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Updated: Mar 25, 2026
13:08
Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
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Stabilizing interactions between D666-S1787 and T657-Y1792 at the A2-A3 interface support factor VIIIa stability in
M Monaghan1, H Wakabayashi1, A E Griffiths1,2
1Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, NY, USA.
Journal of Thrombosis and Haemostasis : JTH
|February 16, 2016
Summary
Factor VIIIa (FVIIIa) stability is enhanced by specific interactions at the A2-A3 interface. Creating a disulfide linkage between D666 and S1788 significantly boosts FVIIIa stability, reducing decay rates.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Stability
Background:
- Factor VIIIa (FVIIIa) activity and stability depend on the A2 subunit's association with the A1/A3C1C2 dimer.
- Interactions stabilizing this association are not fully understood.
- Previous mutations (D666A, Y1792F) increased FVIIIa decay, indicating residue importance.
Purpose of the Study:
- To identify specific interactions involving residues D666 and Y1792 at the FVIIIa A2-A3 interface.
- To elucidate the role of these interactions in FVIIIa stability and function.
Main Methods:
- Site-directed mutagenesis of FVIIIa residues D666 and Y1792.
- Biochemical analysis of FVIII variant stability and activity.
- Introduction of disulfide bonds via cysteine mutations (D666C/S1788C).
Main Results:
- Identified stabilizing hydrogen bonds between D666-S1788 and T657-Y1792.
- Swap variants (D666S/S1787D, T657Y/Y1792T) showed wild-type stability.
- Single mutations (D666S, S1787D, Y1792T) increased FVIIIa decay; T657Y reduced activity.
- A D666C/S1788C variant with a disulfide linkage exhibited a decay rate 1% of wild-type.
Conclusions:
- Specific hydrogen bonds (D666-S1787, T657-Y1792) are crucial for FVIIIa stability.
- Disulfide bond formation across the A2-A3 interface dramatically enhances FVIIIa stability.
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