SERPINC1 p.M313T variant induces aberrant O-Glycosylation and leads to conformational instability-related transient antithrombin deficiency

Backgrounds: Congenital antithrombin (AT) deficiency, primarily caused by SERPINC1 variants, is a major risk factor for venous thromboembolism (VTE). We previously reported the SERPINC1 p.M313T variant in three VTE probands with normal AT activity and antigen levels. This study aims to elucidate its pathogenetic mechanism. Methods: Thrombin generation test (TGT), thermal stability, native-urea PAGE, in vitro protein expression, and enzymatic assays were performed. Glycosylation analysis was conducted using glycosidase treatment, and structural analysis was performed through molecular dynamics simulation. Results: AT in probands' plasma samples exhibited reduced thermostability and increased proportions of denatured and latent forms compared with normal pooled plasma. The recombinant AT-M313T protein exhibited increased inhibitory activity, consistent with findings in proband plasma based on AT activity and TGT. Despite this enhanced activity, the mutant protein demonstrated reduced thermostability and a marked tendency to transition into the latent form, potentially predisposing carriers to thrombosis under stress conditions. These characteristics may result from the introduction of abnormal O-linked glycosylation within the breach region, confirmed in both plasma-derived and recombinant AT. Molecular dynamics simulation revealed a less compact structure, with increased spacing in the shutter region and enhanced flexibility of the reactive center loop. Conclusions: The SERPINC1 p.M313T variant exhibits dual characteristics of high inhibitory activity and low structural stability, which together contribute to the transient AT deficiency. These findings suggest that AT deficiency may be underdiagnosed and highlight the importance of integrating techniques such as native-urea PAGE into standard diagnostic workflows to identify variants associated with structural abnormalities.