Fig. 3. The association of lipid rafts with the actin cytoskeleton is dependent on IIbß3 integrin engagement. Platelets pretreated or not with 500 µM RGDS for 1 minute (A,B,C) or 400 µM SR-121566 for 10 minutes (B,C) were stimulated at the concentration of 2x10⁹ cells/ml, with 5 µM TRAP for 3 minutes and their actin cytoskeleton was isolated. (A) The effect of RGDS treatment on platelet aggregation was assessed using a chrono-Log dual-channel aggregometer with stirring at 900 rpm (left panel). Proteins from the isolated actin cytoskeleton were separated by SDS-PAGE (7.5%) and stained with Coomassie Blue (right panel). (B) The quantification of the major phospholipids and cholesterol associated with the actin cytoskeleton isolated from 3x10⁹ platelets was performed as described in Fig. 1A. Results shown are the mean±s.d. of four independent experiments. (C) Lipid rafts were isolated from whole platelets (2x10⁹ cells) pretreated or not with RGDS or SR-121566 and stimulated or not with 5 µM TRAP for 3 minutes and their position in the sucrose density gradient is shown. The distance `d', indicated by the gray bars, represents the height in cm from the top of gradient where the light refractive band corresponding to the raft fraction was observed. The relative amount of actin present in rafts (pooled fractions 2-4) was determined by western blotting using an anti-actin antibody. As a loading control, the amount of CD36 was also analyzed by western blotting. Data shown are representative of three independent experiments.