JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE) ›› 2021, Vol. 41 ›› Issue (9): 1133-1141.doi: 10.3969/j.issn.1674-8115.2021.09.001

• Innovative research team achievement column •    

A novel antithrombotic antibody targeting the binding sites of the coagulation factor FⅨa-FⅧa complex

Tian-yao SUN1(), Shi-feng JIANG2(), Qin XU2, Jun-ling LIU1, Su-ying DANG1(), Xue-mei FAN1()   

  1. 1.Department of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
    2.Department of Bioengineering, School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China
  • Received:2021-03-09 Online:2021-08-24 Published:2021-08-24
  • Contact: Su-ying DANG,Xue-mei FAN;;;
  • Supported by:
    National Natural Science Foundation of China(81970123);Innovative Research Team of High-level Local Universities in Shanghai(SSMU-ZDCX20180101)

Abstract: Objective

·To prepare a monoclonal antibody targeting coagulation factor Ⅸa (FⅨa), a key factor in endogenous coagulation pathways, and study its antithrombotic roles and mechanisms.


·Immunization of mice, hybridoma technology, cell expression and purification were used to prepare the anti-FⅨa monoclonal antibodies of high purity. The monoclonal antibodies with high affinity for FⅨa were screened by enzyme-linked immunosorbent assay. The activated partial thromboplastin time (APTT) and prothrombin time (PT) were used to evaluate the antithrombotic effects of the monoclonal antibody. And then chromogenic substrate method was used to detect the effect of monoclonal antibody on the enzyme activity of FⅨa. The method of computer simulation of protein-protein docking was adopted to predict the possible binding sites between FⅨa and the antibody, and the binding site, was verified by competitive experiments (indirectly through the chromatic substrate method).


·FⅨa-4, an anti-FⅨa monoclonal antibody with high affinity, was generated. Although FⅨa-4 did not affect PT and the enzyme activity of FⅨa, it significantly prolonged APTT to 88.8 s, which was 3.5 times of the control group (25.5 s), in a concentration-gradient dependence. The protein-protein docking prediction results revealed that FⅨa-4 did not directly bind to substrate catalytic sites of FⅨa, but occupied the binding region of FⅨa and FⅧa. Competitive experiments further verified the above results. FⅨa-4 inhibited FⅩa production in a dose-dependent manner, almost completely inhibiting FⅩa production at the concentrations of 400 pmol/L, and FⅧa could correct the inhibition effect of the antibody up to nearly 50%.


·The monoclonal antibody against FⅨa-4 is obtained. FⅨa-4 competes with FⅧa to bind FⅨa, and inhibits the conversion of FⅩ to FⅩa which is catalyzed by FⅧa-FⅨa complex; it plays an antithrombotic role mainly by inhibiting endogenous coagulation pathway.

Key words: coagulation factor Ⅸa (FⅨa), monoclonal antibody, activated partial thromboplastin time (APTT), enzyme activity, coagulation factor Ⅷa (FⅧa), antithrombosis

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