Antithrombotic and prohemorrhagic actions of different concentrations of apixaban in patients exposed to single and dual antiplatelet regimens

Figures

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  Figure 1. The diagram summarizes the design of the study. APIX was spiked on blood samples from the different study groups: healthy controls, or patients under treatment with aspirin (ASA), aspirin plus clopidogrel (ASA + CLOPI), or aspirin plus ticagrelor (ASA + TICA). APIX was initially solved in dimethyl sulfoxide (DMSO)and further diluted in phosphate-buffered saline (PBS) containing a low concentration of DMSO. Volumes spiked to blood samples for Control or treatment groups were equal (100 µL per 2 mL of blood or equivalent). Concentrations of APIX were corrected for the small volume change. Concentrations of DMSO were kept identical (< 0.05%) in all blood samples.

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  Figure 2. Modifications in clotting times in ROTEM studies. Bars summarize the results of clotting times in seconds (mean ± SEM) for Control and treatment groups. Apixaban caused a dose-related prolongation of clotting times in all study groups. Prolongations in clotting times were more evident with APIX160 and less pronounced in the group exposed to APIX40. *p < 0.05; **p < 0.01; ***p < 0.001; vs. respective APIX0. Control (n = 25); ASA (n = 21) ASA + CLOPI (n = 11), ASA + TICA (n = 21).

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  Figure 3. Modifications in lag times (A) and thrombin peaks (B) in thrombin generation assays. Bars summarize the results expressed as mean ± SEM of lag times (minutes) and thrombin peaks (nM) for Control and treatment groups. Apixaban caused a dose-related prolongation of lag times with a subsequent reduction in thrombin peaks. Prolongations in lag times were superior with APIX160 and less pronounced in the group exposed to APIX40. Reductions in thrombin peaks were more evident with APIX160. *p < 0.05; **p < 0.01; ***p < 0.001; vs. respective APIX0. Control (n = 25); ASA (n = 21) ASA + CLOPI (n = 11), ASA + TICA (n = 21).

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  Figure 4. Areas under the curve (AUC) calculated during the thrombin generation assay for the different study groups. Bars summarize the results of total thrombin generation in the different groups. The solid lines provide a visual estimate of the progressive effects calculated as a percentage of the AUC in the absence of apixaban (APIX0). Reductions in total thrombin generation appear more intense in the group of patients exposed to APIX + TICA, less pronounced in the group of patients treated with ASA, and somewhere intermediate in the group of patients treated with ASA + CLOPI. AUC expressed as arbitrary units *p < 0.05; **p < 0.01; ***p < 0.001; vs. respective APIX0. Control (n = 25); ASA (n = 21) ASA + CLOPI (n = 11), ASA + TICA (n = 21).

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  Figure 5. Percentages of the perfused surface covered by platelets. Bars summarize percentages of the surface covered by platelets in microfluidic studies evaluated by confocal microscopy for the different study groups. The solid lines provide a visual estimate of the progressive inhibitory effects calculated as a percentage of the platelet coverage in the absence of apixaban (APIX0). APIX40 significantly reduced the association of platelets to the forming thrombi. Reductions were more significant in studies with APIX160. Reductions in the combination of APIX160 with antiplatelet regimens seem more intense in the group of patients exposed to APIX + TICA, less prominent in the group of patients treated with ASA, and somewhere intermediate in the group of patients treated with ASA + CLOPI. *p < 0.05; **p < 0.01; ***p < 0.001; vs. respective APIX0. Control (n = 9); ASA (n = 6) ASA + CLOPI (n = 4), ASA + TICA (n = 8).

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  Figure 6. Percentages of the perfused surface covered by fibrin. Bars summarize percentages of the surface covered by fibrin in microfluidic studies evaluated by confocal microscopy for the different study groups. The solid lines provide a visual estimate of the progressive inhibitory effects calculated as a percentage of fibrin coverage in the absence of apixaban (APIX0). APIX40 significantly reduced the association of fibrin to the forming thrombi. Reductions were more significant in studies with APIX160 *p < 0.05; **p < 0.01; ***p < 0.001; vs. respective APIX0. Control (n = 9); ASA (n = 6) ASA + CLOPI (n = 4), ASA + TICA (n = 8).

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  Figure 7. Confocal Images from representative frames from corresponding 3-D image compilations. (A) ASA + TICA APIX0, (B) ASA + TICA APIX40 and (C) ASA + TICA APIX160. Recruitment of platelets was reduced by the antiplatelet agents and further potentiated by the presence of APIX. Structural changes in fibrin fibers and fibrin networks are observed after exposure to APIX40 or APIX160. At the lower concentration (APIX40) apixaban interferes with the thickness and structure of the fibers formed. APIX160 delayed the generation of fibrin and caused more evident disorganization of the fibrin networks formed on thrombogenic surfaces in microfluidic studies. These patterns were consistently observed with the different antiplatelet agents investigated and were maximal for the combination of ASA + TICA.