Fig. 5
Effect of columbianadin (CBN) in cyclic nucleotides and vasodilator-stimulated phosphoprotein (VASP) phosphorylation as well as integrin αIIbβ3 activation. Washed platelets (3.6 × 108 cells/mL) were preincubated with (a) prostaglandin E1 (PGE1; 1 μM), nitroglycerin (NTG; 10 μM), or CBN (80 μM) in the presence of SQ22536 (100 μM) or ODQ (10 μM) for 3 min before addition of collagen (1 μg/mL) to trigger platelet aggregation. (b) For immunoblotting the VASP phosphorylation, washed platelets were stimulated with PGE1 (1 μM), NTG (10 μM), or CBN (60 and 80 μM). (c) For flow cytometry analysis, resting platelets (a, red line) or platelets were preincubated with the solvent control (b, 0.1% DMSO, black line) or CBN (c, 60 μM, blue line; d, 80 μM, green line) and FITC-conjugated anti-PAC-1 mAb (2 μg/mL) was added before the addition of collagen. Profiles in (a) are representative of four independent experiments. Data are presented as the means ± SEM (n = 4). ***p < 0.001, compared with the resting group; ###p < 0.001, compared with the 0.1% DMSO-treated group