2.3. Mepacrin Assay

The mepacrine uptake and release assay in whole blood was assessed by measuring a 60 s baseline fluorescence of resting platelets on a FACSCelesta flow cytometer (Becton Dickinson, Heidelberg, Germany). Platelets were then loaded with mepacrine dihydrochloride dye (Sigma-Aldrich, Darmstadt, Germany) to a final concentration of 25 µM for 30 min at 37 °C and the mepacrine-based fluorescence after sub-gating to CD61-positive platelets (antibody clone Y2/51; Bio-Rad Laboratories, Feldkirchen, Germany) was recorded for further 60 s. Stained platelets were activated using 0.1 U/mL thrombin from human plasma (Roche, Freiburg, Germany) and directly analyzed for additional five minutes. The difference between unloaded and loaded as well as loaded and released platelets was calculated as deltaMFI (mean fluorescence intensity) [10].