A Method for Systematic Purification from Bovine Plasma of Six Vitamin K-Dependent Coagulation Factors: Prothrombin, Factor X, Factor IX, Protein S, Protein C, and Protein Z1

Abstract

A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).

Crystal Structure of Mg2+- and Ca2+-bound Gla Domain of Factor IX Complexed with Binding Protein

“…Protein Purification-Factor IX was purified from frozen bovine plasma following standard protocols (21). Gla domain peptide (residues 1-46, IXGD1-46) was prepared as described (13).…”

Highly sensitive peptide‐4‐methylcoumaryl‐7‐amide substrates for blood‐clotting proteases and trypsin

“…The purity of these synthetic substrates was confirmed by thin-layer chromatography, high-performance liquid chromatography, elemental analysis and amino acid analysis. Factor Xa [13], factor IXaa [13], factor XIIa [14], plasma kallikrein [15], activated protein C [13] from bovine plasma, and a-thrombin [16], factor IXaP 1171 and factor XI [18] from human plasma were highly purified by the published methods. Human factor XI was converted to factor XIa by bovine factor XIIa, according to the method of Kurachi and Davie [19].…”

Bothrops jararaca envenomation: Pathogenesis of hemostatic disturbances and intravascular hemolysis

“…In addition, an aliquot of the control plasma pool was used to deplete VKCDF using BaCl 2 adsorption. 41 To evaluate the coagulant activity of BjV on those different plasma pools, the minimum coagulant dose (MCD)-i.e. the least amount of venom that clotted a solution of plasma or fibrinogen in 60 s at 37 C 43 -was determined.…”