VASP mediates PKCδ-dependent regulation of filopodia formation, actin polymerization, and platelet aggregation. (A) Washed platelets from either wild-type (WT) or VASP^–/– (KO) mice were preincubated with rottlerin (5 μM) or vehicle as control. Platelets were then dispensed onto collagen-coated coverslips and their adhesion and spreading studied by DIC microscopy. (i) An example of platelets 40 minutes after addition to the coverslip, representative of 3 independent experiments. (ii) The perimeter of adherent platelets in the different experimental conditions was measured 40 minutes after dispensing of platelets onto the surface. Data shown are mean ± SEM (n = 4), and the statistical significance was analyzed by 1-way ANOVA (*P < .01, comparison between data in the absence or presence of rottlerin). (B) Washed platelets derived from either wild-type (WT) or VASP^–/– mice were allowed to adhere to collagen-coated coverslips in the absence or presence of rottlerin (5 μM, 15 minutes of preincubation). Platelets were fixed 40 minutes after addition to the coated surfaces, permeabilized, and stained with TRITC-phalloidin. Fluorescence labeling was detected by confocal microscopy and quantified using Leica Confocal Software. Data represent mean ± SEM (n = 3), and the statistical significance of difference was analyzed by 1-way ANOVA (*P < .01, comparison between results in the absence or presence of rottlerin). Images below the graphs show representative examples of platelets obtained under the same conditions indicated in the bar graph and stained with TRITC-phalloidin (top row), with corresponding phase-contrast images (bottom row). (C) Washed platelets were prepared from (i) wild-type (WT) or (ii) VASP^–/– (KO) mice and pretreated either with rottlerin (5 μM) for 15 minutes or DMSO vehicle as control. Platelet aggregation was induced with collagen (30 μg/mL) and monitored by turbidimetric aggregometry over a 10-minute period. Traces shown are representative of 3 independent experiments.