Isorhamnetin Inhibits Platelet Aggregation

Inhibition of platelet aggregation was evaluated by a turbidimetric method using a lumi-aggregometer (Chrono-Log, Havertown, PA, USA) [25 (link),26 (link)]. Washed platelets (3 × 10⁸ platelets/mL) were incubated for 5 min with CaCl₂ (2 mM) plus isorhamnetin (1, 10, 20, 50 and 100 µM) or vehicle (DMSO, 0.2%). Similar concentrations in vitro have been evaluated in other studies [6 (link),27 ]. Platelet aggregation was induced by TRAP-6 (5 µM), collagen (1 mg/mL), and PMA (100 nM). Platelet aggregation (transmittance) was measured for 6 min [23 (link)]. The platelet aggregation percentage was obtained with the AGGRO/LINK software (Chrono-Log, Havertown, PA, USA). Platelet inhibition was calculated as: inhibition of platelet aggregation (%) = 100 − ((platelet aggregation of isorhamnetin/platelet aggregation of negative control) × 100) [28 (link)]. The concentration necessary to reduce platelet aggregation by 50% (IC₅₀) was obtained from isorhamnetin’s concentration curves (1, 10, 20, 50, and 100 µM).