Heparin binding affinity of normal and genetically modified antithrombin III measured using a monoclonal antibody to the heparin binding site of antithrombin III.
The inhibitory activity of the plasma serine proteinase inhibitor antithrombin III (AT III) is enhanced about 1000-fold upon binding to heparin. We have determined the dissociation constants, Kd, of 48.8 nM for the heparin-AT III interaction, of 175 nM for the specific pentasaccharide-AT III interaction, and of 13 microM for the low-affinity heparin-AT III interaction, using a binding assay based on a monoclonal antibody (MAb) that recognizes an epitope at or close to the heparin binding site of AT III. The heparin binding affinities and proportions of normal and variant AT III in plasma from patients with mutations of AT III have been quantitated for the first time using the binding assay. Substitution mutations in three regions of AT III have been investigated: (i) mutations in the reactive site loop affecting Ala382, Arg393, and Ser394 have no discernible effect on heparin binding; (ii) mutations in the previously identified N-terminal heparin binding region, affecting Arg47, Leu99, and Arg129, produce variant AT III molecules with heparin affinities reduced 11-924-fold, the largest reduction being observed for the substitution mutation Arg47-Cys in Padua 2, which has an affinity of 65.6 microM; (iii) mutations in the hydrophobic regions around strand 1C of the C terminus, affecting Phe402, Ala404, Asn405, Pro407, and Pro429, have pleiotropic effects that include the production of reduced amounts of low-affinity AT III with dissociation constants from 6 to 43 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
Watton
J
J
National Institute for Biological Standards and Control, South Mimms, Hertfordshire, UK.
Longstaff
C
C
Lane
D A
DA
Barrowcliffe
T W
TW
D000911:Antibodies, Monoclonal / Q000276:immunology*; D000990:Antithrombin III / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020152:Antithrombin III Deficiency; D001665:Binding Sites; D002850:Chromatography, Gel; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007122:Immunoelectrophoresis; D015592:Immunoradiometric Assay; D009154:Mutation; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D000990:Antithrombin III; D006493:Heparin