The inactivation of thrombin and plasmin by antithrombin III in the presence of sepharose-heparin.

The inhibition of thrombin and plasmin by antithrombin III was studied in the presence of heparin conjugated to Sepharose. When either enzyme was adsorbed to the heparin conjugate in quantities insufficient to occupy all the affinity sites, subsequent passage of antithrombin III through the column invariably produced complete inhibition as measured by the response to the chromogenic substrate S2160. However, when the loading sequence was reversed (i.e. adsorbing non-saturating quantities of antithrombin III before the enzyme), antithrombin III only insignificantly inhibited either enzyme over a 20-hour period. A second load of antithrombin III following the thrombin load resulted in complete inactivation. When thrombin was rendered incapable of binding to heparin by cyclohexanedione treatment and reacted with Sepharose-heparin to which antithrombin III was adsorbed, the protease inhibitor exhibited ‘progressive’ antithrombin activity. These studies may indicate that thrombin and plasmin possess higher affinities for heparin than for the antithrombin III-heparin complex. Furthermore antithrombin III reacts more readily with the enzyme-heparin complex than with heparin. If the relative affinities of heparin, thrombin and antithrombin III in plasma are similar to those observed under these affinity chromatographic conditions, then the present data are consistent with the view that heparin promotes the inactivation of thrombin and plasmin by augmenting their reaction with antithrombin III. An allosteric effect of heparin on thrombin is apparent from the 50% increase in esterase activity of thrombin on α-N-benzoyl-L-arginine ethyl ester which is observed in the presence of optimal concentrations of heparin.