Dabigatran Attenuates the Binding of Thrombin to Platelets—A Novel Mechanism of Action

Figures

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  Fig. 1. Important structural components of human α-thrombin. Human α-thrombin bound to dabigatran in the active site pocket has been shown. The secondary structural components of thrombin are represented as cartoons and dabigatran as sticks. The structural units of thrombin like exosite 1 (red), exosite 2 (blue), active site (orange), and the 60 loop (green), which are considered important for binding, catalytic activity, and substrate specificity, are shown as spheres.

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  Fig. 2. Inhibition of thrombin binding to platelets as measured by microscale thermophoresis (MST). Binding of thrombin to the platelets and the effect of inhibitors were evaluated using MST. In this normalized display, the change in normalized fluorescence (ΔFnorm) of thrombin was plotted against the concentration (conc) of the thrombin receptors on platelets in log scale. Binding curve of thrombin to platelets without any inhibitors (control) is compared with the binding of thrombin (55 nM) to platelets in the presence of ( A ) thrombin exosite specific inhibitors HD1 (1 µM) or HD22 (1 µM) or both and ( B ) thrombin active site inhibitors dabigatran (318 nM), argatroban (318 nM), or PPACK (55 nM). The data was calculated using the one phase decay equation in the exponential model using GraphPad Prism. The results shown are mean with standard deviation (SD) represented as shadow outlines around the binding curves, where number of blood donors ( n ) ≥5.

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  Fig. 3. Inhibition of thrombin binding to platelets and effects on platelet activation by flow cytometry. Thrombin binding to platelets and its effect on platelet activation have been shown in the presence and absence of various inhibitors of the thrombin exosites and the active site. Platelet activation markers P-selectin exposure and fibrinogen binding were measured to confirm inhibitors' efficacy and platelet reactivity. Negative control test without thrombin has been compared and shown here as control. Median fluorescence intensity of thrombin without any inhibitor has been considered as 100% and the relative fluorescence intensity (%) to thrombin without any inhibitor has been shown as bar graph. The data was calculated using a one-way ANOVA test and results shown are the mean with standard error of mean (SEM) where number of blood donors ( n ) ≥5. RFI, relative fluorescence intensity.

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  Fig. 4. Effect of dabigatran on thrombin binding to exosite ligands by microscale thermophoresis (MST). The effect of dabigatran on exosite ligands binding to thrombin without platelets was analyzed by MST. Binding of NHS-labeled thrombin (55 nM) to exosite 1 ligand HD1 ( A ; highest concentration 1 µM), exosite 2 ligand HD22 ( B ; highest concentration 1 µM), or another exosite 2 ligand heparin ( C ; highest concentration 1 U/mL) in the presence (+) or absence (−) of dabigatran (318 nM) was measured by MST. In this normalized display, the change in normalized fluorescence (fraction bound) is plotted against the concentration of the ligand in log scale. All the results shown are mean with standard deviation (SD) where n ≥ 3.

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  Fig. 5. Surface plasmon resonance (SPR) sensogram of thrombin injected onto an SPR chip with a carboxylated dextran with immobilized aptamers. The interactions between thrombin exosites and DNA aptamers were analyzed. The aptamers HD1 and HD22 are immobilized in separate flow channels to an increase in resonance units (RU) of 971 and 1,395, respectively. Thrombin was injected at a concentration of 100 nM in the presence and absence of 300 nM dabigatran. ( A, B ) Sensograms of thrombin in the presence and absence of dabigatran injected over immobilized HD1 and HD22, respectively. To further evaluate the interaction between thrombin and HD22 the injection volume was doubled to 60 uL ( C ). Overall, no significant differences between thrombin in the presence and absence of dabigatran can be observed. Although response curves are of different shapes for the two aptamers, the addition of dabigatran does not alter the response.

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  Fig. 6. IC50 of dabigatran for thrombin binding to platelets by flow cytometry. The inhibition potential of dabigatran for thrombin binding to platelets and platelet activation was assessed using flow cytometry. Increasing concentration of the dabigatran (2–600 nM) as log transformation of concentrations to base 10 is plotted against the median fluorescence intensity (MFI) of thrombin binding, P-selectin exposure, or fibrinogen binding and IC50 values were calculated as described in the methods. The results shown are the mean with standard error of mean (SEM) where number of blood donors, n ≥ 5.