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Adrenaline enhances in vitro platelet activation and aggregation in blood samples from ticagrelor‐treated patients

Abstract Background Temporarily improved platelet reactivity may reduce the bleeding in patients on antiplatelet therapy who have ongoing bleeding or who are in need of acute surgery. Adrenaline can bind to adrenergic α2A‐receptors on platelets and potentially enhance platelet reactivity. Objective To assess if adrenaline can improve adenosine diphosphate (ADP)‐induced platelet aggregation and activation in blood samples from patients on dual antiplatelet therapy with acetylsalicylic acid (ASA) and the ADP‐receptor antagonist ticagrelor. Methods Blood samples were collected from a total of forty acute coronary syndrome patients on dual antiplatelet therapy with ASA and ticagrelor. ADP‐induced platelet aggregation (by impedance aggregometry) and activation (by flow cytometry) were assessed before and after supplementation with adrenaline and/or platelet concentrate. Results Adrenaline supplementation (770 nmol L−1) increased median ADP‐induced aggregation from 15 (25‐75th percentiles: 10‐20) to 26 (18‐38) aggregation units. The effect was independent of concomitant platelet supplementation. Adrenaline also increased ADP‐induced platelet activation: from 40% (36‐54%) to 83% (74‐88%) platelets with active fibrinogen receptor (binding PAC‐1) and from 13% (7‐21%) to 35% (18‐50%) P‐selectin‐expressing platelets. Conclusions Adrenaline potentiated ADP‐induced platelet aggregation and activation in blood samples from ticagrelor‐treated patients. Adrenaline infusion may be a new method to enhance platelet function in ticagrelor‐treated patients who are in need of acute surgery or have ongoing bleeding. In vivo studies are needed to confirm the present results.


| INTRODUCTION
Dual antiplatelet therapy (DAPT) with acetylsalicylic acid (ASA) and an adenosine diphosphate (ADP) receptor (P2Y 12 ) antagonist such as clopidogrel, ticagrelor, or prasugrel reduces the risk of thrombotic events compared to ASA only in patients with acute coronary syndrome (ACS), but it is also associated with an increased risk of spontaneous and perioperative bleeding complications. [1][2][3] Both the efficacy and the bleeding risk are higher when ticagrelor, a P2Y 12 -receptor antagonist of the third generation, is used instead of clopidogrel. 3 If bleeding occurs, or if acute surgery is unavoidable, the effective platelet inhibition in patients with ongoing ticagrelor treatment becomes a significant disadvantage. Accordingly, a high incidence of severe perioperative bleeding complications has been reported in ACS patients with ongoing or recently discontinued DAPT with ASA and ticagrelor. 4,5 The perioperative bleeding risk in cardiac surgery patients, with ongoing or recently discontinued treatment with a P2Y 12 -receptor antagonist, correlates to the residual ADP-dependent platelet aggregability. [5][6][7] In two recent studies, even small differences in residual platelet aggregability were found to have an effect on the bleeding risk. 5,6 In patients on ticagrelor, platelet transfusion has little or no effect, [8][9][10] and there is no currently available antidote to ticagrelor. New alternatives are therefore needed to reduce the risk of excessive bleeding in ticagrelor-treated patients who have ongoing bleeding or who are in need of emergent surgery. One potential method would be to temporarily enhance platelet activation and aggregation during surgery or until the bleeding has stopped. Supplementation with adrenaline, which is commonly used in cardiac surgery during and after cardiopulmonary bypass to maintain blood pressure, has previously been shown to potentiate platelet aggregation induced by ADP in healthy subjects 11,12 and in clopidogrel-treated patients. 13 In the present study, we hypothesized that adrenaline would potentiate ADP-induced platelet aggregation and activation in patients with ongoing ASA and ticagrelor treatment, since the platelet membrane express adrenergic α 2A -receptors. 11,[14][15][16] To test this hypothesis, we performed an in vitro study using blood samples from ACS patients on ongoing DAPT with ASA and ticagrelor. In addition, we also tested the effect of concomitant supplementation with platelet concentrate and adrenaline.

| Ethics approval
The study was approved by the regional research ethics committee and was performed in accordance with the Declaration of Helsinki.
All the patients gave written informed consent.

| Study design
The blood samples were supplemented with adrenaline, platelet concentrates, and/or ADP. Impedance aggregometry was used to assess platelet aggregation and flow cytometry was used to evaluate platelet activation before and after supplementations. Firstly, the effect of five different added concentrations of adrenaline on platelet aggregation was assessed (n = 10). Next, platelet aggregation was measured after addition of adrenaline alone or in combination with platelet concentrate (n = 10). To evaluate whether adrenaline induces aggregation/activation by itself or whether it potentiates ADP-induced aggregation/activation, it was added alone or in combination with ADP and platelet aggregation (n = 10), and activation (n = 10) was assessed. The order in which the different substances/concentrations were tested was alternated to avoid any systematic influence of time.

