Src kinases are crucial for responses induced by 4N1-1 in platelets.
(A) Human platelets were pretreated for 10 minutes with 5, 10, or 20 μM PP1 and stimulated for 2 minutes with 100 μM 4N1-1. Whole-cell lysates were resolved by 12.5% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody. Arrows on the right indicate the position of prominent phosphorylated bands. (B) Human platelets (2 × 108/mL) loaded with tritium–5-HT were preincubated for 10 minutes with 10 μM PP1 and then stimulated for 2 minutes with 4N1-1 (25 μM), convulxin (1 μg/mL), or thrombin (0.1 U/mL). Tritium–5-HT release determined by scintillation spectrometry was expressed as a percentage of total tissue content. Each experiment was done with triplicate samples, and results are expressed as means ± SD. (C) Human platelets (2 × 108/mL) were preincubated for 10 minutes with 0.5, 2, or 10 μM PP1 or 5 μM PD174265. Platelets were then stimulated with 25 μM 4N1-1 or 1 μg/mL convulxin. Aggregation was monitored by light transmission. Arrows indicate addition of agonist. Results are representative of experiments using samples from 3 different donors.
