Purification and properties of the phenprocoumon-induced decarboxyfactor X from bovine plasma

Abstract

1. By a procedure involving adsorption to barium sulfate, chromatography on DEAE-Sephadex and QAE-Sephadex and preparative polyacrylamide gel electrophoresis, decarboxyfactor X was purified from plasma of phenprocoumon-treated cows. No contaminants could be detected in the final preparation by polyacrylamide gel electrophoresis and zone-electrophoresis. 2. The molecular weight of decarboxyfactor X, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis is approximately 55 000, which is equal to that of factor X. The protein consists of two polypeptide chains with molecular weights of 44 000 and 17 000. 3. Decarboxyfactor X has antigenic determinants in common with normal factor X. 4. The amino acid composition and aminoterminal amino acids of normal factor X and decarboxyfactor X are identical. 5. Less than one residue of gamma-carboxyglutamate could be detected per mole of decarboxyfactor X. 6. In the absence of Ca2+, normal factor X has a slightly higher electrophoretic mobility than decarboxyfactor X. In the presence of Ca2+ the mobility of factor X decreases considerably while the mobility of decarboxyfactor X remains unaltered.

Combined Functional and Immunochemical Analysis of Normal and Abnormal Human Factor X

“…The finding that Factor X present in the plasma of patients on warfarin therapy appeared immunochemically identical with normal Factor X agrees with previous reports for bovine Factor X and decarboxy Factor X (13), and implied for human Factor X by immunodiffusion (9) and antibody neutralization (8,12). In those studies on the bovine molecules (13) and in the present study on human Factor X, antisera were generated in rabbits using highly purified and well-characterized Factor X, mitigating the possible contamination by other plasma proteins.…”

“…Antigenic identity observed between purified Factor X and normal human plasma was expected, but has only been reported by far less critical and discriminating immunodiffusion techniques (9,12,13). The finding that Factor X present in the plasma of patients on warfarin therapy appeared immunochemically identical with normal Factor X agrees with previous reports for bovine Factor X and decarboxy Factor X (13), and implied for human Factor X by immunodiffusion (9) and antibody neutralization (8,12).…”

“…In a few studies antisera with specificity to Factor X have been used to immunologically estimate the presence of Factor X protein (7)(8)(9)(10)(11)(12)(13)(14)(15). Whereas qualitative assessment has employed immunoelectrophoresis (9,11,12), quantitation of Factor X antigen has been attempted by antibody neutralization (7,8,10), and radioimmunoassay (14,15).…”

The role of γ-carboxyglutamyl residues in the positive cooperative binding of Ca2+ to blood coagulation factor X

“…Factor X and decarboxyfactor X were purified according to the method described previously [15]. All ehemicals were analytical grade anci obtained from Merck.…”

Factor X Fusion Proteins: Improved Production and Use in the Release in Vitro of Biologically Active Hirudin from an Inactive α-Factor–Hirudin Fusion Protein

“…Although the posttranslational ␥-carboxylation of glutamate residues is essential for the procoagulant activity of FX (18), neither the Gla domain nor the E1 domain is required for the proteolytic activity of FXa (19). In addition to the Gla domain playing a role in intracellular trafficking (2), incomplete ␥-carboxylation can lead to intracellular degradation of Gla-containing proteins (20,21).…”