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

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. This study aimed to characterize the structural and functional consequences of the previously undescribed FXI p.Phe295Cys variant identified in a hemizygous patient with a de novo 7-Mb deletion involving F11 and KLKB1. 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. 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. 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.