Autoactivation of human recombinant coagulation factor VII

Area of Science:

• Biochemistry
• Hematology
• Protein Chemistry

Background:

• Recombinant factor VII (rFVII) is a crucial therapeutic protein.
• Understanding its activation pathway is vital for optimizing its use.
• Previous studies have focused on extrinsic activators, but intrinsic activation remains less understood.

Purpose of the Study:

• To investigate the intrinsic activation properties of single-chain human recombinant factor VII.
• To identify factors influencing the autoactivation of rFVII.
• To elucidate the role of charged surfaces in rFVII activation.

Main Methods:

• Purification of single-chain human rFVII from baby hamster kidney cells.
• Assay of amidolytic activity using a specific peptidylnitroanilide substrate.
• Monitoring of rFVII activation in the presence and absence of charged surfaces (anion-exchange matrix, poly(D-lysine)).
• Enzyme kinetics studies, including benzamidine inhibition.

Main Results:

• Single-chain rFVII exhibited <1% amidolytic activity compared to factor VIIa.
• Purified single-chain rFVII spontaneously activated in vitro.
• Activation was significantly enhanced (>100-fold) by positively charged surfaces, resulting in a sigmoidal progress curve for factor VIIa generation.
• Benzamidine inhibited rFVIIa and factor VII activation with an identical Ki (11 mM), distinct from its inhibition of bovine factors Xa and IIa.
• Activation occurred similarly in rFVII purified with or without bovine serum, ruling out bovine protease contamination.

Conclusions:

• Factor VII undergoes autoactivation in vitro.
• Positively charged surfaces dramatically accelerate factor VII autoactivation.
• This autoactivation mechanism, enhanced by charged surfaces, is intrinsic to factor VII and not dependent on extrinsic activators or contaminants.