Protective Effect of Ticagrelor Against Infective Endocarditis Induced by Virulent Staphylococcus aureus in Mice

Figures

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  Figure 1. Ticagrelor But Not Clopidogrel Prevents S aureus IE Development (A) Experimental setup. Mice received a single oral dose of ticagrelor or clopidogrel or corresponding vehicles before surgical catheter insertion, histamine infusion on the surface of aortic valve and intravenous injection of a S aureus IE CI. This figure was created with Biorender. (B) Number of ticagrelor-treated and control mice that developed IE (∗∗P < 0.01; Fisher’s exact test). (C) Number of clopidogrel-treated and control mice that developed IE (P > 0.05; chi-square test). (D) Ex vivo ADP (10 mmol/L)-induced platelet aggregation analyzed in PRP 1 hour after oral administration of ticagrelor (3 mg/kg, n = 5) or vehicle (n = 9). Data represent median and 25th to 75th percentiles (∗∗∗P = 0.001, Mann-Whitney U test). (E) Ex vivo ADP (10 mmol/L)-induced platelet aggregation analyzed in PRP 24 hours after oral administration of clopidogrel (30 mg/kg, n = 4) or vehicle (n = 4). Data represent median and interquartile range (∗P = 0.03, Mann-Whitney U test). ADP = adenosine diphosphate; AUC = area under the curve; CI = clinical isolate; CFU = colony-forming units; IE = infective endocarditis; PRP =…

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  Figure 2. Ticagrelor Antibacterial Activity (A) Time-kill curve of S aureus IE CI with ticagrelor (20 μg/mL) or vehicle (C: 1% DMSO) (n = 3). (B) Growth of IE CI was followed over time in TSB medium containing the indicated concentrations of ticagrelor or vehicle (C: 1% DMSO). (C) Growth of USA300 JE2 MRSA was followed over time in TSB medium containing the indicated concentrations of ticagrelor or vehicle (C: 1% DMSO). Data are from 3 independent experiments. DMSO = dimethyl sulfoxide; MRSA = methicillin-resistant Staphylococcus aureus; OD = optical density; TSB = Tryptic Soy Broth; other abbreviations as in Figure 1.

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  Figure 3. Conventional Concentrations of Ticagrelor Inhibit Toxin Production by S aureus JE2, JE2 ΔHla, and IE CI bacteria grown in the presence of indicated concentrations of ticagrelor or vehicle (C) (1% DMSO). (A) Western blot detection of α-toxin in bacterial supernatants. Data represent median and 25th-75th percentiles (n = 8; ∗P < 0.05; ∗∗∗P < 0.001, Kruskal-Wallis and Dunn’s multiple comparison tests). Vehicle data are arbitrarily set to 100. (B) Hemolytic activity of bacterial supernatants (IE CI, n = 5: ∗P < 0.05, ∗∗P < 0.01; JE2, n = 4: ∗P < 0.05, ∗∗P < 0.01). (C) Pigment intensity in bacterial pellets (IE CI, n = 7; JE2, n = 6; ∗∗∗P < 0.001). Data in C and D are the mean ± SD (P: ticagrelor vs vehicle, analysis of variance and Dunnett’s multiple comparison test). (D) Quantitative reverse transcriptase polymerase chain reaction analysis of messenger RNA expression in exponentially phase IE CI (n = 4, ∗P < 0.05, ∗P < 0.05, ∗∗∗P < 0.001 vs vehicle, Student’s t-test). Data represent the mean ± SD. Abbreviations as in Figure 1.

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  Figure 4. Conventional Concentrations of Ticagrelor Alter S aureus Adhesive Properties (A) Human whole blood perfused with bacteria over activated HUVEC. Bacteria were pretreated with ticagrelor or vehicle (vehicle, n = 8; 0.75 mg/mL, n = 8; 1.25 mg/mL, n = 8; ∗∗P < 0.01). (B) Ticagrelor or vehicle was added to blood just prior perfusion (vehicle, n = 4; 0.75 mg/mL, n = 4; 1.25 mg/mL, n = 4; ∗P < 0.05, ∗∗P < 0.01). (C) Bacteria suspensions perfused over VWF (IE CI: vehicle, n = 4; 0.75 mg/mL, n = 4; 1.25 mg/mL, n = 4; JE2: vehicle, n = 7; 0.75 mg/mL, n = 3; 1.25 mg/mL, n = 3; JE2 Δcoa, Δvwb, and ΔcoaΔvwb: n = 3; ∗∗P < 0.01, ∗∗∗P < 0.001). (D) Bacteria adhesion on fibrinogen (vehicle, n = 7; 0.75 mg/mL, n = 7; 1.25 mg/mL, n = 7; ∗∗P < 0.01, ∗∗∗P < 0.001). (E) Thrombin activity in bacterial supernatants added to human plasma (IE CI: n = 5, ∗∗P < 0.01; JE2: n = 5, ∗∗∗P < 0.01). All data represent the means ± SD (P: ticagrelor vs vehicle, analysis of variance, and Dunnett’s multiple comparison test). Vehicle control is arbitrarily set to 100. EC = endothelial cells; HUVEC = human umbilical vein endothelial cells; VWF = von Willebrand factor; other abbreviations as in Figure 1.

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  Figure 5. Effect of Ticagrelor Treatment on Toxin-Induced Platelet Aggregation (A) Platelet aggregation in human PRP was induced by supernatants of bacteria that have been pretreated with ticagrelor or vehicle (control). Representative aggregation curves are shown on the left. (Middle) n = 5, ∗∗∗P < 0.001, 2-tailed paired t-test. (Right) n = 3, ∗P < 0.05 vs vehicle; analysis of variance and Dunnett’s multiple comparison test. (B) Platelet aggregation was induced by bacterial cells that have been pretreated with ticagrelor or vehicle (control). Alternatively, ticagrelor was added in PRP before bacteria addition. Representative aggregation curves are shown (left). (Middle) n = 3, ∗P < 0.05 vs vehicle, analysis of variance, and Dunnett’s multiple comparison test. (Right) n = 3, P >0.05, 2-tailed paired t-test. (C) Platelet-killing assay performed with human washed platelets and IE IC. Bacterial survival is plotted as percentage of initial CFU input (n = 3, P > 0.05, 2-tailed paired t-test). All data represent the mean ± SD. Abbreviations as in Figure 1.