Essentials
• Interventions to temporarily improve platelet function may reduce bleeding in ticagrelor-treated patients.
• Adrenaline was added to blood from patients on ticagrelor and platelet function was assessed.
• Adrenaline infusion may be used to enhance platelet function in patients on ticagrelor. and added at volumes ranging between 12 and 118 μL to achieve the calculated concentrations in plasma.

| Platelet concentrates
Apheresis platelet concentrates of blood group O stored for a median of 1 day (range: 1-3) were used. The platelet concentrates were produced by the blood bank at Sahlgrenska University Hospital using an apheresis device (Trima Accel; Terumo BCT Europe, Zaventem, Belgium) in accordance with European guidelines. 17 Autologous plasma was used as storage medium. The mean platelet count in the platelet concentrates was 1747 ± 41 × 10 9 L −1 . In vitro addition of 120 × 10 6 platelets to a 1-mL blood sample was tested. This corresponds to an increase in platelet count achieved by transfusion of approximately three apheresis units to a 70-kg patient. By using the platelet concentration in the individual concentrate, the specific volume to add from the concentrate was calculated. The mean volume of platelet concentrate added was 69 ± 1.6 μL.

| Impedance aggregometry
Blood samples were collected in hirudin tubes (0.15 mg L −1 ; Roche Diagnostics GmbH, Mannheim, Germany). Whole blood impedance aggregometry (Multiplate; Roche Diagnostics, Basel, Switzerland) was used to study platelet aggregation as previously described. 18 The tests used were the ADP HS test (ADP 6.3 μmol L −1 and prostaglandin E1 9.4 nmol L −1 ) which assesses the P2Y 12 receptor-dependent aggregation, and the ASPI test (arachidonic acid AA, 0.5 mmol L −1 ), which assesses cyclooxygenase-dependent aggregation (i.e, is ASAsensitive). When the effect on aggregation of adrenaline alone was studied, no other agonist was added.

| Flow cytometry
Flow cytometry was used to assess platelet activation by measuring the binding of PAC-1 (which binds to the activated fibrinogen receptor glycoprotein IIb/IIIa) 19

| Statistics
Data are presented in boxplots with median and 25th-75th percentiles. Platelet aggregation, before and after addition of adrenaline and/or platelet concentrate and/or ADP, was compared using the

| Effects of adrenaline ± ADP on platelet aggregation and platelet activation
The combination of ADP and adrenaline (770 nmol L −1 ) resulted in the highest expression of P-selectin; 35% (18-50%) (P = 0.005 vs all other).

| DISCUSSION
The main finding of the present study was that addition of adrenaline to blood samples from patients who were on DAPT with ASA and ticagrelor potentiated ADP-induced platelet aggregation and activation.
There is a need for new methods of reducing the risk of excessive bleeding in ticagrelor-treated patients with ongoing bleeding or in need of acute surgery. One option would be to transiently enhance platelet function until bleeding has stopped or until the operation is completed. Platelets express adrenergic receptors, and supplementation with adrenaline has previously been shown to potentiate ADP-induced platelet aggregation in healthy subjects 11 and in clopidogrel-treated patients. 13 In a study conducted on platelet-rich from healthy subjects, the P2Y 12 -receptor antagonists ticagrelor, clopidogrel, prasugrel, and cangrelor were added in vitro and adrenaline supplementation (1 μmol L −1 ) was shown to restore ADP (5 μmol L −1 )-induced aggregation with either P2Y 12 -receptor antagonist present. 12 In the present study, we hypothesized that adrenaline would increase platelet activation and aggregation in blood samples from ACS patients on dual antiplatelet therapy with ASA and ticagrelor which, to our knowledge, has not previously been investigated.
The results of the present study support our hypothesis. Adrenaline in itself had only a mild effect but potentiated ADP-induced platelet activation and aggregation in the blood samples from ASA and ticagrelor-treated patients in a concentration-dependent way.
One could speculate that the potentiating effect of adrenaline may be due to the molecular actions of the substances. ADP binds to the G-protein-coupled receptors P2Y 1 22 and P2Y  In fact, both studies suggested a cut-off level of 22 U for when cardiac surgery may be performed without an increased risk of bleeding, 5,6 which is lower than what was achieved in the present study.
We observed that platelet supplementation alone did not influence ADP-induced aggregation but markedly increased AA-induced aggregation. This is in line with results from previous studies, which showed no effect or only a minor effect of platelet transfusion on ADP-induced aggregation [8][9][10] and an improvement in AA-induced All tested concentrations, except the lowest, were higher than the plasma concentrations achieved by infusion of adrenaline at clinically relevant doses. 30 Accordingly, the results of the present study support the concept that adrenaline enhances ADP-dependent platelet aggregation, but in vivo studies with clinically acceptable adrenaline doses are necessary to validate the present findings. Furthermore, only adrenaline was tested in the present study. Other clinically used adrenergic agents such as noradrenaline also need to be evaluated, although in vitro data suggest that noradrenaline is not as potent as adrenaline in inducing aggregation of untreated platelets. 11,15,16 In conclusion, the results demonstrate that adrenaline potentiates ADP-induced activation and aggregation in blood samples from patients on ongoing treatment with ASA and ticagrelor. Adrenaline could therefore possibly be used to treat and prevent excessive bleeding in ticagrelor-treated patients. In vivo studies are needed to confirm the present results.