A Natural Cysteine Variant Reveals Disulfide-Dependent Structural and Functional Heterogeneity of Factor XI

Background: Factor XI (FXI) is a coagulation protease that contributes to thrombin amplification. FXI circulates as a disulfide-linked homodimer whose structure and function depend on a complex cysteine network comprising 17 intrachain disulfide bonds, free Cys29, and the interchain Cys339-Cys339 disulfide bond. Objectives: . Methods: Clinical, biochemical, genetic, and functional studies were performed in the proband and relatives. Thrombin generation was triggered with tissue factor and silica. Recombinant wild-type (WT) and mutant FXI were expressed in HEK293 cells and evaluated by immunoblotting, functional assays, and mutagenesis, including liquid chromatography-electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) under non-reducing thiol-alkylating conditions. Results: The proband showed a mild prekallikrein deficiency and severe FXI deficiency with residual procoagulant FXI (FXI:C) activity (7%) and two disulfide-linked FXI species with different electrophoretic mobilities in plasma, which collapsed into a single entity under reducing conditions. Recombinant expression reproduced this heterogeneity in dimeric and apparent monomeric species. Mutagenesis did not support Cys295-mediated interchain disulfide formation or involvement of Cys29 in the aberrant dimer. Proteomic analysis discarded interaction with other proteins and identified Carbamidomethylation (CAM)-modified Cys317, Cys321, and Cys193 exclusively in mutant FXI, indicating altered thiol availability. Contact pathway-dependent FIX activation in the proband was associated with impaired thrombin generation, whereas tissue factor-triggered thrombin generation was preserved. The proband had no spontaneous bleeding and underwent two procedures uneventfully with prophylaxis. Conclusion: The p.Phe295Cys variant is associated with disulfide-dependent structural heterogeneity and impaired FXI function. The findings support perturbation of the native FXI disulfide network but do not establish the precise alternative disulfide connectivity.