Model 490 aggregometer

PRP was incubated with 5HT_2A receptor antagonists, cyproheptadine, pizotifen, or EMD 281014 for 1 min prior to experiments, except in control experiments. Platelets were activated with submaximal concentration of ADP (1 µM), in the presence or absence of 15 µM serotonin. Platelet aggregation was measured by the turbidometric method using model 490 aggregometer (Chrono-Log Corporation, Havertown, PA). Each experiment was repeated at least 3 times, with blood collected from three different human donors, or pooled together from at least eight mice for each experiment. (what about in vitro?).

PRP was incubated with TRPC6 inhibitor (10 μM) for 5 min prior to experiments, except in control experiments. Platelets were activated with ADP (5 μM) and U46619 (0.5 μM) in the presence or absence of TRPC6 inhibitor (10 μM). Platelets were also activated with U46619 (0.5 μM) in the presence and absence of the TRPC6 inhibitor (10 μM) with or without the addition of BAPTA (10 μM). Platelet aggregation was measured by the turbidometric method using model 490 aggregometer (Chrono-Log Corporation, Havertown, PA). ATP secretion was monitored in parallel with platelet aggregation after addition of the luciferin-luciferase (1% [v/v] final concentration) reagent to the same PRP suspension used for aggregation and measured for ATP secretion upon adding 1 μM U46619 and 0.1 U/ml thrombin. Each experiment was repeated at least 3 times, with blood collected from three different human donors.