Modulation of human platelet activation and in vivo vascular thrombosis by columbianadin: regulation by integrin αIIbβ3 inside-out but not outside-in signals

Figures

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  Fig. 1. Inhibitory activities of columbianadin (CBN) on platelet aggregation and cytotoxicity stimulated by agonists. Washed human platelets (3.6 × 108 cells/mL) were preincubated with a solvent control (0.1% DMSO), CBN (40–100 μM) (a, chemical structure) or aspirin (20–100 μM) and subsequently treated with 1 μg/mL of collagen, 0.01 U/mL of thrombin, 1 μM U46619, or 60 μM of arachidonic acid (AA) to stimulate platelet aggregation (b). Concentration–response histograms of CBN in inhibition of platelet aggregation (%) (c). To assess the cytotoxicity (d), platelets were preincubated with the solvent control (0.1% DMSO) or CBN (60, 80, and 160 μM) for 20 min, and a 10-μL aliquot of the supernatant was deposited on a Fuji Dri-Chem slide LDH-PIII. Data (c and d) are presented as the means ± SEM (n = 4)

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  Fig. 2. Effectiveness of columbianadin (CBN) on surface P-selectin expression, ATP releases, and relative [Ca2+]i mobilization in human platelets. