Isolation and characterization of thrombin-activated human factor VIII.

Abstract

Recombinant human factor VIII (fVIII) was activated by thrombin at pH 7.4, followed by CM-Sepharose chromatography at pH values ranging from 3.5 to 7.4. Optimal coagulant activity was recovered at pH 5.5 and was associated with the isolation of an A1/A2/A3-C1-C2 heterotrimer. The activity was stable at -80 degrees C, but decayed slowly (t1/2 approximately 1 week) and nonproteolytically at room temperature or 4 degrees C. The coagulant activity of the pH 5.5 fVIIIa preparation assayed in human hemophilia A plasma was only 20% that of porcine factor VIIIa. However, its activity was approximately 75% that of porcine fVIIIa in a plasma-free assay, indicating that human fVIIIa is unstable relative to porcine fVIIIa during the coagulation assay. The first-order rate constant for spontaneous, nonproteolytic loss of activity of human fVIIIa at pH 7.4 was decreased 8-fold by fIXa and phospholipid, indicating that human fVIIIa is stabilized when incorporated into the intrinsic pathway factor X activation complex.

• Authors: J E Curtis, S L Helgerson, E T Parker, P Lollar
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 269 Issue 8 Pg. 6246-51 (Feb 25 1994) ISSN: 0021-9258 [Print] United States
• PMID: 8119969 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Phospholipids
• Recombinant Proteins
• Factor VIIIa
• Factor VIII
• Factor IXa
• Thrombin

Topics

• Animals
• Binding Sites
• Blood Coagulation
• Chromatography, Ion Exchange
• Circular Dichroism
• Electrophoresis, Polyacrylamide Gel
• Factor IXa (metabolism)
• Factor VIII (isolation & purification, metabolism)
• Factor VIIIa (metabolism)
• Humans
• Phospholipids (metabolism)
• Recombinant Proteins (isolation & purification, metabolism)
• Swine
• Thrombin (pharmacology)