Prasugrel

Blood was incubated with vehicle, aspirin (100 µM), the P2Y₁₂ receptor antagonist prasugrel active metabolite (PAM) (3 µM; a gift from AstraZeneca), or aspirin + PAM. Part of the blood was centrifuged (175 g; 15 min) to obtain platelet-rich plasma (PRP). Platelets in both whole blood and PRP were then activated under static conditions (in initial experiments) or under stirring conditions in a light transmission aggregometer by the addition of collagen (30 µg/ml) (Takeda Pharmaceuticals, Deerfield, IL, USA), TRAP-6 (30 µM) (Sigma-Aldrich, St. Louis, MO, USA), or A23187 (50 µM) and incubation for 5 min. Plasma was then quickly separated from the samples by centrifugation (2000 g, 5 min, 4°C) and stored at −80°C for eicosanomic analysis, as previously described (14 (link), 15 (link)). In brief, HyperSep Retain PEP SPE cartridges (Thermo Fisher Scientific, Waltham, MA, USA) were preconditioned with a solution of 0.1% acetic acid/5% methanol and spiked with 30 ng each of internal standards. Plasma (0.25 ml) was diluted in 0.1% acetic acid/5% methanol containing 0.009 mM butylated hydroxytoluene and added to the column. Samples were then washed with two volumes of 0.1% acetic acid/5% methanol, eluted in 1 ml of ethyl acetate and 1 ml methanol, dried by vacuum centrifugation at 37°C, and reconstituted in 30% ethanol. AA-derived metabolites were then separated by reverse-phase HPLC on a 1 × 150 mm, 5 μm Luna C18 (2 (link)) column (Phenomenex, Torrance, CA, USA) and quantified using a MDS Sciex API 3000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA, USA) with negative-mode electrospray ionization and multiple reaction monitoring. Data were captured and analyzed using Analyst 1.5.1 software. Relative response ratios of each analyte were used to calculate concentrations, and extraction efficiency for each sample was calculated based on recovery of the internal standards.

AMI was defined as the presence of acute myocardial injury detected by abnormal levels of cardiac biomarkers and angiographically proven atherothrombotic coronary artery disease (CAD). Cardiology specialists collected the patients’ medication lists and medical histories, such as the presence of hypertension, diabetes mellitus, and dyslipidemia. All laboratory variables were measured upon admission, except for lipid profiles, which were obtained after at least 9 h of fasting within 24 h of hospitalization. The baseline left ventricular ejection fraction (LVEF) was determined by two-dimensional echocardiography performed before or immediately after PCI. Coronary blood flow before and after PCI was classified by the thrombolysis in myocardial infarction (TIMI) score, and coronary lesion complexity was based on the American College of Cardiology (ACC)/American Heart Association (AHA) definitions. Patients who underwent PCI received 300 mg of aspirin and 600 mg of clopidogrel, 60 mg of prasugrel, or 180 mg of ticagrelor as a loading dose before PCI. Doses of 50 to 70 U/kg of unfractionated heparin were used before or during PCI to maintain an activated clotting time of 250 to 300 s. After PCI, 100 to 300 mg of aspirin and 75 mg of clopidogrel, 5 to 10 mg of prasugrel, or 45 to 90 mg of ticagrelor were prescribed daily. All patients had coronary lesions with at least 50% stenosis by quantitative coronary analysis. The study endpoint was vascular events associated with plaque instability, which consisted of recurrent AMI, stent thrombosis, and ischemic cerebral infarction. Recurrent MI was defined as the development of recurrent angina symptoms with new 12-lead electrocardiographic changes or increased cardiac-specific biomarkers.

The study cohort has been described previously [21 (link)]. In total, 206 ACS patients on daily aspirin (100 mg/day), and either prasugrel (10 mg/d, n = 116) or ticagrelor (180 mg/d, n = 90), were included. Pre- and periprocedurally, all patients received weight-adjusted unfractionated heparin (UFH) (70–100 IU/kg, aiming for an activated clotting time > 250 s) [22 (link)]. None of the patients received a GPIIb/IIIa inhibitor. After successful PCI, blood was drawn after an overnight fast. Exclusion criteria included oral anticoagulation with either vitamin K antagonists (warfarin, phenprocoumon and acenocoumarol) or direct oral anticoagulants (edoxaban, dabigatran, apixaban and rivaroxaban), a known aspirin, prasugrel or ticagrelor intolerance (allergic reaction and gastrointestinal bleeding complication), a history of bleeding complications or a positive family history of bleeding complications, treatment with ticlopidine, dipyridamole or nonsteroidal antirheumatic drugs, malignant myeloproliferative disorders or heparin-induced thrombocytopenia, major surgery within one week before enrollment, severe hepatic failure with impaired hepatic synthesis (spontaneous international normalized ratio [INR] ≥1.5 and albumin levels <35 mg/dl) [23 (link)], known qualitative defects in platelet function, a platelet count < 100.000 or > 450.000/µL and a hematocrit < 30%.
The study protocol was in accordance with the criteria of the Declaration of Helsinki and has been approved by the Ethics Committee of the Medical University of Vienna (EC-No. 1940/2013). All study participants gave written informed consent.