Properties of antithrombin-thrombin complex formed in the presence and in the absence of heparin.
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. Moreover, the first-order dissociation rate constants, measured by thrombin release, of these complexes were similar, 2.3 X 10(-6)-3.4 X 10(-6)S-1, regardless of whether newly formed or purified complex was analysed. Similar dissociation rate constants were also obtained for purified complex formed with or without heparin, from analyses by dodecyl sulphate/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. Furthermore, the results indicate that 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 first-order process with a half-life of about 3 days. The inhibition thus resembles a normal proteolytic reaction, one intermediate step of which is very slow.
D000818:Animals; D000991:Antithrombins / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D006493:Heparin / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011487:Protein Conformation; D013917:Thrombin / Q000378:metabolism*
D000991:Antithrombins; D046911:Macromolecular Substances; D006493:Heparin; D013917:Thrombin