Fibrinogen binding and platelet adhesion. (A) Fibrinogen binding. Platelets were preincubated with ADP (10 μM), PMA (100 nM), and thrombin (0.4 U/mL). FITC-labeled fibrinogen was then added at a final concentration of 300 nM for 30 minutes and fibrinogen binding was analyzed by FACS. A total of 20 000 events per sample was recorded. Bars present MFI ± SD of triplicates from 2 representative experiments. (B) Fibrinogen binding in the presence of LY294002. Platelets from 3 pairs of WT (i,iii) and Akt-1 null (ii,iv) mice were stimulated with thrombin at 0.1 U/mL or 0.5 U/mL, respectively, in the presence or absence of LY294002 (80 μM). FITC-labeled fibrinogen at concentration of 300 nM was added. Fibrinogen binding was analyzed by FACS analysis and 10 000 events per sample were recorded. Gray profiles represent fibrinogen binding in the presence of PGE₁; MFIs are shown in response to thrombin in the presence (thin lines) and the absence (thick lines) of LY294002, respectively. Similar results were obtained in an additional 2 experiments. (C-D) Platelet adhesion to collagen. Platelets were resuspended in calcium-free Tyrode buffer containing 2 U/mL apyrase. Platelet suspensions were added to coverslips coated with collagen at 20 μg/mL and incubated for 10 minutes at 37°C. Adherent platelets were fixed and stained with TRITC-phalloidin for actin. The adhesion was visualized and quantified by fluorescence microscopy. Bars present the mean number of platelets (means ± SD) for 6 fields of 3 independent experiments (C). The photograph illustrates adherent or spread platelets in the representative fields (D). ^* indicates significant difference between WT and Akt-1-null platelets (P < .05). Images were acquired using a Leica DMR fluorescence microscope with a × 100 objective lens, oil, and a × 1.6 zoom adaptor, and a Micromax RTE/CCD-1300-V/HS camera.