Cardioprotective Properties of the Platelet P2YSu

Authors: R M Bell V Sivaraman S P Kunuthur M V Cohen J M Downey D M Yellon

Publish Date: 2015/07/16

Volume: 29, Issue: 5, Pages: 415-418

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Abstract

Dual antiplatelet therapy represents a cornerstone of the current management of acute coronary syndromes Combining pharmacological antiplatelet agents the cyclooxygenase COX inhibitor aspirin with a purine P2Y12 receptor inhibitor these pharmacological platelet inhibitors are orally loaded at the time of diagnosis prior to the patient entering the cardiac catheter laboratory Until recently the primary physiological and pharmacological focus of these agents has been appropriately directed towards their ability to alter the rheology of the blood reducing platelet aggregability to attenuate the risk of stent thrombosis However there is now an increasing awareness of the pleotropic properties of antiplatelet therapies particularly of the P2Y12 inhibitors to ameliorate myocardial ischaemia/reperfusion injuryRecently it has been demonstrated that administration of P2Y12 inhibitors prior to the onset of reperfusion can result in a significant reduction of infarct size Interestingly this appears to be a classeffect with a range of chemically distinct thienopyridine and non thienopyridine P2Y12 inhibitors demonstrating the same cardioprotective ability to ameliorating infarct size provided that an adequate circulating concentration of the inhibitor was present at the moment of reperfusion 1 The critical difference between thienopyridine and nonthienopyridine P2Y12 inhibitors concerns the rate of onset The first P2Y12 inhibitor to gain widespread acceptance in the management of acute coronary syndromes was the thienopyridine clopidogrel Clopidogrel like the other widely used thienopyridine prasugrel requires a significant period of time to realise P2Y12 inhibition by virtue of the need for hepatic P450mediated conversion of the prodrug into its active metabolite 2 The requirement for metabolic conversion is not a problem with the nonthienopyridine P2Y12 inhibitors such as Ticagrelor and Cangrelor 2 but the absorption time from the gut—particularly in patients receiving opiate analgesia—will delay the onset of adequate P2Y12 inhibition for drugs with an oral route of administration 3 With a rapid onset of action and the ability to quickly and reliably load the circulation through intravenous bolus and subsequent infusion Cangrelor is perhaps the ideal antiplatelet therapy for use in the emergency management of STsegment elevation myocardial infarction ensuring rapid and effective platelet inhibition at the time of revascularisation and stent deployment The hitherto unexpected and unrealised potential clinical advantage of Cangrelor is the amelioration of ischaemia/ reperfusion injury Not currently targeted as part of routine clinical management the excess myocardial cell death resulting from the restoration of blood and oxygen supply can contribute up to 50  of the final infarct size in experimental and clinical studies 4 Ischaemia/reperfusion injury therefore represents a clear and currently unmet clinical need—a need that could be met through repurposing of a therapeutic intervention that is already in widespread clinical practice for which an intravenous P2Y12 inhibitor would be ideally suitedThe mechanism by which platelet P2Y12 inhibition mediates its protection remains unclear but in this communication we provide further evidence that the protection is dependent upon the presence of blood and that unlike other forms of cardioprotection such as ischaemic conditioning 5 we demonstrate for the first time that P2Y12 inhibitors can result in significant attenuation of injury of myocardium of animals with diabetes an important comorbidity in the cohort of patients presenting with an acute coronary syndromeAll work was conducted in accordance with the Guidelines on the Operation of the Animals Scientific Procedures Act 1986 published by The Stationery Office London UK conforming with National Institute of Health Guidelines for the Care and Use of Laboratory Animals Cangrelor was kindly supplied by the Medicines Company NJ USA and made up into solution in normal salineMale C57BL6 mice 3–4 months of age 20–30 g weight were