Fig. 5.

Effect of columbianadin (CBN) in cyclic nucleotides and vasodilator-stimulated phosphoprotein (VASP) phosphorylation as well as integrin α_IIbβ₃ 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