In vitro and ex vivo platelet aggregation assay

The in vitro platelet aggregation study was performed according to a previously reported method^52,53. Washed human platelets were incubated with the indicated concentration of each compound in DMSO for 1, 3, 5, or 10 min. They were subsequently stimulated by U46619 (2 μM) in 0.9% saline solution at 37 °C for 5 min. Platelet aggregation was recorded using an aggregometer (Chronolog, Havertown, PA, USA). For the ex vivo aggregation assay, male mice were fasted overnight and the indicated concentration of each compound in DMSO was administered by intravenous (i.v.) injection. After 24 h, platelet-rich plasma (10⁹ platelets/mL) in a volume of 240 μL was incubated at 37 °C for 1.5 min in the aggregometer under continuous stirring at 1000 rpm and subsequently stimulated with U46619 (2 μM). Platelet aggregation was recorded as described above.