Discrimination of normal and abnormal prothrombin and protein C in plasma using a calcium ion-inhibited monoclonal antibody to a common epitope on several vitamin K-dependent proteins.
Vitamin K deficiency or administration of vitamin K antagonists results in the biosynthesis of abnormal des-gamma-carboxy forms of the vitamin K-dependent proteins. Monoclonal antibody H-11 binds several vitamin K-dependent proteins at a determinant that includes the first two residues of gamma-carboxyglutamic acid. Antibody H-11 binds fully carboxylated prothrombin and protein C in the presence of EDTA but binding is inhibited by the divalent metal ions, calcium, magnesium, and manganese. By contrast, des-gamma-carboxy prothrombin and protein C bind antibody H-11 the same in the presence of EDTA or calcium ion. Antibody H-11 thus appears to bind a conserved antigenic site containing gamma-carboxyglutamic acid that in the presence of divalent metal ion undergoes a conformational transition. This ability of antibody H-11 to bind des-gamma-carboxy prothrombin and protein C in the presence of calcium ion allowed the development of an immunoassay for these proteins in plasma. Prothrombin and protein C from stably anticoagulated individuals receiving warfarin were characterized by their ability to bind antibody H-11 in the presence of calcium ion. Binding of prothrombin and protein C to antibody H-11 in the presence of calcium correlated temporally with warfarin administration. The inability of calcium ion to inhibit binding of antibody H-11 to abnormal prothrombin and protein C in plasma suggests that the circulating forms of both proteins following warfarin administration cannot undergo the metal ion-dependent conformational transition that includes sequence residues 1 through 12.
Church
W R
WR
Department of Biochemistry, College of Medicine, University of Vermont, Burlington 05405.
Bhushan
F H
FH
Mann
K G
KG
Bovill
E G
EG
D000911:Antibodies, Monoclonal / Q000276:immunology*; D001779:Blood Coagulation Factors / Q000276:immunology*; D001798:Blood Proteins / Q000276:immunology*; D002413:Cations, Divalent / Q000494:pharmacology; D005971:Glutamates / Q000276:immunology; D006801:Humans; D066298:In Vitro Techniques; D011486:Protein C / Q000276:immunology*; D011516:Prothrombin / Q000276:immunology*; D014812:Vitamin K; D014859:Warfarin / Q000627:therapeutic use
D000911:Antibodies, Monoclonal; D001779:Blood Coagulation Factors; D001798:Blood Proteins; D002413:Cations, Divalent; D005971:Glutamates; D011486:Protein C; D014812:Vitamin K; D014859:Warfarin; D011516:Prothrombin