Fig. 6

BML-111 regulates VASP S-157 phosphorylation via cAMP-independent PKA activation. Human isolated platelets (4 × 10⁸ cells/mL), under (a) Resting and (b) (0.1 U/ml) thrombin were pre-treated with BML-111 (12.5, 25 and 50 µM) or a vehicle-control (modified-Tyrode’s HEPES buffer) for 5 min then immunoblotted to detect VASP S-157 phosphorylation, a marker of PKA activity. Platelets were treated with PGI₂ (1 µg/ml) as a positive control to stimulate the activity of PKA. Resting isolated human platelets (4 × 108 cells/mL) under resting conditions were treated with (c) H-89 (10 µM) or (d) SQ 22536 (100 µM) for 5 min before treatment with a vehicle-control (modified-Tyrode’s HEPES buffer) or BML-111(50 µM) for 5 min. Then, the samples were assayed for VASP S-157 phosphorylation. Treatment with PGI₂ (1 µg/mL), which activates PKA by stimulating AC, was used as a positive control. Lysis of the samples was carried out using Laemmli sample buffer prior to separation by SDS-PAGE, then the samples were transferred to PVDF membranes. 14-3-3-ζ was detected by immunoblotting as a loading control. The impact of BML-111 on VASP S-157 phosphorylation is shown in representative blots from three distinct experiments. Data represent the mean ± SEM (n = 4). *P ≤ 0.05, ***P ≤ 0.001 and ****P ≤ 0.0001 were as calculated by One-way ANOVA