Isolation and characterization of thrombin-activated human factor VIII.
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.
Curtis
J E
JE
Baxter Healthcare Corp., Baxter Biotech Group, Duarte, California 91010.
Helgerson
S L
SL
Parker
E T
ET
Lollar
P
P
D000818:Animals; D001665:Binding Sites; D001777:Blood Coagulation; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D015949:Factor IXa / Q000378:metabolism; D005169:Factor VIII / Q000302:isolation & purification* / Q000378:metabolism; D015944:Factor VIIIa / Q000378:metabolism; D006801:Humans; D010743:Phospholipids / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine; D013917:Thrombin / Q000494:pharmacology*
D010743:Phospholipids; D011994:Recombinant Proteins; D015944:Factor VIIIa; D005169:Factor VIII; D015949:Factor IXa; D013917:Thrombin