A new recombinant procoagulant protein derived from the cDNA encoding human factor VIII

Area of Science:

• Biochemistry
• Molecular Biology
• Hematology

Background:

• Human factor VIII (FVIII) is a critical protein for blood coagulation.
• The B domain of FVIII is known to be involved in its processing and activity.
• Recombinant DNA technology allows for the engineering of FVIII variants.

Purpose of the Study:

• To construct and characterize a novel B domain-deleted human factor VIII derivative (FVIII delta II).
• To investigate the impact of B domain deletion on FVIII processing, expression, and procoagulant activity.
• To compare the properties of FVIII delta II with recombinant (r)FVIII and plasma-derived (pd)FVIII.

Main Methods:

• Recombinant DNA techniques were used to create the FVIII delta II cDNA, deleting amino acids 771-1666.
• Expression systems including vaccinia virus (VV) in baby hamster kidney (BHK) 21 cells and Chinese hamster ovary (CHO) cells were utilized.
• Characterization involved assessing procoagulant activity, expression levels, SDS-PAGE migration, thrombin activation, and von Willebrand factor (vWf) binding.

Main Results:

• FVIII delta II was successfully constructed and expressed, exhibiting FVIII procoagulant activity.
• Expression levels of FVIII delta II were approximately 5-fold higher than the full-length molecule.
• FVIII delta II migrated as a single major band on SDS-PAGE, showed enhanced thrombin activation, and retained vWf binding capacity.

Conclusions:

• The B domain deletion in FVIII delta II removes a key processing site, leading to improved expression and specific activity.
• FVIII delta II demonstrates favorable characteristics, including high expression and enhanced activation, suggesting its potential as a therapeutic agent.
• This engineered FVIII variant offers a promising alternative for treating factor VIII deficiencies.