).

Methods: FXII activity was measured by chromogenic substrate, activated partial thromboplastin time (aPTT) and thrombin generation assay. Redox state of FXII was determined by 3-(N-maleimido-propionyl) biocytin (MPB) label. Recombinant FXII mutants that disrupt disulfide bonds were expressed in 293T cells and secreted FXII proteins activity was screened. Mechanism-based kinetic trapping was used to identified FXII as a substrate of endoplasmic reticulum protein 46 (ERp46). Molecular dynamics (MD) analysis was employed to analyze the structural effects of disulfide bond disruption. Furthermore, in vivo murine venous thrombosis studies were conducted to evaluate the redox state of FXII and its prothrombotic activity.

Results: A thiol blocker N-ethylmaleimide (NEM) significantly prolonged APTT and inhibited FXIIa chromogenic activity. The reducing reagent dithiothreitol (DTT) inhibited the cleavage of high-molecular-weight kininogen (HK) during contact activation. The oxidized form of ERp46 (ERp46-oxi), but not other PDIs, strongly enhanced FXII activity, and reduced free thiols in FXII. Only FXII/C519S and FXII/C513S were secreted, and FXll/C513S-C529S completely lost FXII activity. ERp46-oxi did not further enhance the activity of FXll/C513S-C529S. In an inferior vena cava stenosis model, ERp46-/- mice showed a significant reduction in venous thrombus weight, along with increased free thiols of FXII at the stenosis site, suggesting that ERp46 is needed for FXII disulfide formation in vivo.

Conclusion(s): ERp46 enhances FXII activation, possibly by catalyzing FXII C513-C529 disulfide formation. The ERp46-FXII coupling could represent a novel pathway in the mechanism of thrombosis.