anaesthetized with an intraperitoneal injection of 60 mg/kg pentobarbitone The heart was harvested via a paramedial thoracotomy and rapidly transferred to a dissection dish filled with ice cold Krebs–Henseleit buffer and the aorta cannulated with a 21gauge cannula The heart was transferred and retrogradely perfused on a Langendorff apparatus using a modified Krebs–Henseleit buffer concentrations in mmol/L NaCl 1185 NaHCO3 25 glucose 11 KCl 47 KH2PO4 12 MgSO4 12 CaCl2 18 at 80 mmHg pressure Each Langendorff perfused mouse heart was randomly assigned to one of the reperfusion protocols with the reperfusion perfusate fortified with either NaCl vehicle control or 20 200 or 400 nmol/L Cangrelor for the duration of reperfusion All hearts are stabilized for 20 min prior to being subjected to 35 min global normothermic 370 ± 02 °C ischaemia and 30 min reperfusion prior to determination of infarct size by triphenyl tetrazolium chloride TTC staining as described belowC57BL6 male mice age/weight characteristics as above were anaesthetized by intraperitoneal injection with a combination of ketamine xylazine and atropine 001 ml/g to produce final concentrations of 10 mg/ml 2 mg/ml and 006 mg/ml respectively and the body temperature maintained at 37 °C The external jugular vein and carotid artery were isolated and cannulated for drug administration and mean arterial blood pressure MABP measurement respectively A tracheotomy was performed for artificial respiration at 120 strokes/min and 200 μl tidal volume using a rodent Minivent type 845 Harvard Apparatus Kent UK and supplemental oxygen supplied A limb lead I electrocardiogram ECG LabChart AD Instruments UK was recorded A left anterior thoracotomy and a chest retractor were used to expose the heart Ligation of the left anterior descending LAD coronary artery was performed using an 8/0 prolene monofilament polypropylene suture Successful LAD coronary artery occlusion was confirmed by the presence of STsegment elevation on ECG and a decrease in MABP Ischaemic conditioning was used as a positive control comprising of a single cycle of 5 min ischaemia and 5 min reperfusion was effected by temporary LAD ligation All hearts were subjected to 35 min injurious regional ischaemia followed by 2 h reperfusion Ten minutes prior to the onset of reperfusion the animals were treated with an intravenous 10 min bolus infusion of either vehicle normal saline or Cangrelor 60 μg/kg followed by a continuous infusion of either the vehicle or Cangrelor 6 μg/kg/min upon the onset and for the duration of reperfusion At the end of the reperfusion the heart was isolated the aortic root was cannulated and infarct size and risk zone determined by TTC and Evans blue staining respectively as described belowMale diabetic GotoKakizaki rats 3–4 months of age 250–300 g University College London UK were used in this study Once anesthetized with sodium pentobarbital 50 mg/kg ip the rats were intubated and ventilated with a Harvard ventilator 70 strokes/min tidal volume 8–10 ml/kg Body temperature was maintained at 37 ± 1 °C by means of a rectal probe thermometer attached to a temperature control system CMA450 Once the animal was stable a left lateral thoracotomy was performed to expose the heart and a 60 suture placed around the LAD The suture was tightened using a loop system to create an LAD occlusion and regional ischaemia which was confirmed by ST elevation on ECG and blanching of the myocardium Following 30 min of ischaemia the myocardium was reperfused for 2 h As in the mouse protocol described above the rats were randomly allocated into vehicle control or Cangrelor treatment groups Cangrelor was administered as a bolus infusion 60 μg/kg 10 min before reperfusion followed by continuous infusion 6 μg/kg/min for the whole of the reperfusion period At the end of reperfusion the heart was excised and the aorta cannulated for identification of risk zone Evans blue dye and determination of infarct size by TTC staining as described belowFor invivo regional ischaemia experiments at the end of reperfusion the heart was excised and aorta cannulated to flush the coronary circulation of blood and the LAD then permanently occluded and the heart then perfused with 05  Evans blue dye to delineate the area at risk AAR

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