INHIBITION OF THE HEPARIN-ANTITHROMBIN-III-THROMBIN REACTION BY ACTIVE-SITE BLOCKED-THROMBIN

Active site blocked-thrombin, prepared by reacting thrombin with valyl-isoleucyl-prolyl-arginine chloromethyl ketone, inhibits the heparin enhanced-antithrombin III/thrombin reaction. Since active site blocked-thrombin does not interact with antithrombin III it was concluded that active site blocked-thrombin was competing for heparin in the reaction system. The heparin concentration dependence for maximum enhancement of the antithrombin III/thrombin reaction in the presence and absence of active site blocked-thrombin indicated that heparin was binding to thrombin to enhance the reaction rate. A dissociation constant value of 6.4×10-9M was estimated for the heparin·thrombin complex which is similar to the value of 5.8×10-9M previously reported (Griffith M.J. (1979)J. Biol. Chem. in press). Antithrombin III·thrombin complexes were also found to bind heparin with an affinity equivalent to thrombin. The results were interpreted to indicate that heparin binds to thrombin as the first step in the mechanism of action of heparin in enhancing the antithrombin III/thrombin reaction. © 1979.