Isolation and characterization of thrombin-activated human factor VIII

Area of Science:

• Biochemistry
• Hematology

Background:

• Recombinant human factor VIII (fVIII) is crucial for treating hemophilia A.
• Understanding fVIII stability and activity is vital for therapeutic applications.

Purpose of the Study:

• To characterize the optimal conditions for recombinant human factor VIII (fVIII) activity.
• To assess the stability and comparative activity of human fVIIIa versus porcine fVIIIa.
• To investigate the stabilization of human fVIIIa within the intrinsic coagulation pathway.

Main Methods:

• Recombinant human factor VIII activation by thrombin at pH 7.4.
• CM-Sepharose chromatography across a pH range (3.5–7.4).
• Coagulant activity assays in hemophilia A plasma and plasma-free conditions.
• Assessment of human fVIIIa stabilization by factor IXa and phospholipid.

Main Results:

• Optimal coagulant activity of human fVIIIa was recovered at pH 5.5, forming an A1/A2/A3-C1-C2 heterotrimer.
• Human fVIIIa exhibited significant instability at room temperature and 4°C (t1/2 ≈ 1 week).
• Human fVIIIa showed reduced activity in plasma compared to plasma-free assays, indicating instability during coagulation.
• Factor IXa and phospholipid decreased the rate of human fVIIIa activity loss by 8-fold.

Conclusions:

• Recombinant human factor VIII (fVIII) requires specific pH conditions for optimal activity and exhibits limited stability.
• Human fVIIIa is less stable than porcine fVIIIa in coagulation assays.
• Incorporation into the factor X activation complex significantly stabilizes human fVIIIa, highlighting its role in the intrinsic pathway.