Expression, purification, and characterization of inactive human coagulation factor Xa (Asn322Ala419).
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.
Sinha
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COR Therapeutics, Inc., South San Francisco, California 94080.
Hancock
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Lin
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Hollenbach
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Wolf
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D016466:CHO Cells; D006224:Cricetinae; D015951:Factor Xa / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013925:Thromboplastin / Q000187:drug effects
D011498:Protein Precursors; D011994:Recombinant Proteins; D013925:Thromboplastin; D015951:Factor Xa