Related Experiment Video
Updated: Jun 1, 2025
Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes
Published on: June 3, 2014
Structural Conformation and the Binding of Factor VIII R2159C (FVIII-Ise) Mutated in the C1 Domain to Phospholipid
Kuniyoshi Mizumachi1, Masahiro Takeyama1,2, Kaoru Horiuchi1
1Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.
Abstract:
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.To investigate functional and structural characteristics of the FVIII-R2159C mutant.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.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.
Insights
The R2159C mutation in Factor VIII (FVIII) affects its binding to phospholipids and antibodies, despite normal interactions with other clotting factors. This suggests subtle conformational changes influence FVIII function in hemophilia A.
Area of Science:
- Biochemistry
- Molecular Biology
- Hematology
Background:
- A previously identified Factor VIII (FVIII) mutation (R2159C) in the C1 domain was associated with undetectable FVIII antigen (FVIII:Ag).
- This mutation, termed FVIII-Ise, presented with mild hemophilia A, where FVIII:C correlated with FVIII:Ag measured by specific monoclonal antibodies (mAbs).
- The findings suggested the R2159C mutation altered the antigenicity of the FVIII C2 domain.
Purpose of the Study:
- To investigate the functional and structural characteristics of the FVIII-R2159C mutant.
- To understand how the R2159C mutation impacts FVIII's interactions and overall function.
Main Methods:
- Recombinant FVIII-R2159C mutant was prepared and analyzed using enzyme-linked immunosorbent assays (ELISAs) with specific antibodies.
- Kinetic parameters (Km) for interactions with Factor IXa (FIXa) and Factor X (FX) were determined.
- Cleavage reactions by thrombin and FXa were assessed.
- Binding affinity to phospholipids and von Willebrand factor (vWF) was measured.
- In silico molecular dynamic simulations were performed.
Main Results:
- ELISAs confirmed a 56% lower antigen level for FVIII-R2159C compared to wild-type (WT) using an anti-C2 domain alloantibody.
- The anti-C2 alloantibody binding was competitively inhibited by FVIII and an anti-C1 domain mAb, indicating shared epitopes.
- FVIII-R2159C exhibited similar Km values for FIXa and FX, and comparable cleavage patterns to FVIII-WT.
- A moderately increased dissociation constant (Kd) for phospholipid binding was observed for FVIII-R2159C, while vWF binding remained unchanged.
- Molecular dynamic simulations revealed subtle structural differences between FVIII-WT and FVIII-R2159C.
Conclusions:
- The R2159C mutation does not alter FVIII's interactions with FIXa, FX, or thrombin.
- Reduced binding to phospholipids and a specific anti-C1/C2 domain alloantibody is associated with the R2159C mutation.
- These functional alterations are likely due to subtle changes in the conformational structure of FVIII.
More Related Videos
07:26High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment
Published on: July 18, 2017
13:08Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Related Concept Videos
01:49Conserved Binding Sites
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally...
01:22Protein Folding
01:39Mechanism of Filopodia Formation
Their main function is to guide migrating cells during normal tissue morphogenesis or cancer metastasis by recognizing and making initial contacts with the extracellular matrix. However, they can also act as stationary cell anchors or help to establish communication...
01:30Assembly of Signaling Complexes
Interaction domains in cell signaling
Interaction domains recognize exposed features of their binding partners containing post-translationally modified sequences,...
01:42Phosphoinositides and PIPs
Different phosphoinositides are synthesized and recruited on the cytosolic face of the plasma membrane. The localization of specific phosphoinositides concentrated in separate membrane...