Platelet-Mediated Transfer of Cardioprotection by Remote Ischemic Conditioning and Its Abrogation by Aspirin But Not by Ticagrelor

Abstract

Purpose: The role of platelets during myocardial ischemia/reperfusion (I/R) is ambivalent. They contribute to injury but also to cardioprotection. Repeated blood flow restriction and reperfusion in a tissue/organ remote from the heart (remote ischemic conditioning, RIC) reduce myocardial I/R injury and attenuate platelet activation. Whether or not platelets mediate RIC's cardioprotective signal is currently unclear.

Methods and results: Venous blood from healthy volunteers (without or with pretreatment of 500/1000 mg aspirin or 180 mg ticagrelor orally, 2-3 h before the study, n = 18 each) was collected before and after RIC (3 × 5 min blood pressure cuff inflation at 200 mmHg on the left upper arm/5 min deflation). Washed platelets were isolated. Platelet-poor plasma was used to prepare plasma-dialysates. Platelets (25 × 10³/µL) or plasma-dialysates (1:10) prepared before and after RIC from untreated versus aspirin- or ticagrelor-pretreated volunteers, respectively, were infused into isolated buffer-perfused rat hearts. Hearts were subjected to global 30 min/120 min I/R. Infarct size was stained. Infarct size was less with infusion of platelets/plasma-dialysate after RIC (18 ± 7%/23 ± 9% of ventricular mass) than with platelets/plasma-dialysate before RIC (34 ± 7%/33 ± 8%). Aspirin pretreatment abrogated the transfer of RIC's cardioprotection by platelets (after/before RIC, 34 ± 7%/33 ± 7%) but only attenuated that by plasma-dialysate (after/before RIC, 26 ± 8%/32 ± 5%). Ticagrelor pretreatment induced an in vivo formation of cardioprotective factor(s) per se (platelets/plasma-dialysate before RIC, 26 ± 7%/26 ± 7%) but did not impact on RIC's cardioprotection by platelets/plasma-dialysate (20 ± 7%/21 ± 5%).

Conclusion: Platelets serve as carriers for RIC's cardioprotective signal through an aspirin-sensitive and thus cyclooxygenase-dependent mechanism. The P2Y₁₂ inhibitor ticagrelor per se induces a humoral cardioprotective signal.

Keywords: Aspirin; Cardioprotection; Ischemia/reperfusion; Remote ischemic conditioning; Ticagrelor.

Fig. 1

Infusion of saline, aspirin, ticagrelor, or Tyrode buffer supplemented with apyrase and prostaglandin E₁ does not impact on infarct size. Solutions were infused into isolated perfused rat hearts subjected to 30-min global ischemia and 120-min reperfusion. The concentrations for infusion with aspirin (40 µmol/L, n = 8) and ticagrelor (10 µmol/L, n = 8) were chosen to reflect the expected maximum plasma concentration 2–3 h after oral ingestion of aspirin and ticagrelor, respectively, in healthy volunteers. The concentration of apyrase and prostaglandin E₁ (0.1 U/mL and 1 µmol/L, n = 8) was equal to that used for the preparation of washed platelet solutions. Infusion of saline (n = 10) served as control

Fig. 2

Turbidometric light transmission aggregometry of platelets sampled from volunteers before and after remote ischemic conditioning (RIC). Platelets were sampled before and after RIC from the same volunteers (n = 18): without pretreatment (n = 18), with aspirin pretreatment (n = 18), and with ticagrelor pretreatment (n = 18), respectively. White color indicates data before RIC, gray after RIC. PPP, platelet-poor plasma; PRP, platelet-rich plasma. a ADP-induced aggregation of platelet-rich plasma. *p < 0.001 vs. without pretreatment; #p < 0.001 vs. with aspirin pretreatment; Kruskal–Wallis one-way ANOVA on Ranks and Dunn’s multiple comparison procedures. b Arachidonic acid-induced aggregation of platelet-rich plasma. *p < 0.001 vs. without pretreatment; #p < 0.001 vs. with aspirin pretreatment; Kruskal–Wallis one-way ANOVA on Ranks and Dunn’s multiple comparison procedures. c Thrombin-induced aggregation of washed platelets

Fig. 3

Platelets serve as transmitters of remote ischemic conditioning’s (RIC)s cardioprotective signal through an aspirin-sensitive mechanism. Platelets were sampled before and after RIC from the same volunteers (n = 18): without pretreatment (n = 18), with aspirin pretreatment (n = 18), and with ticagrelor pretreatment (n = 18), respectively. Platelets were infused into isolated perfused rat hearts, which were then subjected to 30-min global ischemia/120-min reperfusion. In the aspirin pretreatment group, gray symbols indicate volunteers who were pretreated with 500 mg aspirin (n = 12) and black symbols those with 1000 mg aspirin (n = 6). *p < 0.001 vs. before RIC without pretreatment; **p < 0.002 vs. before RIC with ticagrelor pretreatment; #p = 0.002 vs. before RIC without pretreatment and p = 0.011 vs. before RIC with aspirin pretreatment; †p < 0.001 vs. after RIC without pretreatment and vs. after RIC with ticagrelor pretreatment; two-way repeated measures ANOVA with Fisher’s least significant differences post hoc tests

Fig. 4

Pretreatment with aspirin or ticagrelor does not impact on the cardioprotective transfer of remote ischemic conditioning (RIC) via plasma-dialysate. Plasma-dialysates were prepared before and after RIC from the same volunteers (n = 18): without pretreatment (n = 18), with aspirin pretreatment (n = 18), and with ticagrelor pretreatment (n = 18), respectively. Plasma-dialysates before and after RIC were infused into isolated perfused rat hearts, respectively, which were then subjected to 30-min global ischemia/120 min reperfusion. In the aspirin pretreatment group, gray symbols indicate volunteers who were pretreated with 500 mg aspirin (n = 12) and black symbols (n = 6) those with 1000 mg aspirin.*p < 0.001 vs. before RIC without pretreatment; †p = 0.030 vs. before RIC with aspirin pretreatment; **p = 0.044 vs. before RIC with ticagrelor pretreatment and p = 0.041 vs. after RIC with aspirin pretreatment; ‡p = 0.010 vs. before RIC without pretreatment and p = 0.029 vs. before RIC with aspirin pretreatment; two-way repeated measures ANOVA with Fisher least significant differences post hoc tests

Fig. 5

In response to remote ischemic conditioning (RIC), platelets transfer a cardioprotective signal through an aspirin and thus Cox-sensitive mechanism. Aspirin pretreatment of healthy volunteers abrogated RIC’s transfer of cardioprotection through washed platelets but not that through plasma-dialysate, which supports a platelet-specific component of RIC’s humoral transfer of cardioprotection. Ticagrelor pretreatment obviously did not impact on protection by RIC, since the level of infarct size reduction achieved with platelets or plasma-dialysate from blood after RIC was not different in the absence or presence of ticagrelor. Washed platelets or plasma-dialysates from volunteers before and after RIC (without or with aspirin/ticagrelor pretreatment) were infused into an isolated perfused rat heart with 30-min global ischemia and 120-min reperfusion with infarct size as endpoint