Antiplatelet Agents Have a Distinct Efficacy on Platelet Aggregation Induced by Infectious Bacteria

Figures

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  Figure 1. Representative measurement of light transmission over time in response to platelet aggregation. Untreated and aspirin (2 mM), ticagrelor (10 µM), association of both and tirofiban (25 ng/ml) treated PRP, infected by (A) S. aureus P2188 or (B) S. sanguinis P8633. NaCl and TRAP (10 µM) treated PRP were used as controls.

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  Figure 2. Effect of antiplatelet drugs on bacterial induced platelet aggregation. (A) Lag time for the onset of aggregation and percentage of maximum aggregation as measured by LTA of PRP activated by TRAP (10 µM) or infected with six bacterial strains belonging to two species, S. aureus and S. sanguinis. Results are expressed as mean ± SD. (B) Final aggregation as measured by LTA of untreated PRP and PRP treated with aspirin (Asp, 2 mM), ticagrelor (Tcg, 10 µM), association of both (Asp + Tcg) and tirofiban (Tfb, 0.5 µM) and then infected by three different strains of S. aureus. Results are expressed as mean ± SD. n = 5–6; one-way ANOVA, Bonferroni’s multiple comparison test. *: p < 0.05; **: p < 0.01; ***: p < 0.001. (C) Final aggregation as measured by LTA of untreated PRP and PRP treated with aspirin (Asp, 2 mM), ticagrelor (Tcg, 10 µM), association of both (Asp + Tcg) and tirofiban (Tfb, 0.5 µM) and then infected by three different strains of S. sanguinis. Results are expressed as mean ± SD. n = 6; one-way ANOVA, Bonferroni’s multiple comparison test. *: p < 0.05; **: p < 0.01; ***: p <0.001.

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  Figure 3. Measurement of CD62P surface exposure by flow cytometry. (A) Surface exposure of CD62P on native platelets, platelets activated by TRAP (10 µM) and platelets infected with S. aureus P6142 or S. sanguinis P8633. The MFI of untreated platelets was used as 100%. The MFI of each experiment was calculated as follow: MFI × 100/MFI of untreated and uninfected platelets. Results are expressed as mean ± SD. n = 6; Paired t test. *: p < 0.05. (B) Surface exposure of CD62P on untreated platelets and platelets treated with aspirin (Asp, 2 mM), ticagrelor (Tcg, 10 µM), association of both (Asp + Tcg) and tirofiban (Tfb, 0.5 µM). Untreated or treated platelets were then infected with S. aureus P 6142. The MFI of untreated platelets was used as 100%. The MFI of each experiment was calculated as follow: MFI × 100/MFI of untreated and uninfected platelets. Results are expressed as mean ± SD. n = 6; one-way ANOVA, Bonferroni’s multiple comparison test. **: p < 0.01; ***: p < 0.001. (C) Surface exposure of CD62P on untreated platelets and platelets treated with aspirin (Asp, 2 mM), ticagrelor (Tcg, 10 µM), association of both (Asp + Tcg) and tirofiban (Tfb, 0.5 µM). Untreated or treated platelets…

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  Figure 4. Scanning electron microscopy of uninfected platelets and platelets–bacteria aggregates. The whole deposited glass slide was analyzed for each condition with a TM4000Plus™ (Hitachi, Tokyo, Japan) scanning electron microscope operated at 10 and 15 kV in BSE mode at magnifications ranging from ×200 to ×3,000. Compared to uninfected control PRP (A, B), the addition of S. aureus P6142 induced a change in the ultrastructure with a denser organization and the presence of an intense network with fibrillar organization. Also, several well-defined bags of bacteria were observed (C, D). Clots obtained from PRP infected with S. sanguinis P8633 was organized in a form of a compact pack of amorphous clusters until an absence of cellular integrity (E, F).

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  Figure 5. Scanning electron microscopy of aggregates formed from treated platelets and infected with S. aureus P6142 or S. sanguinis P8633. Aspirin treated PRP and infected with (A) S. aureus or (B) S. sanguinis. Ticagrelor treated PRP and infected with (C) S. aureus or (D) S. sanguinis. Aspirin-ticagrelor combination treated PRP and infected with (E) S. aureus or (F) S. sanguinis. Tirofiban treated PRP and infected with (G) S. aureus or (H) S. sanguinis. The whole deposited glass slide was analyzed for each condition with a TM4000Plus™ (Hitachi, Tokyo, Japan) scanning electron microscope operated at 10 and 15 kV in BSE mode at magnifications ranging from ×200 to ×3,000. The use of different antiplatelet molecules was accompanied by a slight decrease in the filamentous networks as well as in the organization of bagged bacteria in the case of S. aureus. For S. sanguinis, the use of antiplatelet agents was accompanied by a decrease in clot density and the possibility of detecting cellular elements within it.

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  Figure 6. Interpretative scheme. Summarized results show here the effects of bacteria with or without antiplatelet drugs on platelets aggregation. Results of LTA and scanning electron microscopy are illustrated by three parameters: number of aggregates, their size and their ultrastructure. Antiplatelet agents significantly reduced aggregation induced by the two bacterial species. Among the antiplatelet agents taken orally and for long term, ticagrelor and the combination (aspirin–ticagrelor) showed the greatest decrease with S. aureus and S. sanguinis respectively (framed aggregates). The ultrastructure was different according to the two species tested.