PB0315 - Structural instability of antithrombin caused by SERPINC1 p.Met313Thr variant is associated with venous thromboembolism

Sunday, June 23, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Changming chen, PhD candidate

Technician
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
Shanghai, Shanghai, China (People's Republic)

Co-Author(s)

Xi Wu, MD

Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Shanghai, Shanghai, China (People's Republic)
Lei Li, Ph.D

Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Shanghai, Shanghai, China (People's Republic)
JD

Jing Dai

Professor; engaged in the study of the mechanism of hemorrhage and thrombosis
Department of Clinical Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Shanghai, Shanghai, China (People's Republic)
XW

Xuefeng Wang

Professor
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
Shanghai, Shanghai, China (People's Republic)
QD

Qiulan Ding

Professor
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Shanghai, Shanghai, China (People's Republic)

Background: Inherited antithrombin (AT) deficiency, caused by mutations in SERPINC1, is associated with venous thromboembolism (VTE). Routine functional methods may miss certain pathogenic variants, emphasizing the importance of genetic diagnosis. In this study, 3 unrelated VTE patients with normal AT activities and antigen levels were identified with SERPINC1 p.Met313Thr (c.938T>C) variant, which was previously reported to be associated with VTE, but the detailed mechanism had not been illustrated.

Aims: To determine the pathological VTE mechanism of p.Met313Thr variant through in vitro experiments.

Methods: The Ethics Committee of Ruijin Hospital approved the study. All related individuals gave their informed consent. Anti-FXa, anti-FIIa activity and antigen levels of AT were tested in plasma from probands. Wild-type AT (WT-AT) and mutant-AT (M313T-AT) proteins were expressed in vitro and analyzed by SDS-PAGE under non-reduced condition. The second-order association rate constants (k2) and stoichiometry of inhibition (SI) towards thrombin or FXa by WT-AT and M313T-AT in the absence and presence of pentasaccharide (H5) or heparin were measured. The thermal stability was tested after incubating at 39℃. Latent AT formation was also performed at 50℃ and detected by Western Blot (WB).

Results: Three probands all showed normal AT activities and antigen levels (Ag 109.7±6.4%, Anti-FXa 106.7±10.1%, Anti-FIIa 92.0±5.8%). M313T-AT migrated slightly slower than WT-AT in non-reduced SDS-PAGE and showed higher k2 in both the absence and presence of H5 or heparin (Table 1). The SI values showed no significant difference. The activity of M313T-AT decreased dramatically over time at 39℃, while that of WT-AT remained stable (Figure 1). WB showed more latent AT formation by M313T-AT as well.

Conclusion(s): p.Met313Thr variant showed thermal instability and propensity for latent AT formation. This might suggest a transient AT deficiency, leading to VTE under certain stress conditions, such as fever or infectious diseases.