4N1-1 induces phosphorylation events independently of secondary platelet activation.
(A) Washed human platelets (2 × 108/mL) were stimulated with increasing concentrations of 4N1-1. (B) Washed human platelets were stimulated with 25 μM 4N1-1, 50 μM 4NGG, and 50 μM 4N1K. Aggregation was monitored by light transmission. Arrows indicate addition of agonist. (C) Platelets were stimulated with increasing concentrations of 4N1-1. (D) Human platelets were pretreated for 10 minutes with 2 U/mL apyrase (apy), 10 μM indomethacin (indo), 1 mM EGTA, or all 3. Mix platelets were then stimulated with 100 μM 4N1-1 for 2 minutes. (E) Human platelets, preincubated as described in the legend for panel D with apyrase, indomethacin, and EGTA, were stimulated with 100 μM 4N1-1, 100 μM 4NGG, and 100 μM 4N1K for 2 minutes. (C-E) Platelets were lysed by addition of NP-40 buffer. Proteins of whole-cell lysate (35 μL) were resolved by 12.5% SDS-PAGE and analyzed with Western blotting using an antiphosphotyrosine antibody (WB: P-Y). Molecular-weight markers are on the left. Arrows on the right indicate the position of prominent phosphorylated bands. Results are representative of an experiment using samples from a minimum of 3 different donors.
