Thromb Haemost
DOI: 10.1055/s-0039-1683442
Coagulation and Fibrinolysis
Georg Thieme Verlag KG Stuttgart · New York

The Disulfide Bond between Cys22 and Cys27 in the Protease Domain Modulate Clotting Activity of Coagulation Factor X

Fang Li*
1  State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
,
Changming Chen*
2  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
,
Si-Ying Qu
1  State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
,
Ming-Zhu Zhao
3  Instrumental Analysis Center, Shanghai Jiao Tong University, Shanghai, China
,
Xiaoling Xie
2  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
,
Xi Wu
2  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
,
Lei Li
2  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
,
Xuefeng Wang
2  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
4  Faculty of Laboratory Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China
,
Qiulan Ding
2  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
,
Qin Xu
1  State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
,
Dong-Qing Wei
1  State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China
,
Wenman Wu
2  Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
4  Faculty of Laboratory Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China
5  Collabortive Innovation Center of Hematology, Shanghai Jiao Tong University School of Medicine, Shanghai, China
› Author Affiliations
Funding This study was supported by the National Natural Science Foundation of China (81570115, 31770772, 61832019 and 61503244), the Key Research Area Grant 2016YFA0501703 of the Ministry of Science and Technology of China, and Joint Research Funds for Translational Medicine at Shanghai Jiao Tong University (ZH2018ZDA06).
Further Information

Publication History

10 October 2018

30 January 2019

Publication Date:
27 March 2019 (eFirst)

Abstract

The Cys22-Cys27 disulfide bond of factor X (FX) protease domain is not conserved among coagulation factors and its contribution to the physiological haemostasis and implication in the pathogenesis of haemostatic and thrombotic disorders remain to be elucidated. Mutation p.Cys27Ser was identified in a pedigree of congenital FX deficiency and fluorescence labelling study of transiently transfected HEK293 cells showed accumulation of FX p.Cys27Ser within cell, indicating incompetent secretion partially responsible for the FX deficiency. The clotting activity of FX p.Cys27Ser was decreased to about 90% of wild-type, while amidolytic and pro-thrombinase activities (kcat/Km) determined with recombinant FXa mutant were 1.33- and 4.77-fold lower. Molecular dynamic simulations revealed no major change in global structure between FXa p.Cys27Ser and wild-type FXa; however, without the Cys22-Cys27 disulfide bond, the insertion of newly formed N terminal of catalytic domain after the activation cleavage is hindered, perturbing the conformation transition from zymogen to enzyme. The crystal structure of FXa shows that this disulfide bond is solvent accessible, indicating that its stability might be subject to the oxidation/reduction balance. As demonstrated with FX p.Cys27Ser here, Cys22-Cys27 disulfide bond may modulate FX clotting activity, with reduced FX pertaining less pro-coagulant activity.

Authors' Contributions

F.L. performed molecular dynamic simulations and analysis; C.C. performed protein expression collection and purification and enzyme kinetical analysis; S.Q. and M.Z. contributed to discussion and manuscript revision of MD results; X.W. performed clinical data acquisition; X.X. and L.L. performed phenotype and genetic analysis; X.W. and Q.D. supervised studies conducted in Ruijin Hospital, Shanghai Jiao Tong University; D.W. supervised studies in School of Life Sciences and Biotechnology, Shanghai Jiao Tong university; and Q.X. and W.W. designed experiments, analysed the data and wrote the manuscript. All authors approved the final version of this manuscript.


* Fang Li and Changming Chen equally contributed to this work.


Supplementary Material