Curtis J E, Helgerson S L, Parker E T, Lollar P
Baxter Healthcare Corp., Baxter Biotech Group, Duarte, California 91010.
J Biol Chem. 1994 Feb 25;269(8):6246-51.
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.
重组人凝血因子VIII（fVIII）在pH 7.4条件下被凝血酶激活，随后在pH值为3.5至7.4范围内进行CM-琼脂糖凝胶层析。在pH 5.5时回收了最佳凝血活性，且与分离出的A1/A2/A3-C1-C2异源三聚体相关。该活性在-80℃下稳定，但在室温或4℃下缓慢衰减（半衰期约为1周）且无蛋白水解作用。在人甲型血友病血浆中检测的pH 5.5 fVIIIa制剂的凝血活性仅为猪凝血因子VIIIa的20%。然而，在无血浆测定中其活性约为猪fVIIIa的75%，表明在凝血测定过程中，人fVIIIa相对于猪fVIIIa不稳定。在pH 7.4条件下，fIXa和磷脂使人类fVIIIa自发非蛋白水解失活的一级速率常数降低了8倍，表明当人类fVIIIa掺入内源性途径因子X激活复合物时会得到稳定。
