The γ-chain of fibrinogen exists in two forms, γA and γ′, such that circulating fibrinogen consists of two populations, γA/γA (90%) and γA/γ′ (10%). The 16 amino acid extension at the COOH-terminus of the γ′ chain contains numerous negatively-charged residues. This alteration endows γA/γ′-fibrin (Fn) with a greater capacity to bind thrombin (IIa), a feature that may render thrombi prothrombotic. The purpose of this study was to explore how the various mechanisms by which IIa binds to Fn impact on IIa protection from inactivation by antithrombin/heparin. IIa binds weakly to γA/γA-Fn utilizing exosite 1. IIa binds with higher affinity to γA/γ′-Fn due to the additional exosite 2/γ′ interaction. In the presence of heparin, IIa can bind to γA/γA-Fn with high affinity by forming a ternary complex wherein heparin bridges IIa to Fn via exosite 2, thereby heightening the interaction of IIa with Fn via exosite 1. Consequently, the amount of IIa bound to γA/γA-Fn clots increases in the presence of heparin. Formation of the γA/γA-Fn/heparin/IIa ternary complex reduces the second order rate constant of IIa inhibition by antithrombin (AT) 11-fold compared with the heparin-catalyzed rate of inhibition of free IIa (1.1x108 M−1 min−1). This reduction reflects the inability of AT-bound heparin to access exosite 2. When γA/γ′-Fn is used in place of γA/γA-Fn, the heparin-catalyzed rate is reduced 55-fold. The enhanced protection with γA/γ′-Fn is due to the exosite 2-mediated interaction of IIa with the γ′-chain because addition of an antibody against the γ′ sequence that blocks this interaction reduces the protection from AT to the level seen with γA/γA-Fn. Thus, both γA/γ′-Fn/IIa and γA/γA-Fn/heparin/IIa complexes restrict the access of heparin to exosite 2, thereby impairing inhibition by heparin/AT. Heparin cofactor II (HCII) also utilizes heparin to bridge to IIa, but, unlike AT, HCII must also directly engage exosite 1 to effect IIa inhibition. Consequently, the heparin-catalyzed rate of IIa inhibition by HCII is reduced 27-fold in the presence of γA/γA-Fn compared with the 11-fold reduction with AT. To examine the contribution of the heparin-Fn interaction to this phenomenon, dermatan sulfate (DS) was used to catalyze HCII because, unlike heparin, DS does not bind Fn. γA/γA-Fn produced only a 5-fold reduction in the DS-catalyzed rate of IIa inhibition by HCII. This suggests that in the absence of heparin, occupation of exosite 1 by γA/γA-Fn only modestly impairs inhibition by HCII. In contrast, γA/γ′-Fn produced a 28-fold reduction in the DS-catalyzed rate of IIa inhibition by HCII. This protection is abolished by addition of the antibody that blocks binding of IIa to the γ′ chain. Therefore, although exosite 1 mediates IIa binding to both γA/γA- and γA/γ′-Fn, interaction of exosite 2 with the γ′-chain in γA/γ′-Fn heightens exosite 1-mediated binding. These findings provide independent confirmation that ligation of both exosites on IIa accentuates the affinity of the individual exosite 1 and 2 interactions. Thus, reactants that require access to either exosite will be restricted when IIa binding is mediated by both exosites as occurs with γA/γA-Fn/heparin or with γA/γ′-Fn. These results confirm that Fn can serve as a reservoir of active IIa and that bound IIa is protected from inhibition by circulating inhibitors. Our data also highlight the limitations of physiological inhibitors of IIa and validate the need for development of direct thrombin inhibitors.