Fig. 6
Endothelial cell-derived NO is the only source of platelet PKG activation. Western blot analysis of VASPS239 phosphorylation in platelets (3x108/ml). Washed platelets were coincubated with WBCs (7x106/ml) for 10 min, or added to the confluent HUVECs cultured in 24 well plate for 10 min then L-arginine or L-NAME (200 μM each, 10 min) were added to the samples. After 10 min, incubation, WBCs were separated from platelets by centrifugation and suspensions of non-adhered platelets were collected from HUVECs for Western blot analysis. As a control washed platelets and WBCs (7x106/ml) alone were stimulated with DEA-NO (1 μM, 1 min). In addition, WBCs were incubated with the same concentration/time with L-Arginine and L-NAME. Shown are representative blots of four independent experiments. Total VASP served as loading control. For the bar graphs, immunoblots were scanned and quantified by the Image J program. The intensity of the VASPSer239 signal was normalized to the total VASP signal, which was designated as 1 in control samples. Results are means ± SEM, n = 4, + significant difference from control samples