Short-acting P2Y12 blockade to reduce platelet dysfunction and coagulopathy during experimental extracorporeal circulation and hypothermia  S. Krajewski, J. Kurz, B. Neumann, T.O. Greiner, A. Stolz, B. Balkau, K. Peter, K. Unertl, H.P. Wendel, A. Straub  British Journal of Anaesthesia  Volume 108, Issue 6, Pages 912-921 (June 2012) DOI: 10.1093/bja/aer518 Copyright © 2012 The Author(s) Terms and Conditions

Fig 1 ECC- and hypothermia-induced platelet granule release, platelet–granulocyte binding, and platelet loss is inhibited by ADP receptor blockade ex vivo. Human blood was left untreated (baseline) or treated ex vivo with PBS (‘control’), cangrelor, or 2-MeSAMP (2-MeS). All treated samples were circulated in an ECC model for 30 min at 28 or 37°C. The percentage of platelets with expression of the platelet activation marker P-selectin was evaluated in flow cytometry (a, n=8). β-TG plasma concentrations (IU ml−1) were measured using ELISA (b, n=4). To evaluate ADP plasma concentrations (μM), a bioluminometric assay was used (c, n=8). The formation of platelet–granulocyte aggregates was measured in flow cytometry (geo mean fluorescence intensity; d, n=5). Platelet counts (×103 μl−1) were measured in all blood samples (e, n=8). Data are given as mean and sem; mean values of all baseline samples were transformed to 100% and data measured post-ECC are given as percentages of adjusted baseline values of each temperature group; groups were compared using RM-anova with Bonferroni’s multiple comparison test (*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001). British Journal of Anaesthesia 2012 108, 912-921DOI: (10.1093/bja/aer518) Copyright © 2012 The Author(s) Terms and Conditions

Fig 2 Platelet interaction with the ECC surface during hypothermic ECC is not inhibited by ADP receptor blockade ex vivo. Human blood was left untreated (baseline) or treated ex vivo with either vehicle (PBS) as a control, the P2Y12 antagonist cangrelor, or the P2Y12 antagonist 2-MeSAMP (2-MeS). All treated samples were circulated ex vivo in the Chandler loop ECC model for 30 min at 28 or 37°C. Platelet binding to ECC surface was measured in optical density units using a specialized ELISA (a; n=5). Binding of anti-CD42bα-PE mAb (geo mean fluorescence intensity; b; n=8) was measured using flow cytometry. Data are given as mean and sem; mean baseline values were transformed to 100% and data measured post-ECC are given as percentages of adjusted baseline values of each temperature group; groups were compared using RM-anova with Bonferroni’s multiple comparison test (*P<0.05). British Journal of Anaesthesia 2012 108, 912-921DOI: (10.1093/bja/aer518) Copyright © 2012 The Author(s) Terms and Conditions

Fig 3 Timeline of hypothermic CPB in pigs. T1–T6 indicate blood sampling time points: T1, skin incision before median sternotomy; T2, 10 min after initiation of cangrelor or placebo infusion and heparin application directly before the start of CPB; T3, 45 min after the start of CPB; T4, directly before the end of CPB; T5, 10 min after the end of CPB; T6, 60 min after the end of CPB. *Central venous catheter for administration of medication; arterial catheter for blood sampling and arterial pressure management. British Journal of Anaesthesia 2012 108, 912-921DOI: (10.1093/bja/aer518) Copyright © 2012 The Author(s) Terms and Conditions

Fig 4 Haematocrit and haemoglobin values and platelet counts. Before, during, and following hypothermic CPB in vehicle-treated (placebo) and cangrelor-treated (0.075 µg kg−1 min−1) pigs, changes in haematocrit (a) and haemoglobin concentration (b) were measured. Data are given as mean and sem; in both groups, baseline values (T1) were compared with T2–T6 of the respective group using RM-anova with Bonferroni’s multiple comparison test (**P<0.01; ***P<0.001; ****P<0.0001; n=5). Platelet counts (c) were measured at all time points (T1–T6). Data are given as mean and sem. For platelet counts, a haematocrit correction was performed for values measured after the start of CPB (T2–T6) to adjust for haemodilution caused by the priming volume of the HLM. The mean baseline values were adjusted to 100% and data measured during and after CPB are given in relation to the adjusted baseline value in each treatment group; groups were compared using RM-anova with Bonferroni’s multiple comparison test. T1, skin incision before median sternotomy; T2, 10 min after initiation of cangrelor or placebo infusion and heparin application directly before the start of CPB; T3, 45 min after the start of CPB; T4, directly before the end of CPB; T5, 10 min after the end of CPB; T6, 60 min after the end of CPB. British Journal of Anaesthesia 2012 108, 912-921DOI: (10.1093/bja/aer518) Copyright © 2012 The Author(s) Terms and Conditions

Fig 5 ADP receptor blockade with cangrelor prevents an increase in platelet aggregation. Whole-blood samples were taken at different time points before, during, and after hypothermic CPB (T1–T6) from vehicle-infused (placebo) and cangrelor-infused (0.075 µg kg−1 min−1) pigs. Platelet-rich plasma was prepared from all blood samples and ADP-induced (20 µM) platelet aggregation was analysed. Data are given as mean and sem. In placebo- and cangrelor-treated groups, baseline values (T1) were adjusted to 100% and groups were compared using RM-anova with Bonferroni’s multiple comparison test (*P<0.05; **P<0.01; ***P<0.001; n=5). T1, skin incision before median sternotomy; T2, 10 min after initiation of cangrelor or placebo infusion and heparin application directly before the start of CPB; T3, 45 min after the start of CPB; T4, directly before the end of CPB; T5, 10 min after the end of CPB; T6, 60 min after the end of CPB. British Journal of Anaesthesia 2012 108, 912-921DOI: (10.1093/bja/aer518) Copyright © 2012 The Author(s) Terms and Conditions

Fig 6 ADP receptor blockade with cangrelor prevents an increase in fibrinogen binding. Platelet–fibrinogen binding was measured in flow cytometry in the vehicle-treated (placebo) and cangrelor-treated (0.075 µg kg−1 min−1) groups at different time points during hypothermic CPB in pigs. Data are given as mean and sem. In both groups, baseline values (T1) were adjusted to 100% and groups were compared using RM-anova with Bonferroni’s multiple comparison test (*P<0.05; n=5). T1, skin incision before median sternotomy; T2, 10 min after initiation of cangrelor or placebo infusion and heparin application directly before the start of CPB; T3, 45 min after the start of CPB; T4, directly before the end of CPB; T5, 10 min after the end of CPB; T6, 60 min after the end of CPB. British Journal of Anaesthesia 2012 108, 912-921DOI: (10.1093/bja/aer518) Copyright © 2012 The Author(s) Terms and Conditions