Thrombin-Induced Platelet TxB2 Levels

The level of TxB₂ was measured with an enzyme immunoassay kit (No. 501020; Cayman Chemical, Ann Arbor, MI, USA) as previously described [32 (link)]. A volume of 250 µL of washed human platelets (2.5 × 10⁸/mL) was pre-warmed to 37 °C along with the Rhosin-analogs, Indomethacin, or DMSO (0.1%) for 2 min before being stimulated with thrombin (0.1 U/mL) for 4 min. The reactions were terminated by adding 250 µL of HEPES Tyrode’s buffer (pH 7.4) containing 2 mM EDTA. The platelets were centrifuged for 2 min, and the level of TxB₂ in the supernatant was measured with the ELISA kit according to the manufacturer’s instructions.