The study had an observational, non-randomized open label design. The study population consisted of 160 ACS patients on daily aspirin (100 mg/day), and either prasugrel (10 mg/day, n = 80), or ticagrelor (180 mg/day, n = 80) therapy. All patients were Caucasians from the Vienna urban area and were recruited within 2 years for the study. Blood sampling was performed on day 3 after acute successful PCI with stent implantation after an overnight fast, 12 h after the last intake of the respective P2Y₁₂ antagonist in all patients.

Exclusion criteria were a known aspirin, prasugrel, or ticagrelor intolerance (allergic reactions, gastrointestinal bleeding); a therapy with vitamin K antagonists (warfarin, phenprocoumon, acenocoumarol) or direct oral anticoagulants (rivaroxaban, apixaban, dabigatran, edoxaban); treatment with ticlopidine, dipyridamol, or nonsteroidal anti-inflammatory drugs; a family or personal history of bleeding disorders; malignant myeloproliferative disorders or heparin-induced thrombocytopenia; severe hepatic failure; known qualitative defects in platelet function; a major surgical procedure within 1 week before enrolment; a platelet count < 100.000 or > 450.000/μl; and a hematocrit < 30%.

The study protocol was approved by the Ethics Committee of the Medical University of Vienna (Ethics commitee number: 1940/2013) in accordance with the Declaration of Helsinki and its later amendments, and written informed consent was obtained from all study participants.

All laboratory personnel were blinded to study treatment. Blood was drawn by aseptic venipuncture from an antecubital vein using a 21-gauge butterfly needle (0.8 × 19 mm; Greiner Bio-One, Kremsmünster, Austria) on day 3 after PCI. To avoid procedural deviations, all blood samples were taken by the same physician applying a light tourniquet, which was immediately released and the samples were mixed adequately by gently inverting the tubes. After the initial 3 ml of blood had been discarded to reduce peri-procedural platelet activation, blood was drawn into hirudin-coated tubes (Roche Diagnostics, Mannheim, Germany) for multiple electrode aggregometry (MEA) and into 3.8% sodium citrate Vacuette tubes (Greiner Bio-One; 9 parts of whole blood, 1 part of sodium citrate 0.129 M/L) for evaluations by flow cytometry and light transmission aggregometry (LTA).

The expression of P-selectin and the binding of the monoclonal antibody PAC-1 to activated glycoprotein (GP) IIb/IIIa were determined in citrate-anticoagulated blood, as previously published [40, 41]. In brief, whole blood was diluted in phosphate-buffered saline to obtain 20 × 10³/μL platelets in 20 μL, and incubated for 10 min with the platelet-specific monoclonal antibody anti-CD42b (clone HIP1, allophycocyanin labelled; Becton Dickinson (BD), San Jose, CA, USA), without agonists, and after in vitro exposure to suboptimal concentrations of ADP (final concentration 1 μM; Roche Diagnostics GmbH, Mannheim, Germany), the TLR-1/2 agonist Pam3CSK4 (final concentration 8.9 μg/mL; InvivoGen, San Diego, USA), the TLR-4 agonist lipopolysaccharide (LPS; final concentration 1429 μg/mL; InvivoGen, San Diego, USA), the PAR-1 agonist SFLLRN (final concentration: 14.25 μM; Roche Diagnostics GmbH, Mannheim, Germany), or the PAR-4 agonist AYPGKF (final concentration 714 μM; Roche Diagnostics GmbH, Mannheim, Germany), each 10 μL for 10 min. The concentrations of all agonists were determined in previous titration experiments with increasing dosages of each agonist in 10 healthy controls. The selected concentrations of agonists induced about 60–70% of the maximal achievable increase in median fluorescence intensity (MFI) in healthy controls. Samples were then incubated for another 10 min with a mixture of antibodies against activated GPIIb/IIIa (the monoclonal antibody PAC-1-fluorescein (BD)) and P-selectin (anti-CD62p-phycoerythrin, clone CLB-Thromb6; Immuno-tech, Beckman Coulter, Fullerton, CA, USA). Isotype matched control antibodies (BD) were used for the determination of non-specific binding. After 15 min of incubation in the dark, the reaction was stopped by adding 500 μl phosphate-buffered saline (PBS) and samples were acquired immediately on a FACSCanto II flow cytometer (BD). At acquisition, the platelet population was identified by its characteristics in the forward scatter versus side scatter plot (Fig. 1a). A total of 10,000 events were acquired within this gate. This population was further identified by platelets stained with the platelet-specific monoclonal antibody anti-CD42b versus side scatter (Fig. 1b). Binding of the antibodies against activated GPIIb/IIIa and P-selectin was determined in histograms for PAC-1 and P-selectin, respectively (Fig. 1c, d). Cytometer Setup and Tracking beads (BD), which consist of FITC, PE, PERCP-CY5.5, PE-CY7, APC, APC-H7, V450, and V500-C labelled beads, were used for daily calibration of the cytometer applying the Diva software. The MFI based on all events was used for statistical calculations.

Whole blood impedance aggregometry was performed with the Multiplate analyzer (Roche Diagnostics, Mannheim, Germany) as previously described [42]. One Multiplate test cell contains 2 independent sensor units and 1 unit consists of 2 silver-coated highly conductive copper wires with a length of 3.2 mm. After dilution (1:2 with 0.9% NaCl solution) of hirudin-anticoagulated whole blood and stirring in the test cuvettes for 3 min at 37 °C, ADP (6.4 μM, Roche Diagnostics, Mannheim, Germany) or arachidonic acid (AA; final concentration of 0.5 mM; Roche Diagnostics, Mannheim, Germany) was added and aggregation was continuously recorded for 6 min. The adhesion of activated platelets to the electrodes led to an increase of impedance, which was detected for each sensor unit separately and transformed to aggregation units (AU) that were plotted against time. The AU at 6 min were used for calculations. One AU corresponds to 10 AU*min (area under the curve of AU).

Light transmission aggregometry (LTA) was performed on a PAP-8E aggregometer (Bio-Data, Horsham, PA USA) as previously described [42, 43]. Citrate-anticoagulated whole blood was allowed to “rest” in a tilt position at room temperature for 20 min before centrifugation. Blood tubes were centrifuged at 150×g for 10 min (min) at room temperature to acquire platelet-rich plasma (PRP). To obtain platelet-poor plasma (PPP), the remaining specimen were re-centrifugated at 2.000×g for 10 min. Platelet counts were not adjusted as the median platelet count was 198 G/L (range 166–230 G/L) [44]. The optical density of PPP was set as 100% aggregation. Platelet aggregation was initiated by addition of ADP (5 μM) or AA (1600 μM) as agonists to PRP. Optical density changes were recorded photoelectrically for 10 min as platelets began to aggregate to obtain maximal aggregation %. Maximal aggregation % was automatically calculated by the PAP-8E aggregometer by comparing the increase of light transmission through platelet-rich plasma after addition of an agonist to the baseline optical density that was set with platelet-poor plasma and considered as 100% platelet aggregation.

Statistical analysis was performed using the Statistical Package for Social Sciences (IBM SPSS version 25, Armonk, New York, USA). Median and interquartile range of continuous variables are shown. Categorical variables are given as number (%). We performed Mann Whitney U tests to detect differences in continuous variables. The chi-square test was used to assess differences in categorical variables, respectively. Two-sided p values < 0.05 were considered statistically significant.