Immunoaffinity purification of bovine factor VII

Area of Science:

• Biochemistry
• Hematology
• Protein Chemistry

Background:

• Factor VII is a crucial coagulation factor involved in the extrinsic pathway of hemostasis.
• Previous purification methods for Factor VII often resulted in contamination with its active form, Factor VIIa, affecting activation studies.
• Understanding the properties of highly purified Factor VII is essential for accurate hemostasis research.

Purpose of the Study:

• To develop and implement a robust purification protocol for homogeneous bovine Factor VII.
• To assess the purity and activation characteristics of the purified Factor VII.
• To determine the plasma concentration of Factor VII in bovine plasma.

Main Methods:

• Purification of Factor VII from bovine plasma utilizing affinity chromatography with an immunoadsorbent column.
• Quantification of Factor VII recovery using coagulant assays and liquid scintillation counting with a radiolabeled internal standard.
• Assessment of homogeneity and molecular weight via sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis.
• Determination of Factor VII activation by Factor Xa and measurement of coagulant activity.

Main Results:

• Achieved a purification factor of approximately 120,000-fold with an 18% yield.
• Confirmed homogeneity by SDS-PAGE, showing a single polypeptide chain of 53,000 daltons.
• Demonstrated quantitative conversion of the purified Factor VII zymogen to its active two-chain form, Factor VIIa.
• Observed up to a 119-fold activation of coagulant activity, closely matching theoretical predictions for pure Factor VII.
• Estimated bovine plasma Factor VII concentration to be between 10.1 nM and 18.5 nM.

Conclusions:

• Developed a highly effective affinity purification method yielding homogeneous bovine Factor VII.
• The purified Factor VII is essentially free of Factor VIIa, enabling accurate studies of its activation.
• The enhanced activatability observed provides strong evidence for the high purity of the isolated Factor VII.
• Established a reliable method for Factor VII purification and characterization relevant to hemostasis research.