Adenosine Receptor Agonists Exhibit Anti-Platelet Effects and the Potential to Overcome Resistance to P2Y12 Receptor Antagonists

Figure 1. Inhibition of ADP-induced platelet aggregation by AR agonists. Data shown as median ± interquartile ranges, with dose-response plots based on the AUC values using non-linear regression analysis (NECA (A) n = 5, regadenoson (B) n = 5; LUF5834 (C) n = 4). Changes in platelet aggregation were measured in whole blood in response to 10 μM ADP after 3 min preincubation at 37 °C with AR agonist. Data was analysed for statistical significance using repeated measures ANOVA with Geisser-Greenhouse correction and Holm-Sidak’s multiple comparisons test. * indicates statistical significance p < 0.05 or lower.

Figure 2. AR agonists intensify the anti-aggregatory effect of P2Y12 antagonists: NECA (A), regadenoson (B), and LUF5835 (C). Data are presented as median, interquartile range and minimum and maximum values (n = 5 for each AR agonist; totally n = 15). Changes in platelet aggregation were measured in whole blood in response to 10 μM ADP after 3 min preincubation at 37 °C with AR agonist and cangrelor, or 15 min preincubation at 37 °C with PM. Statistical significance was estimated by repeated measures ANOVA with Bonferroni’s multiple comparison test, or Friedman’s test with Dunn’s multiple comparison test depending on data distribution. * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 3. Anti-platelet effect of AR agonists in high- and low-responders to P2Y12 antagonists. Combinations of cangrelor (A,B,E,F,I,J), and PM (C,D,G,H,K,L) with NECA (A–D) regadenoson (E–H), and LUF5835 (I–L) are shown. Framed numbers denote the mean inhibition increase factor (arithmetic mean of ratios calculated individually for each donor). Data are shown as pairs of data points (without and with AR agonist) for each blood donor (n = 10 in each group). Changes in platelet aggregation were measured in whole blood in response to 10 μM ADP after 3 min preincubation at 37 °C with AR agonist and cangrelor, or 15 min preincubation at 37 °C with AR agonist and PM. Statistical significance between P2Y12 inhibitor alone and P2Y12 inhibitor with AR agonist groups for each agonist-antagonist pairing estimated by two-tailed Wilcoxon signed rank test. * p < 0.05, ** p < 0.01.

Figure 4. AR agonists deepen the P2Y12 inhibitory effect on platelet reactivity as measured by P-selectin expression (A) and PAC-1 binding (B). Data are given as median, interquartile range, and minimum and maximum values (n = 5). Changes in platelet reactivity were measured in whole blood in response to 20 μM ADP after 3 min preincubation at 37 °C with AR agonist and cangrelor, or 15 min preincubation at 37 °C with PM. Statistical significance estimated by repeated measures ANOVA with Bonferroni’s multiple comparison test or Friedman’s test with Dunn’s multiple comparison test depending on the data distribution. * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 5. Anti-platelet effect of AR agonists and P2Y12 antagonists on thrombus formation under flow conditions. Representative images (A) and clot formation inhibition rates (decrease of total clot volume) (B). Data are given as median, interquartile range, and minimum and maximum values (n = 5). Changes in thrombus formation were analysed in whole blood in response to 60 dyne/cm2 flow after 3 min preincubation at 37 °C with an AR agonist and cangrelor, and 15 min preincubation at 37 °C with PM (n = 5). The samples were imaged by confocal microscopy and analysed using FIJI image analysis software to obtain summarized clot volumes (for details see Materials and methods). Statistical significance of differences between summarized clot volumes for various AR agonists, P2Y12 antagonists and combinations thereof was estimated by the bootstrap-boosted unpaired Student’s t-test. ** p < 0.01, *** p < 0.005.