Bivalent binding to γa/γ′ fibrin engages both exosites of thrombin and protects it from inhibition by the antithrombin-heparin complex

Thrombin exosite 1 binds the predominant gamma(A)/gamma(A)-fibrin form with low affinity. A subpopulation of fibrin molecules, gamma(A)/gamma '-fibrin, has an extended COOH terminus gamma '-chain that binds exosite 2 of thrombin. Bivalent binding to gamma A/gamma '-fibrin increases the affinity of thrombin 10-fold, as determined by surface plasmon resonance. Because of its higher affinity, thrombin dissociates 7-fold more slowly from gamma(A)/gamma '-fibrin clots than from gamma(A)/gamma '-fibrin clots. After 24 h of washing, however, both gamma(A)/gamma '- and gamma(A)/gamma(A)-fibrin clots generate fibrinopeptide A when incubated with fibrinogen, indicating the retention of active thrombin. Previous studies demonstrated that heparin heightens the affinity of thrombin for fibrin by simultaneously binding to fibrin and exosite 2 on thrombin to generate a ternary heparin-thrombin-fibrin complex that protects thrombin from inhibition by antithrombin and heparin cofactor II. In contrast, dermatan sulfate does not promote ternary complex formation because it does not bind to fibrin. Heparin-catalyzed rates of thrombin inhibition by antithrombin were 5-fold slower in gamma(A)/gamma-fibrin clots than they were in gamma(A)/gamma(A)-fibrin clots. This difference reflects bivalent binding of thrombin to gamma(A)/gamma '-fibrin because (a) it is abolished by addition of a gamma '-chain-directed antibody that blocks exosite 2-mediated binding of thrombin to the gamma '-chain and (b) the dermatan sulfate-catalyzed rate of thrombin inhibition by heparin cofactor II also is lower with gamma(A)/gamma '-fibrin than with gamma(A)/gamma '-fibrin clots. Thus, bivalent binding of thrombin to gamma(A)/gamma '-fibrin protects thrombin from inhibition, raising the possibility that gamma(A)/gamma '-fibrin serves as a reservoir of active thrombin that renders thrombi thrombogenic.