Purification and properties of human coagulation factor VII

Area of Science:

• Biochemistry
• Hematology
• Molecular Biology

Background:

• Blood coagulation Factor VII is a crucial protein in the extrinsic pathway of hemostasis.
• Understanding Factor VII's structure and activation is vital for comprehending blood clotting disorders.

Purpose of the Study:

• To purify and characterize human plasma Factor VII.
• To investigate the activation mechanism of Factor VII to its enzymatic form, Factor VIIa.
• To analyze the inhibitory properties of Factor VII and Factor VIIa.

Main Methods:

• Purification of Factor VII from human plasma using a multi-step process achieving 100,000-fold enrichment.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular weight determination.
• Amino-terminal sequencing to identify the polypeptide chain.
• Enzymatic assays to study activation by Factor Xa and Factor XIIa, and inhibition by diisopropyl fluorophosphate and antithrombin III.

Main Results:

• Factor VII was purified to apparent homogeneity with a 30% yield.
• Estimated molecular weight of 48,000 Da for a single polypeptide chain.
• Conversion to a two-chain Factor VIIa form by Factor Xa or Factor XIIa, resulting in a 20- to 25-fold increase in activity.
• Factor VIIa exhibited faster inactivation by diisopropyl fluorophosphate than Factor VII, accelerated by tissue factor and CaCl2.
• Heparin significantly increased the inhibition rate of Factor VIIa by antithrombin III compared to Factor VII.

Conclusions:

• Human Factor VII is a single-chain protein that undergoes activation to a two-chain form, Factor VIIa, with significantly enhanced coagulant activity.
• The activation process and inhibition kinetics provide insights into the regulation of blood coagulation.
• Factor VIIa's interaction with inhibitors, particularly in the presence of heparin, highlights its role in hemostatic control.