TY - JOUR
T1 - Human Factor VII
AU - Broze, George J.
AU - Majerus, Philip W.
N1 - Funding Information:
Studies reported from the authors' laboratory were done during the tenure of a Clinician-Scientist Award from The American Heart Association and with funds contributed in part by The St. Louis Heart Association, and was also supported by Grants HLBI 14147 (Specialized Center in Thrombosis), HL 07088, and HL 16634 from The National Institutes of Health.
Funding Information:
This research was supported by Grants HLBI 14147 (Specialized Center in Thrombosis), HL 07088, and HL 16634 from The National Institutes of Health, and in part by NIH Research Service Award GM 07200, Medical Scientist, from the National Institute of General Medical Sciences.
PY - 1981/1/1
Y1 - 1981/1/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=77957026932&partnerID=8YFLogxK
U2 - 10.1016/S0076-6879(81)80021-3
DO - 10.1016/S0076-6879(81)80021-3
M3 - Article
AN - SCOPUS:77957026932
SN - 0076-6879
VL - 80
SP - 228
EP - 237
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -