Platelets store proangiogenic and antiangiogenic regulators in separate α-granules. For immunofluorescent staining, PRP (2 × 108 cells/mL) was subjected to the treatment with vehicle (resting), PAR1-activating peptide (PAR1-AP 10μM; SFLLRNPNDKYEPF-OH from Calbiochem), PAR4-AP (100μM; AYPGKF-NH2, from Sigma-Aldrich), ADP (10μM, Sigma-Aldrich), and CRP (1 μg/mL, from Dr R. Farndale, Cambridge, United Kingdom) for 5 minutes at room temperature. Thereafter, PRP was fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS), applied to 0.01% poly-L-lysine-coated coverslips, and then permeabilized with 0.5% Triton X-100. After blocking with 1% bovine serum albumin-PBS, the samples were incubated overnight at 4°C with respective primary antibodies: mouse anti–human SDF-1α/chemokine (C-X-C motif) ligand 12; MAB350 from R&D Systems at 1:20 (Ai-v,E,F); rabbit anti–human PF4/CXCL4 Ab sc-50301 from Santa Cruz Biotechnology at 1:25 (Bi-v,E,F); rabbit anti–human VEGF Ab-1 from Thermo Scientific at 1:500 (Ci-v); and rabbit anti–human endostatin Ab53702 from AbCam at 1:50 (Di-v). After washing, they were incubated with corresponding fluorescent secondary antibodies (DyLight 549 goat antimouse IgG at 1:50, Jackson ImmunoResearch Laboratories; AlexaFluor-488 goat anti–rabbit IgG at 1:100, AlexaFluor-546 goat anti–rabbit IgG at 1:500, and AlexaFluor-488 goat anti–rabbit IgG at 1:100/AlexaFluor-647 donkey anti–mouse IgG at 1:100 for double labeling, all from Invitrogen) for 2 hours at room temperature. After thorough washings with PBS containing 0.3% Triton X-100 and 0.1% Tween-20, the coverslips were mounted with Prolong Gold antifade mounting medium (Invitrogen). Single immunofluorescence (A-D) and SDF-1α/PF4-double immunofluorescence (E-F) platelet images were acquired using a Leica confocal microscope TCS SP2 equipped with an 100× NA1.4 objective. The digital images were assembled into composite images using Adobe PhotoShop Version 10.0.1. The arrows indicate angiogenic factor-staining that was marginized to form a ring-like distribution on platelet activation. The arrowheads indicate the colocalization (yellow) of SDF-1α and PF4. The images are representatives from 4 or 5 experiments. Single immunogold labeling of SDF-1α (MAB350 at 1:20; followed by 5-nm gold protein A probing, arrows; G) and PF4 (sc-50 301 antibody at 1:25, followed by 10-nm gold protein A probing, arrowheads; H) was performed on cryo-ultrathin sections of unstimulated platelets. SDF-1α-PF4 double immunogold labeling (I) was first probed with the SDF-1α antibody, which was indicated by 5-nm gold protein A (arrows) and then probed with the PF4 antibody, which was indicated with 10-nm gold protein A (arrowheads). The samples were visualized with a Leo 906 transmission electron microscope (Leo GmbH) operating with an accelerating voltage of 80 kV and at 60 000× original magnification. Digital images were taken with a Morada camera (SiS Münster). (I) SDF-1α–containing (numbered 1), PF4–containing (2), and SDF-1α-PF4–containing (3) α-granules within 1 platelet. The results shown are representative images from 3 separate experiments.
