Human Factor VII

This chapter presents the procedure for purification and assaying of human Factor VII. Three major problems are encountered in the purification of human factor VII: (1) it is a trace protein in human plasma; (2) activation and subsequent degradation occur during the purification procedure; and (3) loss of activity occurs at protein concentrations less than approximately 30 μg/ml. To prevent proteolysis, soybean-trypsin inhibitor and high levels of benzamidine have been employed. In the purification procedure, factor VII and the other vitamin K-dependent coagulation factors are separated from the bulk of unrelated plasma proteins by absorption to barium citrate. Factor VII in turn is separated from factors lI, IX, X, and protein C by anion-exchange chromatography on DEAE-Sepharose. Much of the remaining contaminants are removed in a QAE-Sephadex anion-exchange step by taking advantage of the fact that factor VII elutes at a much lower ionic strength in the presence of Ca ²⁺. The remaining contaminants are removed by gel filtration on ACA-44. Two methods are available for the determination of factor VII activity. A one-stage clotting assay is routinely used, employing tissue thromboplastin and factor VII-deficient plasma as substrate. Another assay employ a two-stage system in which the sample, tissue thromboplastin, and a source of factor X are incubated in the first stage and followed by the addition of the chromagenic substrate S-2222 to assay the degree of factor X activation.