Figure 2
Effects of CORM-2 on secretion and α-granule distribution in LPS-stimulated platelets.
Platelets were stimulated and treated as described in Fig. 1. LPS stimulation resulted in a significant increase in ATP release from platelets. Treatment of platelets with CORM-2 significantly reduced the ATP release in response to LPS stimulation (B). Similar results were found in the CORM-2 pre-conditioned (A) and delayed-treatment groups (C). P-selectin expression was detected via flow cytometry. The results indicate that LPS stimulation resulted in a significant increase in P-selectin expression, whereas treatment with CORM-2 significantly reduced this increase (E). Similar results were found in the CORM-2 pre-conditioned (D) and delayed-treatment groups (F). Samples were fixed for transmission electron microscopy (TEM) and immunofluorescence analysis. Platelets were labeled with antibodies to VAMP8 followed by staining with secondary antibodies labeled with Alexa Fluor 594. Images were taken at the indicated times. A series of representative images demonstrate LPS-induced platelet α-granule distribution at different time points: 0 min, 5 min, 10 min, 15 min, 30 min, 45 min and 60 min (G). Representative electron microscopy images (I) and immunofluorescence images (H) show clear changes in platelet α-granule distribution under LPS stimulation and co-incubation with iCORM-2 (50 μmol/L). After treatment with CORM-2 (10 or 50 μmol/L), the amount of α-granules fused with platelet membranes decreased. Representative α-granules are indicated by arrows. Scale bars indicate 0.5 μm for the electron microscopy images and 2 μm for the immunofluorescence images. The results are the mean ± SE (n = 5). *P < 0.01 compared with control, #P < 0.05 compared with LPS.
