Production in vitro and properties of a modified form of bovine antithrombin, cleaved at the active site by thrombin.

A modified, two-chain form of antithrombin, cleaved at the active site, is produced by stoichiometric amounts of thrombin in a reaction that competes with the formation of the inactive antithrombin-thrombin complex.The factors influencing the in vitro production of this modified antithrombin were investigated and the properties of the modified inhibitor were compared to those of intact antithrombin.Maximally, about 5% of the amount of inhibitor allowed to react with the enzyme was converted to the modified form under most conditions studied.However, the presence of high affin- ity heparin increased this conversion up to about 30%.In contrast, low affinity heparin had only little effect.This indicates that the increased proteolytic cleavage of the active site occurs as a result of the conformational change of antithrombin induced by the binding of high affinity heparin.The two polypeptide fragments of the modified antithrombin were shown to be attached to each other by both a disulfide bond and noncovalent interactions.The odified inhibitor had no detectable protease-inhibiting activity.Several properties such as the frictional ratio and the circular dichroism of the modified antithrombin differed from those of the intact inhibitor in a manner suggesting a conformational change following scission of the active site bond.Notably, the heparin affinity of the modified inhibitor was found to be about 500 times lower than that of intact antithrombin.This observation, together with the fact that binding of heparin to the intact inhibitor induces a conformational change affecting the reactivity of the active site, indicates that the conformations of the heparin-binding site and the active site of antithrombin are intimately linked.Antithrombin inhibits all the serine proteases of the intrinsic coagulation system by forming highly stable, inactive complexes with the enzymes.The rates of these inactivation reactions are greatly accelerated by heparin (for reviews, see Refs.1-3).Recent studies with three coagulation proteases, thrombin, Factor X,, and Factor IX,, have shown that the formation of each of the three antithrombin-protease complexes is accompanied by the rapid appearance of the same noncomplexed, proteolytically modified form of the inhibitor,