Figure 5
Effects of CORM-2 on cGMP secretion and signal molecule expression/phosphorylation in LPS-stimulated platelets.
Platelets were stimulated by LPS and treated with CORM-2 as described in Fig. 1. Platelet cGMP accumulation was measured using a standard ELISA kit. LPS stimulation resulted in a significant decrease in platelet cGMP, whereas CORM-2 treatment in LPS-stimulated platelets significantly abolished this decrease in a concentration-dependent manner in the CORM-2 pre-conditioned (A), co-incubation (B) and delayed-treatment groups (C). Platelets were lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails and signal molecule expression and phosphorylation were assessed. LPS stimulation resulted in a significant increase in the PI3Kβ level and this increase was abolished in LPS-stimulated platelets treated with CORM-2 (E). After treatment with exogenous CO, Akt production (H) and phosphorylation (N) in LPS-stimulated platelets were markedly inhibited. The same results were found for GSK-3β production and phosphorylation (K,Q). Similar results were also found in the CORM-2 pre-conditioned (D,G,J,M,P) and delayed-treatment groups (F,I,L,O,R). Representative experiments are shown in panels (D–R). The average ratios of the level of protein expression to phosphorylation are shown in Supplementary Figure 3. The results are presented as the mean ± SE (n = 5). *P < 0.01 compared with the control, #P < 0.05 compared with the LPS treatment. Note that LPS-induced signal molecule expression and phosphorylation were downregulated by CORM-2 in a dose-dependent manner.
