Mechanistic Basis for the Differential Effects of Rivaroxaban and Apixaban on Global Tests of Coagulation

Figures

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  Fig. 1. Comparison of the effects of rivaroxaban and apixaban on the prothrombin time (PT) and activated partial thromboplastin time (aPTT). The ( A ) PT and ( B ) aPTT were determined in human plasma in the presence of increasing concentrations of apixaban ( closed ) or rivaroxaban ( open ). Symbols reflect the mean ± SD of four experiments.

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  Fig. 2. Comparison of the effects of apixaban and rivaroxaban on thrombin generation in plasma. Thrombin generation in plasma containing 4 µM PCPS was triggered by addition of 3 pM tissue factor and 7.5 mM CaCl 2 and quantified by monitoring the hydrolysis of 0.5 mM Z-Gly-Gly-Arg-AMC in the absence or presence of ( A ) apixaban, or ( B ) rivaroxaban, at the indicated concentrations. Using the thrombin generation profiles, ( C ) lag time, ( D ) peak thrombin concentration, and ( E ) ETP are measured and plotted versus the concentrations of apixaban ( closed ) or rivaroxaban ( open ). Data reflect the mean ± SD of three experiments. ETP, endogenous thrombin potential.

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  Fig. 3. Comparison of the effects of rivaroxaban and apixaban on factor Xa chromogenic activity. Inhibition constants (K i ) were determined by incubating 5 nM factor Xa with rivaroxaban ( open ) or apixaban ( closed ) at the indicated concentrations, and residual active factor Xa concentration was determined by monitoring hydrolysis of 500 μM S-2765. Symbols reflect the mean ± SD of three to four experiments. Lines are fit to a rectangular hyperbola equation by nonlinear regression analysis.

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  Fig. 4. Comparison of the effects of rivaroxaban and apixaban on prothrombin activation by prothrombinase. Inhibition constants (K i ) were determined by incubating increasing concentrations of rivaroxaban ( open ) or apixaban ( closed ) with 0.5 nM factor Xa, 5.5 nM factor Va, 50 µM PCPS, 500 nM prothrombin, and 5 mM CaCl 2 . After 20 s, reactions were stopped by addition of EDTA to 10 mM and generated thrombin was quantified by chromogenic assay with Chz-Th. Rates of prothrombin activation are plotted versus inhibitor concentration. Symbols reflect the mean ± SD of three to four experiments. Lines are fit to a rectangular hyperbola equation by nonlinear regression analysis.

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  Fig. 5. Inhibition of factor Xa amidolytic activity in the presence of apixaban and rivaroxaban. Cleavage of 100 µM Pefafluor Xa by 0.25 nM factor Xa was monitored in a fluorescent plate reader at 2-s intervals. Reactions were performed at 37°C in HBSTC containing 2.5 nM apixaban ( closed ) or rivaroxaban ( open ). Fluorescence intensity values were converted to concentration of 4-amino methyl coumarin (product) and plotted versus time. Data were fit to a mixed inhibition model (lines). The inset shows nominal residual factor Xa concentration determined from the fitted data plotted versus time.

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  Fig. 6. Inhibition of factor Xa assembled in the prothrombinase complex by the presence of apixaban and rivaroxaban. Activation of 500 nM prothrombin by 0.5 nM prothrombinase (0.5 nM factor Xa, 5 nM factor Va, 50 µM PCPS, 5 mM CaCl 2 ) containing ( A ) apixaban or ( B ) rivaroxaban at varying concentrations (2, 10, or 30 nM) was monitored using DAPA in a fluorescent plate reader at 37°C. Thrombin generation was monitored at 2-s intervals. Data were fit to a mixed inhibition model (lines).

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  Fig. 7. rMZ was used instead of prothrombin to monitor activation cleavage at Arg320 by prothrombinase using DAPA in the absence or presence of apixaban or rivaroxaban (30 nM). rMZa generation was monitored at 2-s intervals in a fluorescent plate reader at 37°C. Data were fit to a mixed inhibition model (lines).

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  Scheme 1. Mixed inhibition of factor Xa by apixaban or rivaroxaban. This model depicts two ways in which factor Xa may bind its inhibitors. FXa, factor Xa; S, substrate (Pefafluor Xa); I, inhibitor (apixaban or rivaroxaban).

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  Scheme 2. Mixed inhibition of factor Xa in prothrombinase by apixaban or rivaroxaban during prothrombin activation. This model depicts three ways in which prothrombinase may bind its inhibitors. Ratcheting is included in this model, which signifies a conformational change of the intermediate, meizothrombin, so that it is able to be further processed to thrombin. E, prothrombinase; P, prothrombin; M, meizothrombin; M2, ratcheted meizothrombin; T, thrombin; I, inhibitors (apixaban or rivaroxaban).