Adenosine Receptor Agonists Increase the Inhibition of Platelet Function by P2Y12 Antagonists in a cAMP- and Calcium-Dependent Manner

Figure 1. Chemical structures of selected adenosine receptor agonists and P2Y12 antagonists.

Figure 2. AR agonists intensify the inhibitory effect of P2Y12 antagonists on platelet reactivity, as measured by P-selectin expression (n = 5). (A) Effect of AR agonist on platelets, (B) effect on platelets antagonised with the P2Y12 inhibitor cangrelor, (C) effect on platelets antagonised with the P2Y12 inhibitor prasugrel metabolite (PM). Data are presented as median, interquartile range (box), and minimum and maximum values (whiskers). Platelet reactivity was measured after activation with 20 μM ADP, in whole blood. Samples were preincubated at 37 °C for 3 min with AR agonist and cangrelor, or 15 min with PM, all in their previously determined IC50. Statistical significance was estimated by one-way ANOVA for repeated measurements with the post hoc Bonferroni’s multiple comparisons test or Friedman’s test with Dunn’s correction for multiple comparisons. Groups containing AR agonists are compared to control samples: untreated samples for AR agonists alone (A), P2Y12 inhibitor-treated samples for combined systems (B,C). * p < 0.05, ** p < 0.01, *** p < 0.005. UK—UK423,097, HN—HE-NECA, MRE—MRE0094, 2 chloro -2-chloroadenosine, PSB—PSB0777, CGS—CGS21680, rega—regadenoson, CV—CV1808,…

Figure 3. AR agonists intensify the inhibitory effect of P2Y12 antagonists on platelet reactivity, as measured by expression of GPIIb-IIIa active form (PAC-1 antibody binding) (n = 5). (A) Effect of AR agonist on platelets, (B) effect on platelets antagonised with the P2Y12 inhibitor cangrelor, (C) effect on platelets antagonised with the P2Y12 inhibitor prasugrel metabolite (PM). Data are shown as median, interquartile range (box), and minimum and maximum values (whiskers). Platelet reactivity was assessed after activation with 20 μM ADP, in whole blood. Samples were preincubated at 37 °C for 3 min with AR agonist and cangrelor, or 15 min with PM, all in their previously determined IC50. Statistical significance estimated by one-way ANOVA for repeated measurements with the post hoc Bonferroni’s multiple comparisons test or Friedman’s test with Dunn’s correction for multiple comparisons (groups containing AR agonist are compared to control samples: untreated sample for AR agonists alone (A) or P2Y12 inhibitor-treated samples for combined version (B,C)). * p < 0.05, ** p < 0.01, *** p < 0.005. UK—UK423,097, HN—HE-NECA, MRE—MRE0094, 2 chloro—2-chloro- adenosine, PSB—PSB0777, CGS—CGS21680,…

Figure 4. AR agonists intensify the inhibitory effect of P2Y12 antagonists on platelet reactivity as measured by exogenous fibrinogen binding (n = 5). Panels present data on AR agonist impact on platelets (A), platelets antagonised with P2Y12 inhibitor: cangrelor (B) or prasugrel metabolite (PM) (C). Data are shown as median, interquartile range (box), and minimum and maximum values (whiskers). Platelet reactivity was assessed after activation with 20 μM ADP, in whole blood. Samples were preincubated at 37 °C for 3 min with AR agonist and cangrelor or 15 min with PM, all in their previously determined IC50. Statistical significance estimated by one-way ANOVA for repeated measurements with the post hoc Bonferroni’s multiple comparisons test or Friedman’s test with Dunn’s correction for multiple comparisons (groups containing AR agonist are compared to control samples: untreated sample for AR agonists alone (A) or P2Y12 inhibitor-treated samples for combined version (B,C)). * p < 0.05, ** p < 0.01, *** p < 0.005. UK—UK423,097, HN—HE-NECA, MRE—MRE0094, 2chloro—2-chloroadenosine, PSB—PSB0777, CGS—CGS21680, rega—regadenoson, CV—CV1808, C—cangrelor, PM—prasugrel metabolite R-138727. IC50 values:…

