Replacing the factor VIII C1 domain with a second C2 domain reduces factor VIII stability and affinity for factor IXa

Area of Science:

• Biochemistry
• Molecular Biology
• Hematology

Background:

• Factor VIII (FVIII) is a critical coagulation factor essential for hemostasis.
• The FVIII molecule comprises heavy and light chains with distinct functional domains, including the C1 and C2 domains.
• Understanding the specific roles of FVIII domains is vital for comprehending coagulation mechanisms and developing targeted therapies.

Purpose of the Study:

• To investigate the functional significance of the FVIII C1 domain by creating a mutant lacking this domain.
• To elucidate the contribution of the C1 domain to FVIII stability, phospholipid binding, and interaction with other components of the FXase complex.
• To assess the impact of C1 domain deletion on FVIII cofactor activity and antibody inhibition.

Main Methods:

• Construction and characterization of a FVIII mutant (FVIIIC2C2) where the C1 domain was replaced by the C2 domain.
• Assessment of FVIII stability using thermal decay assays.
• Measurement of phospholipid binding affinity via fluorescence resonance energy transfer.
• Evaluation of FVIIIa cofactor activity using FXa generation assays in the presence of specific anti-FVIII antibodies.
• Analysis of factor IXa (FIXa) binding affinity to FVIII variants.

Main Results:

• The FVIIIC2C2 mutant exhibited significantly reduced thermal stability (~11-fold decrease) compared to wild-type (WT) FVIII.
• Phospholipid binding affinity of FVIIIC2C2 was modestly reduced (~2.8-fold) compared to WT FVIII.
• The FVIIIC2C2 mutant showed impaired interaction with FIXa (~4-fold reduction in binding affinity) and differential inhibition by antibodies compared to WT FVIII.
• Anti-C1 antibody (GMA8011) inhibited WT FVIIIa activity and FIXa binding, while anti-C2 antibodies (ESH4, ESH8) inhibited FVIIIC2C2 activity and FIXa binding.

Conclusions:

• The FVIII C1 domain plays a critical role in maintaining FVIII stability and is essential for optimal interaction with factor IXa within the FXase complex.
• The C1 domain likely resides in close proximity to FIXa, contributing significantly to the structural integrity and functional efficiency of the FXase complex.
• These findings provide novel insights into the structure-function relationships of FVIII and may inform the development of therapeutic strategies for bleeding disorders.