Measurement of Intracellular Calcium

Intracellular calcium mobilization was assessed by a spectrofluorometric method using an Enspire™ fluorescence plate reader (PerkinElmer, Waltham, US). Isolated platelets were diluted to 50 × 10⁹ cells/L in HBS-buffer and incubated with the calcium indicator Fluo-4 AM (5 μmol/L) for 20 min at RT. Inhibitors were added in specified combinations and experiments were performed at 37°C in a Nunc™ 384-well clear polystyrene microtiter plate (Thermo Fisher Scientific, Waltham, MA, US). After registering an initial baseline, platelets were activated by automatic dispensation of α-thrombin, PAR1-AP or PAR4-AP at indicated concentrations. Fluorescence was measured in 5 s intervals for 120 or 480 cycles. Excitation was set to 495 nm and emission was recorded at 535 nm.