Figure 5. AR agonists strengthen the inhibitory effect on calcium ion mobilisation by of P2Y12 antagonists (n = 5). Panels present data on the effect of AR agonist on platelets (A), platelets antagonised with P2Y12 inhibitor: cangrelor (B) or prasugrel metabolite (PM) (C). Data are presented as median, interquartile range (box), and minimum and maximum values (whiskers). Platelet reactivity was assessed after activation with 20 μM ADP, in whole blood. Samples were preincubated at 37 °C for 3 min with AR agonist and cangrelor, or 15 min with PM, all in their previously determined IC50. Statistical significance estimated by one-way ANOVA for repeated measurements with the post hoc Bonferroni’s multiple comparisons test or Friedman’s test with Dunn’s correction for multiple comparisons (groups containing AR agonist are compared to samples: untreated sample for AR agonists alone (A) or P2Y12 inhibitor-treated samples for combined version (B,C)). * p < 0.05, ** p < 0.01, *** p < 0.005. UK—UK423,097, HN—HE-NECA, MRE—MRE0094, 2chloro—2-chloroadenosine, PSB—PSB0777, CGS—CGS21680, rega—regadenoson, CV—CV1808, C—cangrelor, PM—prasugrel metabolite R-138727. IC50 values: UK423,097 1 µM, HE-NECA 0.2…

Figure 6. AR agonists strengthen the P2Y12 inhibitory effect on platelet reactivity as measured by cAMP formation in resting platelets (n = 5). Panels present data on AR agonist impact on platelets in comparison to unstimulated platelets (ctrl) (A), platelets antagonised with P2Y12 inhibitor: cangrelor (B). Data are presented as median, interquartile range (box), and minimum and maximum values (whiskers). cAMP level was measured in isolated platelets. Samples were preincubated at 37 °C for 3 min with AR agonist and cangrelor. Statistical significance estimated by one-way ANOVA for repeated measurements with the post hoc Bonferroni’s multiple comparisons test or Friedman’s test with Dunn’s correction for multiple comparisons (samples containing AR agonist are compared to control samples with P2Y12 inhibitor within pertinent group). * p < 0.05, ** p < 0.01, *** p < 0.005. UK—UK423,097, HN—HE-NECA, PSB—PSB0777, C—cangrelor. IC50 values: UK423,097 1 µM, HE-NECA 0.2 µM, NECA 0.5 µM, and cangrelor 17 nM.

Figure 7. AR agonists strengthen the P2Y12 inhibitory effect on platelet reactivity as measured by measured by cAMP formation in ADP- activated platelets (n = 5). Figures present data on AR agonist impact on platelets in comparison to platelets stimulated with 20 µM ADP (ctrl) (A), platelets antagonised with P2Y12 inhibitor: cangrelor (B). Data are shown as median, interquartile range (box), and minimum and maximum values (whiskers). cAMP level was measured in isolated platelets after activation with 20 μM ADP. Samples were preincubated at 37 °C for 3 min with AR agonist and cangrelor. Statistical significance estimated by one-way ANOVA for repeated measurements with the post hoc Bonferroni’s multiple comparisons test or Friedman’s test with Dunn’s correction for multiple comparisons (groups containing AR agonist are compared to control group with P2Y12 inhibitor) (samples containing AR agonist are compared to control samples with P2Y12 inhibitor within pertinent group). * p < 0.05, ** p < 0.01, **** p < 0.001. UK—UK423,097, HN—HE-NECA, PSB—PSB0777, C—cangrelor, IC50 values: UK423,097 1 µM, HE-NECA 0.2 µM, NECA 0.5 µM, and cangrelor 17 nM.

Figure 8. Comparison of AR agonist, P2Y12 antagonist or AR agonist and P2Y12 antagonist pairing inhibitory effectiveness of ADP-induced platelet reactivity assessed by P-selectin expression, GPIIb-IIIa activation, fibrinogen binding, and calcium ion mobilization. Black boxes indicate statistically significant inhibition for P2Y12 antagonist alone and AR agonist alone compared to control non-treated sample, as well for AR agonist and P2Y12 antagonist pairs compared to the sample with P2Y12 inhibitor only. UK—UK423,097, HN—HE-NECA, MRE—MRE0094, 2chloro—2-chloroadenosine, PSB—PSB0777, CGS—CGS21680, rega—regadenoson, CV—CV1808