Self-association of antithrombin III relates to multimer formation of thrombin-antithrombin III complexes.
During the reaction of antithrombin III (AT III) with target proteases the inhibitor serves as pseudo-substrate and undergoes profound conformational changes, becomes incorporated into a covalent stoichiometric enzyme-inhibitor complex which is, in contrast to native AT III, recognized by monoclonal antibody 4C9. In the absence of the target enzyme thrombin, incubation of AT III with 1-2 M guanidine, 0.6% deoxycholate, heating to 56 degrees C, or buffer at pH 4 resulted in inactivation of the inhibitor with concomitant exposure of the epitope for 4C9 and formation of AT III multimers (from 3.9 S to 7.1-7.4 S). Loss of activity, formation of multimers and exposure of neoepitope(s) of AT III occurred in a concerted fashion and followed second order kinetics with an activation energy of Ea = 31 kcal/mol. AT III-multimerization induced by treatment with 1 M guanidine (mainly AT III-tetramers with M(r) of 250,000) and formation of the binary AT III-thrombin complex revealed similar self-association patterns as judged by gel electrophoresis under non-denaturing conditions. In the presence of heparin, even higher multimers of AT III-thrombin complexes were noted. Moreover, self-association products of the ternary vitronectin-thrombin-AT III complex, which is the ultimate reaction product following thrombin inhibition in the circulation, could be recognized and quantitated due to exposure of the 4C9 epitope on AT III, indicating that AT III exists in multimeric forms within binary and ternary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
Preissner
K T
KT
Haemostasis Research Unit, Kerckhoff-Klinik, MPG, Bad Nauheim, Germany.
D000911:Antibodies, Monoclonal / Q000276:immunology; D000990:Antithrombin III / Q000737:chemistry* / Q000276:immunology; D006801:Humans; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000276:immunology; D011108:Polymers; D011485:Protein Binding; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; D011108:Polymers; C046193:antithrombin III-protease complex; D000990:Antithrombin III; D010447:Peptide Hydrolases