Vascular protective effect of aspirin and rivaroxaban upon endothelial denudation of the mouse carotid artery

Figures

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  Figure 1. Temporary ligation of the carotid artery provokes consistent sub-occlusive thrombus formation in vivo. Scanning electron microscopy (SEM) images of thrombi (A) 1, (B) 3, (C) 6, (D) 24 and, (E) 72 h after temporary ligation of the carotid artery in control WT mice. (A–E) In all samples sub-occlusive platelet-rich thrombus formation is observed on the injured vessel wall. Damaged endothelium is indicated by a red arrow and undamaged endothelium by a yellow arrow. (F) No platelet adhesion was observed in absence of temporary ligation (3 mm upstream). Representative SEM images of 6 independent ligations are depicted. Scale bar is 80 μm, or 10 μm for zoomed images. (G) Thrombus size was scored by examining SEM images obtained from ligated carotid arteries that were collected at 1, 3, 6, 24 or 72 h after ligation. Scoring scale: 0 = no platelet adhesion, 1 = platelet monolayer, 2 = small thrombi (< 15 layers), and 3 = larger thrombi (> 15 layers).

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  Figure 2. Mouse treatment with aspirin and/or rivaroxaban alleviates ligation-induced vascular stiffening. Carotid vessel wall extension was measured in the four groups of mice using ultrasound (Vevo 2100) at baseline (day 0), 7 and 14 days after temporary ligation. Extension is expressed as % of lumen with quantification done in M-mode. (A) Representative images acquired in M-mode, illustrating extension of the carotid artery during three heart beats. Extension was measured from the difference between the yellow and light blue bars. (B) Graph of right carotid artery (RCA) extension at site of temporary ligation in control mice or mice treated with aspirin (ASA), rivaroxaban (RIVA) or the combination of both. Also indicated in B are values at three millimetres upstream of injury (control group). Mean ± SEM (n = 11–12), *p < 0.05, **p < 0.01, two-way ANOVA.

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  Figure 3. Mouse treatment with aspirin or rivaroxaban reduces post-ligation intima-media thickening. (A) Haematoxylin and eosin staining of paraffin sections of the right carotid artery of mice treated with vehicle, aspirin (ASA), rivaroxaban (RIVA) or both, obtained 14 days after temporary ligation. Contours of media are indicated by the yellow dotted line. Bar = 50 µm. Boxplot (min/max) with quantification of media thickness at the site of ligation for the right carotid artery (RCA) (B) and left carotid artery (LCA) (C). N = 11–12, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005, one-way ANOVA.

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  Figure 4. Mouse treatment with aspirin and/or rivaroxaban can inhibit the number of bound platelets per leukocyte. Blood was collected from mice at 2 weeks after temporary ligation of the carotid artery. During the post-ligation period, mice were treated with aspirin (ASA), rivaroxaban (RIVA), ASA + RIVA, or saline (control). Using flow cytometry, platelet-leukocyte aggregates (PLAs) were measured in whole blood by dual staining with APC-αCD45 and DyLight 488-labelled αGPIbβ antibodies. Platelets were unstimulated or stimulated with 2-MeSADP (5 μM) or AYPGKF (30 μM). (A) Flow cytometric events were gated for CD45 fluorescence, and size anomalies were excluded by forward sideward scatter analysis. Shown is fluorescence in control blood from all leukocytes without (left of line) and with (right of line) bound platelets as defined by DyLight 488-labelled αGPIbβ fluorescence. (B) Calculated number of bound platelets per PLA. Data represented as mean ± SEM, n = 11 (control), 11 (ASA), 12 (RIVA) and 6 (ASA + RIVA) animals per group, *p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA.

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  Figure 5. Rivaroxaban treatment results in lower murine thrombi in vitro. (A–D) Representative brightfield images after 4 min of whole blood perfusion from WT mice. Whole blood was pre-treated with (A) saline/DMSO, (B) 100 µM aspirin, (C) 500 ng/mL rivaroxaban or (D) 100 µM aspirin and 500 ng/mL rivaroxaban. Bar = 25 µm. (E) Subtraction heatmap of normalised parameters (0–10). Parameters include platelet surface area coverage (%SAC), thrombus morphological score (0–5), multilayer score (0–3) and, contraction score (0–3). N = 9 (control), 4 (ASA), 6 (RIVA) and 6 (ASA + RIVA) animals per group, *p < 0.05 vs. control, Kruskal–Wallis test.

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  Figure 6. Rivaroxaban treatment results in lower human thrombi in vitro. (A–F) Representative brightfield images after 4 min of whole blood perfusion from healthy volunteers. Whole blood was pre-treated with (A) saline/DMSO, (B) 100 µM aspirin, (C) 500 ng/mL rivaroxaban, (D) 10 ng/mL rivaroxaban, (E) 100 µM aspirin and 500 ng/mL rivaroxaban and (F) 100 µM aspirin and 10 ng/mL rivaroxaban. Bar = 25 µm. (G) Subtraction heatmap of normalised parameters (0–10). Parameters include platelet surface area coverage (%SAC), thrombus morphological score (0–5), multilayer score (0–3) and, contraction score (0–3). N = 14 (control), 5 (ASA), 5 (RIVA 500 ng/mL), 5 (RIVA 10 ng/mL), 5 (ASA + RIVA 500 ng/mL) and 5 (ASA + RIVA 10 ng/mL) per group *p < 0.05 vs. control, Kruskal–Wallis test.

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  Figure 7. Rivaroxaban inhibits thrombus growth of human thrombi in vitro. Human whole blood was perfused over a combined collagen type I plus tissue factor surface to study thrombus growth under coagulating conditions. Thrombus growth was visualized by DiOC6 (0.1 µg/mL) labelled whole blood of the same donor which was pre-treated with 10 ng/mL or 500 ng/mL rivaroxaban with (B,C) or without (D,E) 100 µM aspirin or vehicle (A). Quantification (%SAC) of platelet deposition for initial thrombus was assessed by brightfield and for thrombus growth by fluorescence microscopy (F). Black bar: initial thrombus formation, grey bar: thrombus growth. AR: 100 µM aspirin with 10 or 500 ng/mL rivaroxaban, Ctrl: control, R10: 10 ng/mL rivaroxaban, R500: 500 ng/mL rivaroxaban. Data are represented as bar graphs (median + IQR), n = 4–12, *p ≤ 0.05, **p ≤ 0.01, **p ≤ 0.001, Kruskal–Wallis test.