Fig. 5
Deoxygenated RBCs loaded with NO did not stimulate platelet PKG. a Western blot analysis of VASPS239 phosphorylation in platelets (3x108/ml) stimulated by DEA-NO (1 μM, 1 min), or incubated (10 min) with indicated concentrations of NaNO2. Experiment was done under argon. b Washed platelets and RBCs were deoxygenated by argon. RBCs (1x 108/ml) were washed 3 times by deoxygenated HEPES buffer, deoxygenated for additional 30 min and then incubated with DEA-NO (50 μM, 30 min) and added (1x108/ml in 10 μl) to platelets together with indicated concentrations of NaNO2 for 10 min. 10 μl of supernatant from the last wash was used as control for free Hb. Total VASP served as loading control. Shown are representative blots of four independent experiments. c sGC activity was measured under anaerobic conditions after incubation with 100 μM sodium nitrite and indicated amount of RBCs, as described in Method section. Data are presented as mean ± SEM, n = 4. d UV-visible spectra of oxyHb, deoxyHb, and deoxyHb loaded with NO. Hb spectra were taken from, oxygenated, deoxygenated (under argon), or deoxy-RBCs incubated for 15 and 30 min with 50 μM DEA-NO. Shown are representative spectra from four independent experiments