Expression, purification, and characterization of inactive human coagulation factor Xa (Asn322Ala419)
Related Experiment Videos
U Sinha1, T E Hancock, P H Lin
1COR Therapeutics, Inc., South San Francisco, California 94080.
Abstract:
We have expressed in Chinese hamster ovary cells a catalytically inactive form of human factor Xa (factor rXai). A recombinant precursor of human factor Xa was inactivated by two point mutations in the serine protease catalytic triad, Asp322Asn and Ser419Ala. A two-step purification to homogeneity of the secreted material involved immunoaffinity followed by heparin-agarose chromatography. Two forms were identified; a fully processed dimer (70%) and a partially processed monomer (30%). Limited N-terminal amino acid sequencing of factor rXai detected the predicted residues and gamma-carboxyglutamic acid content was 90% of human plasma control. Although devoid of measurable proteolytic activity, factor rXai competitively inhibited plasma factor Xa assembly into functional prothrombinase complexes (Ki = 3 x 10(-10) M). Factor rXai also inhibited plasma clotting in a dose-dependent manner. The possible use of recombinant catalytically inactive proteins as a general approach for pharmacological regulation of human diseases is discussed.
Insights
We developed a catalytically inactive form of human factor Xa (factor rXai) for potential therapeutic use. This recombinant protein effectively inhibits blood clotting by blocking prothrombinase complex formation.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Factor Xa is a critical enzyme in the blood coagulation cascade.
- Developing inhibitors of Factor Xa is a key strategy for anticoagulation therapy.
Purpose of the Study:
- To create and characterize a catalytically inactive recombinant human Factor Xa (factor rXai).
- To evaluate the potential of factor rXai as a pharmacological agent for regulating coagulation.
Main Methods:
- Recombinant expression of inactive human Factor Xa in Chinese hamster ovary cells.
- Two-step purification involving immunoaffinity and heparin-agarose chromatography.
- Biochemical assays to determine proteolytic activity, complex formation inhibition, and clotting inhibition.
Main Results:
- Successfully produced and purified homogeneous factor rXai, existing as a dimer and monomer.
- Factor rXai exhibited no measurable proteolytic activity but competitively inhibited Factor Xa assembly into prothrombinase complexes (Ki = 3 x 10(-10) M).
- Factor rXai demonstrated dose-dependent inhibition of plasma clotting.
Conclusions:
- Catalytically inactive recombinant Factor Xa (factor rXai) can effectively inhibit coagulation.
- Recombinant inactive enzymes represent a promising general strategy for pharmacological regulation of human diseases.