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Thromb Haemost
DOI: 10.1055/a-2509-0511
Coagulation and Fibrinolysis

Structural Conformation and the Binding of Factor VIII R2159C (FVIII-Ise) Mutated in the C1 Domain to Phospholipid

Kuniyoshi Mizumachi
1   Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
,
Masahiro Takeyama 
1   Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
2   Division of Hemophilia, National Hospital Organization, Osaka National Hospital, Osaka, Japan
,
Kaoru Horiuchi
1   Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
,
Keiji Nogami
1   Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
› Institutsangaben Funding This research was supported by the Japan Agency for Medical Research and Development (AMED) under Grant Number 21fk0410037, supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) to KN (21K07804, 24K10935).
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Abstract

Background We previously identified a factor (F)VIII molecular defect associated with an R2159C mutation in the C1 domain (named “FVIII-Ise”) together with undetectable FVIII antigen (FVIII:Ag) levels measured by two-site sandwich ELISA using an anti-C2 domain alloantibody (alloAb). The patient had clinically mild hemophilia A, and his reduced FVIII:C correlated with FVIII:Ag measured by ELISA using monoclonal antibodies (mAbs) with A2 and A2/B domain epitopes, suggesting that the R2159C mutation modified C2 domain antigenicity.

Aim To investigate functional and structural characteristics of the FVIII-R2159C mutant.

Methods and Results ELISAs using a previous anti-C2 domain alloAb confirmed that the antigen level of recombinant FVIII-R2159C mutant prepared in BHK cells was 56% lower relative to wild-type (WT), consistent with our earlier reports. This anti-C2 domain alloAb competitively inhibited FVIII and anti-C1 domain mAb binding, indicating the involvement of specificity for C1 and C2 epitopes. The K m for FVIII-R2159C with FIXa or FX in the tenase complex was similar to that of FVIII-WT. Thrombin- and FXa-catalyzed cleavage reactions of FVIII-R2159C were similar to those of WT. The K d for FVIII-R2159C binding to phospholipids was moderately greater than for FVIII-WT, however, while there were no significant differences in von Willebrand factor binding. In silico molecular dynamic simulation analyses revealed subtle differences between FVIII-WT and FVIII-R2159C.

Conclusion The FVIII-R2159C mutation was not different from FVIII-WT in interactions with FIXa, FX, and thrombin, but reduced binding potential to phospholipids and to an anti-C1/C2 domain alloAb was evident apparently due to subtle changes in conformational structure.

Keywords

factor VIII - C1 domain - phospholipid - binding site - structure

Authors' Contribution

K.M. performed the experiments, analysis, interpreted the data, and made the figures; M.T. designed the research, performed the experiments, analyzed and interpreted the data, made the figures, wrote the paper, and approved the final version to be published; K.H. performed the experiments; K.N. designed the research, analyzed and interpreted the data, made the figures, wrote the paper, and edited the manuscript.


Supplementary Material



Publikationsverlauf

Eingereicht: 02. Juli 2024

Angenommen: 30. Dezember 2024

Artikel online veröffentlicht:
21. Januar 2025

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