An engineered interdomain disulfide bond stabilizes human blood coagulation factor VIIIa

Area of Science:

• Biochemistry
• Hematology
• Protein Engineering

Background:

• Hemophilia A results from a deficiency in coagulation Factor VIII (FVIII).
• Current FVIII therapies are limited by the instability of activated FVIII (FVIIIa) due to A2 subunit dissociation.
• This instability reduces the efficacy of FVIII in treating hemophilia A.

Purpose of the Study:

• To engineer a more stable FVIIIa variant.
• To prevent A2 subunit dissociation by introducing a disulfide bond between the A2 and A3 domains.
• To evaluate the stability and activity of the engineered FVIII mutant.

Main Methods:

• Structural analysis of FVIII A domain homology model to identify potential disulfide bond residues.
• Site-directed mutagenesis to create a FVIII mutant (C664-C1826) with cysteine residues at positions 664 and 1826.
• Production, purification, and activation of recombinant C664-C1826 FVIII.
• Immunoblotting to confirm disulfide bond formation.
• Assays to measure the stability and clotting activity of wild-type and mutant FVIIIa.

Main Results:

• A disulfide bond was successfully engineered between the A2 and A3 domains of FVIII (C664-C1826 FVIII).
• The engineered mutant FVIIIa exhibited significantly increased stability compared to wild-type FVIIIa.
• C664-C1826 FVIIIa retained over 90% of its clotting activity, while wild-type FVIIIa lost over 90% under similar conditions.

Conclusions:

• The engineered C664-C1826 FVIIIa demonstrates remarkable stability, overcoming the limitations of wild-type FVIIIa.
• This stable FVIIIa variant offers a promising avenue for improved hemophilia A therapy.
• The engineered FVIIIa also serves as a valuable tool for studying FVIIIa cofactor activity.