Figure 3.

Cysteine is required for the bioactivation of nitrite to NO in human platelets. Washed human platelets (3 × 10⁸/mL, 100 µL) were incubated with indicated compounds. The concentration of NaNO₂ was 10 µM where added. L-Cysteine (Cys) was added at final concentrations of 100 µM or 200 µM. The incubation time was 5 min. Sodium nitroprusside (SNP) was used at 1 µM (1 min) and served as positive control. Some samples were pre-incubated for 10 min with the CA inhibitor acetazolamide (ACZ) at 200 µM, or with the sGC inhibitor ODQ at 20 µM; then NaNO₂ and Cys were added and the samples were incubated for 10 min. All samples were processed for Western blot analysis of VASP^Ser239 phosphorylation, with actin blots serving as loading control. Blots were scanned and analysed densitometrically using NIH Image J software for uncalibrated optical density (graph). Results are presented as mean with standard deviation from quadruplicate analyses. Asterisk (*) indicate p values < 0.05 compared to control. Statistical analysis was performed by one-way ANOVA and Student’s t-test.