The isolation and characterization of a specific antibody population directed against the thrombin antithrombin complex.

Abstract

We have raised antisera against human thrombin.antithrombin complex in rabbits as well as in goats and have chromatographed the respective y-immunoglobulin fractions on thrombin-antithrombin-Sepharose as well as on antithrombin-Sepharose.The specific antibody population obtained was utilized to construct a double antibody radioimmunoassay capable of measuring as little as 30 ng/ml of this component.The immunoreactivities of the major related plasma components, prothrombin and antithrombin, were, respectively, -13,000-fold and -100,000-fold less than that of the thrombin-antithrombin complex on a molar basis.Reactivities of prothrombin activation intermediates as well as thrombin which are present in only trace amounts within the blood were also minimal.With the exception of Factor Xa 9 antithrombin, other hemostatic enzymeginhibitor complexes exhibited either no, or barely detectable levels of immunoreactivity.The Factor Xa-antithrombin interaction product was -320 times less reactive than the thrombin.antithrombin complex on a molar basis.The radioimmunoassay was employed to assess the importance of antithrombin as a plasma thrombin inhibitor under conditions approaching the in vivo situation.The results demonstrated that this component is responsible for the neutralization of virtually all of the added thrombin.However, the rate of this interaction within plasma is -21/2-fold less than that predicted from purified systems.The generation of thrombin represents a pivotal event in the coagulation of blood.Once formed, this serine protease may act upon its multiple protein substrates such as Factor V, Factor VIII, Factor XIII, and fibrinogen, or upon critical cellular elements such as platelets or endothelial cells (1-5).Alternately, the enzyme can be neutralized by naturally occurring protease inhibitors.The latter process has been examined in considerable detail by a variety of laboratories (6- 8).Most investigators have concluded that the plasma protein, antithrombin, is the major antagonist of thrombin action (6, 9, 10).However, other groups have suggested that various additional plasma proteins may play a primary role in the