Protamine sulfate (salmine), a basic protein with a molecular weight of 4,626 +/- 109, is a known antiheparin agent which in the absence of heparin demonstrates an anticoagulant activity. To date, much work has been done to elucidate the interaction of heparin with thrombin and its physiologic inhibitor, Antithrombin III (ATIII). Little is known, however, about the mechanism of anticoagulant action of protamine sulfate and its mode of thrombin inactivation. We provide information about the interaction of protamine sulfate with purified, labeled thrombin and ATIII through binding experiments in which protamine is shown to inhibit the inactivation of thrombin by ATIII. Furthermore, we show in clotting assays that protamine sulfate has an inhibitory effect on thrombin in the conversion of fibrinogen to fibrin, and that this inhibition is concentration dependent, partial, and reversible.

Protamine sulfate, a simple protein M.W. 4,600, is a known antiheparin agent which, in the absence of heparin, demonstrates an anticoagulant activity both in vitro and in vivo. Much work has been done to elucidate the interactions of heparin with thrombin and ATIII, however, little is known about the mechanism of protamine sulfate anticoagulant activity and its effect on thrombin.In binding studies, radiolabeled thrombin was reacted with protamine for 1 hour and ATIII was then used as a probe to test the availability of the thrombin active site. ATIII/*thrombin complex was separated from free *thrombin on a Sephacryl S200 molecular sieve column. As a result of preincubating thrombin with protamine, ATIII was unable to bind thrombin. Functional assays for thrombin activity, were carried out in a system where time taken for formation of a thrombin catalyzed fibrin clot was the measure used for thrombin activity. Effect of protamine on thrombin activity when tested in this system demonstrated that with increasing molar concentrations of protamine, there is a corresponding decrease in thrombin activity. Maximum inhibition of activity occurred at a protamine concentration of 200 μM. This inhibition was concentration dependent, partial and reversible. In contrast the amydolysis of the synthetic substrate H-D-Phe-Pip-Arg-p-nitroanilide by thrombin was unaffected by protamine.Results of these studies have confirmed the anticoagulant role of protamine sulfate and provided information on its interaction with purified thrombin. Since the drug did not totally inhibit functional activity of thrombin, we feel that it binds the enzyme, partially blocking the active site.

Prothrombin complex concentrates containing FVIII bypassing activity have been employed effectively in the management of patients with factor VIII and IX inhibitors. The active component has not been identified although the protease forms of factor IX and X, as well as VII exist in variable concentrations in these products.As a means of establishing the role of each factor in the concentrates, monospecific antibodies to prothrombin, FX and FVII were coupled to Sepharose 4B by means of cross linking reagent cyanogen bromide. These insol- ubilized antibodies were used to selectively remove all prothrombin FX or FVII from the therapeutic concentrate, Autoplex (570 u/vial) supplied by Hyland Labs, Costa Mesa, by batch adsorption. A control was prepared by reacting the concentrate with cyanogen bromide treated beads whose active sites had been blocked with ethanolamine. Successful removal of specific factors from concentrates was evaluated by immunodiffusion assays and by standard clotting assays using substrate deficient plasma. Thrombin was not found to be present in any of the products following extraction. Thrombogenicity was tested in an animal model. Concentrates reacted with cyanogen bromide beads as a control and those lacking prothrombin were found to shorten clotting time of FVIII deficient plasma and to trigger intravascular coagulation in the animals (D.I.C.). Removal of FVII produced a concentrate that was more active than the control with respect to FVIII correction and induction of D.I.C. Concentrates depleted of FX, on the other hand, lost their capacity to correct FVIII deficient plasma and to initiate D.I.C. Our findings would suggest that the content of FX and its active form FXa plays a major role in the concentrates FVIII correcting activity and most likely is responsible for its thrombogenic characteristics.