Fig. 6
Effect of columbianadin (CBN) on platelet adhesion, spreading on immobilized fibrinogen and fibrin clot retraction as well as integrin αIIbβ3 binding. (a) Washed platelets allowed to spread on the (a) BSA- or (b–d) fibrinogen-coated surfaces in the presence of the (b) solvent control (0.1% DMSO) or CBN (c, 60 μM; d, 80 μM) and subsequently labeled with FITC-conjugated phalloidin as described in the Materials and methods section. Plot of (b) the number of adherent platelets per 0.01 mm2 and (c) the average spreading surface area of individual platelets in six sight views. (d) Washed platelets suspended in 2 mg/mL fibrinogen with the solvent control (0.1% DMSO) or CBN (60 and 80 μM) before the thrombin (0.01 U/mL) stimulation. Images have been photographed at 15- and 30-min intervals. (e) For flow cytometry analysis, washed platelets were preincubated with solvent control (a, 0.1% DMSO, black line), EDTA (b, 2 mM, red line), and CBN (c, 60 μM, blue line; d, 80 μM, green line), followed by the addition of FITC-triflavin (2 μg/mL). Profiles in (d) are representative of four similar experiments. Data are presented as means ± SEM (n = 4). **p < 0.01, compared with the immobilized BSA group (b, c) or 0.1% DMSO-treated group