Properties of antithrombin-thrombin complex formed in the presence and in the absence of heparin

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Abstract
Purification of antithrombin-thrombin complex by ion-exchange chromatography on DEAE-agarose resulted in predominantly monomeric complex, whereas purification on matrix-linked heparin produced large amounts of aggregated complex. Monomeric antithrombin-thrombin complexes formed in the presence and in the absence of heparin had similar conformations and heparin affinities. The 1st-order dissociation rate constants, measured by thrombin release, of these complexes were similar, 2.3 .times. 10-6-3.4 .times. 10-6 s-1, regardless of whether newly formed or purified complex was analyzed. Similar dissociation rate constants were also obtained for purified complex formed with or without heparin, from analyses by dodecyl sulfate/polyacrylamide-gel electrophoresis of the release of modified antithrombin, cleaved at the reactive-site bond. No dissociation of intact antithrombin from the complex was detected by activity measurements or by gel electrophoresis. Aggregation of the complex was found to be accompanied by a decrease in apparent dissociation rate. The similar properties of antithrombin-thrombin complexes formed with or without heparin support the concept of a catalytic role for the polysaccharide in the antithrombin-thrombin reaction. Apparently, the reaction between enzyme and inhibitor involves the rapid formation of an irreversible, kinetically stable, complex that dissociates into active thrombin and modified, inactive, antithrombin by a 1st-order process with a half-life of .apprx. 3 days. The inhibition thus resembles a normal proteolytic reaction, 1 intermediate step of which is very slow.