14C-serotonin release

¹⁴C-serotonin release was performed, as has been described.²⁹ PRP from WT and Ask1^−/− mice was incubated with 0.1 μCi of ¹⁴C-serotonin for 1 h at 37°C. Incorporation was stopped by adding imipramine (1 mM). An aliquot of 100 μL (2.5 × 10⁸ platelets/mL) was stimulated with or without various concentrations of thrombin for 5 minutes, and the reaction was stopped by adding equal volume of stopping solution (2 mL of 1× PBS, 0.25 mL 37% formaldehyde, 2.25 mL of 77-mM EDTA pH 7.5). Another set of unstimulated aliquots of 100 μL were lysed with an equal volume of 2% Triton-X (control for serotonin uptake in platelets). An aliquot (150 μL) of individual sample supernatants was counted in a liquid scintillation counter. The percentage of ¹⁴C-serotonin release was calculated using the following formula: