Figure 2: Inhibitory effects of 11F7t and apixaban and anticoagulant reversal.

(a-b): Thrombin formation was measured following addition of 0.2 nM FXa to reaction mixtures containing 1.4 μM prothrombin, 30 μM PCPS, 9 nM FVa, and either no anticoagulant or one of the following anticoagulant strategies: 20 nM apixaban (apix) alone, 2 μM 11F7t alone, or 20 nM apixaban plus 2 μM 11F7t in n = 2 independent experiments. Panel (a) illustrates progress curves for thrombin formation over time, and panel (b) displays the initial velocities of thrombin generation in the presence of each anticoagulation strategy. The latter results are presented after normalization as V/V_o, where V_o is the initial rate observed in the absence of an anticoagulant. (c-f): Thrombin formation was measured following addition of 2 nM FIXa to reaction mixtures containing 1.4 μM prothrombin, 30 μM PCPS, 9 nM FVa, 0.9 nM FVIII, 110 nM FX, and either no anticoagulant (n = 15) or the same anticoagulation strategies tested in the FXa-initiated system (a-b): 20 nM apixaban alone (n = 6), 2 μM 11F7t alone (n = 5), or 20 nM apixaban plus 2 μM 11F7t (n = 6). Panel (c) depicts progress curves for thrombin generation, while panels (d) and (e) respectively compare the time required for the thrombin concentration to reach 10 nM and the total thrombin concentration generated after 10 minutes in the presence of each anticoagulation strategy within the FXa- and FIXa-initiated systems. Panel (f) displays the effect of GD-FXa^S195A (2 μM) or the 11F7t-specific antidote oligonucleotide AO5-2 (10 μM) on neutralization of 2 μM 11F7t plus 20 nM apixaban in the FIXa-initiated system of thrombin generation in n = 2 independent experiments. The same concentration of GD-FXa^S195A did not appreciably affect thrombin formation when added in the absence of anticoagulants. In all panels, error bars indicate standard error. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test.