Fig. 6.

Effect of columbianadin (CBN) on platelet adhesion, spreading on immobilized fibrinogen and fibrin clot retraction as well as integrin α_IIbβ₃ 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