Isolation and characterization of human factor VII. Activation of factor VII by factor Xa.
A procedure has been developed for the isolation of human Factor VII to apparent homogeneity as judged by the analytical disc electrophoretic system of Davis (pH 8.9) and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The isolated procedure involves adsorption of Factor VII onto barium citrate, ammonium sulfate fractionation, DEAE-Sephadex chromatography, and preparative polyacrylamide gel electrophoresis. The overall yield of Factor VII is 15 to 20% of starting plasma and the purified protein has a specific activity of 1800 to 2200 units/mg in a clotting assay. Factor VII protein obtained by this method has only 1.3 to 1.5 times more activity in a one-stage clotting assay than in a coupled amidolytic assay (Seligsohn, U., Osterud, B., and Rapaport, S. I. (1978) Blood 52, 978-988), which is taken to mean than it contains less than 5% of activated Factor VII. Human Factor VII is a single chain glycoprotein with an apparent molecular weight of 50,000 +/- 2,000 as determined by SDS-polyacrylamide gel electrophoresis. It has alanine as an NH2-terminal amino acid residue and contains 8.8 gama-carboxyglutamic acid residues/mol of protein. Incubation of purified Factor VII with Factor Xa in the presence of Ca(II) and phospholipid results in a rapid up to 25-fold increase in its clotting activity. Factor VII activity in the coupled amidolytic assay remains unchanged throughout the incubation. Activation of Factor VII by Factor Xa is associated with cleavage of the Mr = 50,000 native protein to a protein consisting of two chains of Mr = about 26,000 and 22,000 as determined by SDS-polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. Other properties of human Factor VII, including its amino acid composition and its reactions with an anti-Factor VII antibody, are described.
D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D004789:Enzyme Activation; D005167:Factor VII / Q000302:isolation & purification* / Q000378:metabolism; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa; D006801:Humans; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D008970:Molecular Weight
D000596:Amino Acids; D005167:Factor VII; D005170:Factor X; D015951:Factor Xa