Fig. 4. Lipid rafts do not associate with the actin cytoskeleton of platelets from patients with Glanzmann thrombasthenia. Platelets from healthy volunteer or from patients with type I Glanzmann thrombasthenia were stimulated or not with 1 IU/ml thrombin for 3 minutes in an aggregometer with stirring at 900 rpm (2x10⁹ cells/ml). (A) Proteins from the isolated cytoskeleton were separated by 7.5% SDS-PAGE followed by Coomassie Blue staining (upper panel) or by western blotting (bottom panel) to determine the relative amount of the raft markers CD36 and LAT using specific antibodies. (B) The amount of cholesterol in cytoskeleton isolated from 3x10⁹ platelets was determined as described in Fig. 1. Results shown are representative of three independent experiments. (C) Rafts were isolated from resting or 5 µM TRAP-stimulated control platelets and the relative amount of ß3 subunits in rafts was quantified by western blotting and densitometric analysis and compared to the total amount of ß3 in the same number of platelets. In the western blot shown, rafts isolated from 1.5x10⁹ platelets and a homogenate from 4x10⁸ platelets were analyzed. Results shown are mean±s.d. of three independent experiments.