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 wa s associated with the isolation of an A1/A2/A3-C1-C2 heterotrimer. The activity was stable at -80 degrees C, but decayed slowly (t(1/2) appr oximate to 1 week) and nonproteolytically at room temperature or 4 deg rees C. The coagulant activity of the pH 5.5 fVIIIa preparation assaye d in human hemophilia A plasma was only 20% that of porcine factor VII Ia. However, its activity was approximately 75% that of porcine fVIIIa in a plasma-free assay, indicating that human fVIIIa is unstable rela tive to porcine fVIIIa during the coagulation assay. The first-order r ate constant for spontaneous, nonproteolytic loss of activity of human fVIIIa at pH 7.4 was decreased 8-fold by fIXa and phospholipid, indic ating that human fVIIIa is stabilized when incorporated into the intri nsic pathway factor X activation complex.