Figure 7.
SR12813 negatively regulate GPVI-mediated signalling. Platelets (4 × 108 cells/ml) were pre-treated with vehicle (DMSO 0.1% v/v) or SR12813 (0, 50 and 100 μM) for 20 minutes and stimulated with CRP-XL (1 μg/ml) for 90 seconds in the presence of indomethacin (20 μM), cangrelor (1 μM), MRS2179 (100 μM) and EGTA (1 mM). Samples were tested for (A) Total tyrosine, (B) Syk (Y525/526), (C) LAT (Y200), (D) PLCγ2 (Y1217) and (F) PKC substrate phosphorylation. Representative immunoblots are shown. Levels of phosphorylation were quantified and expressed as a percentage of untreated (vehicle) controls. 14–3–3-ζ or actin was used as a loading control. Full length blots are shown in supplementary Fig. 9 (E) Calcium mobilisation was evaluated in Fura-2AM loaded platelets (4 × 108 cells/ml) incubated with SR12813 (50 and 100 μM) or vehicle-control (DMSO 0.1% v/v) for 20 min prior to stimulation with CRP-XL (0.25 μg/ml). Traces of CRP-XL-stimulated calcium mobilisation over a period of 5 minutes are shown. Cumulative data (peak calcium levels) of calcium mobilisation. Data represent mean ± SD (n ≥ 3) where *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 was determined by One-way ANOVA. Figure adapted from corresponding PhD thesis48.