Fig. 4

a Effects of PM 21 on cAMP level. Rat platelets were pre-incubated for 2 min with PM21 (100 and 200 μg/mL) or aspirin (50 μg/mL) with or without IBMX (50 μg/mL). 0.1% (v/v) DMSO was used for vehicle. Then they were stimulated with 5 μg/mL collagen in the presence of Ca ²⁺ (1 mM) for 5 min. After termination of aggregation reactions, the samples were centrifuged and cAMP level of supernatants was determined with cAMP EIA Kit. b Effects of PM21 on thromboxane B₂ formation. Rat platelets were pre-incubated for 2 min with PM21 (50, 100, 200 μg/mL) or aspirin (50 μg/mL), and stimulated with 5 μg/mL collagen in the presence of Ca ²⁺ (1 mM) for 5 min. After termination of aggregation reactions, the samples were centrifuged and TXB₂ level of supernatants was determined with TXB₂ EIA kit. c Effects of PM21 on serotonin release. Rat platelets were pre-incubated for 2 min with PM21 (50, 100, 200 μg/mL) or aspirin (50 μg/mL), and stimulated with 5 μg/mL collagen in the presence of Ca ²⁺ (1 mM) for 5 min. After termination of aggregation reactions, the samples were centrifuged and serotonin concentration of supernatants was determined with serotonin ELISA kit. d Effects of PM21 on intracellular calcium concentration. Rat platelets were incubated with 5 mM Fura-2/AM for 60 min at 37 °C. The Fura-2-loaded platelets were then pre-incubated for 2 min with PM21 (50, 100, 200 μg/mL) or aspirin (50 μg/mL), and stimulated with 5 μg/mL collagen in the presence of Ca ²⁺ (1 mM) for 5 min. Fura-2 fluorescence was measured by spectrofluorometer at the emission wavelength of 510 nm. From the spectrofluorometric measurements, intracellular concentration of calcium ion was calculated. Data show the mean ± SEM of at least three independent experiments. ** p < 0.01, *** p < 0.001 versus vehicle control