Dabigatran Inhibits Both Clot-Bound and Fluid Phase Thrombin In Vitro: Effects Compared to Heparin and Hirudin.

Thrombin bound to fibrin either in an existing thrombus or on an artificial surface is thought to be protected from inhibition by heparin/ATIII. Thus, this surface can remain “active” despite heparin / LMWH therapy. As long as anticoagulant levels are maintained, fluid phase thrombin inhibition is sufficient to prevent progression of thrombosis. However, during times of trough levels or upon discontinued therapy, this active thrombin could initiate further prothrombotic events. Dabigatran is a reversible, univalent, direct inhibitor of thrombin. Considering its specificity and selectivity for the active site, it is to be expected that it can inhibit thrombin equally, both when bound to a clot and in fluid phase. Thus, this study investigated the ability of dabigatran to inhibit thrombin bound to fibrin, or thrombin in plasma as compared to heparin and hirudin in vitro. Clot-bound thrombin: Clots were generated in human platelet rich plasma by adding CaCl₂ and incubating 2 hrs at 37°C (1). Clots were then washed to remove trapped FPA. Washed clots were then transferred to 0.5 ml platelet poor plasma (PPP) containing either dabigatran, heparin, hirudin or buffer and incubated at 37°C. Fluid-phase thrombin: Thrombin (20 pM) was added to PPP containing either dabigatran, heparin, hirudin or buffer and incubated for 1 hr at 37°C. Fibrinopeptide A (FPA) Measurement: Uncleaved fibrinogen was precipitated and fibrin formation was measured as FPA release using ELISA. The IC₅₀ (concentration required to inhibit FPA release by 50%) in fluid phase and clot-bound conditions was calculated using nonlinear curve-fitting. Dabigatran, hirudin and heparin inhibited fluid and clot-bound thrombin with increasing concentrations. Dabigatran inhibited both clot-bound and fluid phase thrombin with similar affinity, IC₅₀ values of 200 nM and 186 nM, respectively. The IC₅₀ of heparin-induced inhibition of clot-bound thrombin was 98.7 nM and in fluid phase 8.4 nM. This is consistent with published results indicating that heparin is not as efficient in inhibiting thrombin bound to a clot as in the fluid phase. Hirudin is a potent, irreversible inhibitor of thrombin and in this study it inhibited both free and bound thrombin equally in low nM ranges, the IC₅₀ of clot-bound thrombin was 0.59 nM and fluid phase thrombin 1.63 nM. The IC₅₀ values were converted into a ratio of clot-bound vs fluid phase thrombin to allow a more direct comparison and are listed in the table.

Dabigatran is equally effective in inhibiting both clot-bound and fluid phase thrombin. These data are consistent with targeted inhibition of the active site of thrombin by a small molecule, irrespective of whether thrombin is bound via the exosite to fibrin or is present as free enzyme in plasma. In contrast, heparin is less effective in inhibiting of clot-bound than fluid phase thrombin, consistent with its mechanism of thrombin inhibition via ATIII. Hirudin more potently inhibits clot-bound than free thrombin at lower concentrations, at higher concentrations inhibition of fluid phase thrombin is favoured. Thus these data suggest that dabigatran could also be effective in clinical situations where surface-bound thrombin could play a role, such as during catheterization procedures or the treatment of established thrombosis.