Conformational change in antithrombin induced by heparin probed with a monoclonal antibody against the 1C/4B region.
A murine monoclonal antibody (MAb) raised against a covalent antithrombin-heparin complex was used to probe the conformational change resulting when the serpin antithrombin binds to heparin. This MAb completely inhibited the progressive activity of antithrombin against thrombin. However, although the MAb remained bound to antithrombin in the presence of heparin, it did not significantly inhibit heparin cofactor activity against thrombin, and increasing concentrations of the antithrombin-binding pentasaccharide progressively unblocked the inhibitory action of the MAb. The MAb bound to antithrombin without affecting either heparin-binding affinity or heparin-induced fluorescence enhancement, and it did not convert antithrombin from inhibitor to substrate. The MAb failed to interact with reduced and S-carboxymethylated antithrombin, indicating the conformational nature of its epitope. Antithrombin variants with N-terminal substitutions (Arg47-->Cys or His, Leu99-->Phe, Arg129-->Gln) modifying heparin binding, and C-terminal substitutions affecting the reactive site (Arg393-->Cys) or resulting in substrate-variant antithrombin (Ala384-->Pro), were all recognized normally, as were normal reactive site cleaved antithrombin and the thrombin-antithrombin complex. However, interaction of the MAb with antithrombin was reduced by several substitution mutations (Phe402-->Cys, Phe402-->Ser, Phe402-->Leu, Ala404-->Thr, Pro407-->Thr) in the 402-407 sequence which codes for amino acid residues of strand 1C and the polypeptide leading to strand 4B. Pro429-->Leu also blocks recognition [Olds et al. (1992) Blood 79, 1206-1212], and this residue is believed to be spatially approximated to strand 1C.(ABSTRACT TRUNCATED AT 250 WORDS)
Dawes
J
J
Heart Research Institute, Camperdown, NSW, Australia.
James
K
K
Lane
D A
DA
D000818:Animals; D000911:Antibodies, Monoclonal; D000921:Antibody-Producing Cells; D000991:Antithrombins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D002999:Clone Cells; D000939:Epitopes; D006493:Heparin / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D051379:Mice; D011485:Protein Binding; D011487:Protein Conformation; D013917:Thrombin / Q000037:antagonists & inhibitors
D000911:Antibodies, Monoclonal; D000991:Antithrombins; D000939:Epitopes; D006493:Heparin; D013917:Thrombin