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Fig. 6. {alpha}IIbß3 integrin-dependent upregulation of PtdIns(4,5)P2 in rafts and concomitant recruitment of cytoskeleton regulatory proteins. (A) [32P]-labeled platelets (1.5x109 cells) preincubated or not with 400 µM SR-121566 for 10 minutes (white bars) or with 500 µM RGDS for 1 minute (gray bars) were stimulated with 10 µM TRAP for the indicated time. Lipids were extracted from isolated rafts or from whole cells and the amount of [32P]PtdIns(4,5)P2 was quantified by HPLC. ([32P]PtdIns(4,5)P2 concentration in rafts and platelets after RGDS treatment and 6.5-minute TRAP stimulation have not been determined.) Results are the mean±s.d. of four independent experiments and are expressed as fold increase compared to the level of [32P]PtdIns(4,5)P2 under resting conditions in the absence of RGDS or SR-121566 treatment (corresponding to 7.9x104 cpm of [32P]PtdIns(4,5)P2 in rafts isolated from 1.5x109 platelets and 1.1x106 cpm in whole cells). *P<0.05; **P<0.01. (B) Rafts were isolated from 3x109 platelets pretreated or not with 500 µM RGDS for 1 minute and stimulated or not with 10 µM TRAP for 3 minutes. Isolated rafts were submitted to an in vitro PtdIns(4)P kinase assay as described in Materials and methods. The level of [32P]PtdIns(4,5)P2 produced was quantified by HPLC. Results are expressed as cpm of [32P]PtdIns(4,5)P2 produced and are the means of two independent experiments with similar results (upper panel). The amount of Rac associated with rafts (isolated from 1.5x109 cells) or present in 4x108 platelets was estimated by western blotting and densitometry analysis (lower panel). (C) Rafts were isolated from platelets pre-incubated or not with 500 µM RGDS for 1 minute and stimulated or not with 5 µM TRAP for 3 minutes. Proteins from rafts (isolated from 1.5x109 cells) and from whole platelets lysate (from 4x108 cells) were analyzed by western blotting. Results are representative of two to three independent experiments. CD36 was used as a loading control. The amount of this raft marker was constant under the different conditions tested as shown in Fig. 3C.