Production By Thrombin of A Proteolytically Modified Form of Antithrombin and Release of The Same Form from the Antithrombin-Thrombin Complex

Abstract

In addition to the in vitro formation of an inactive, stable complex (ATT) between thrombin (T) and the plasma serine protease inhibitor, antithrombin (AT), a sizeable proportion of AT is converted by T to a form which has lost the ability to inhibit T and which exhibits a reduced affinity for heparin. Under non-denaturing conditions, the isolated modified AT (ATm) could not be differentiated from native AT by hydrodynamic, electrophoretic or immunological analyses. Under reducing/denaturing conditions, ATm yielded two polypeptides, one of about 50000 and one of about 5000 daltons. Preliminary studies indicate that these are the result of a proteolytic cleavage by T of an arg-ser bond in the C-terminal end of the single-chain AT molecule. Dissociation of ATT by prolonged treatment in hot SDS or by treatment with hydoxylamine or ammonia in SDS produced only T and ATm. Under nondenaturing conditions, dissociation by the two latter agents released ATm and up to 40% of the potential T activity from ATT. However, under no conditions tested could intact AT be shown to be produced from dissociation of ATT. These results suggest that scission by T of a specific arg-ser bond in the active site region of AT, or, alternatively, conversion of this bond to a tetrahedral intermediate state, occurs during the formation of the inactive enzyme-inhibitor complex.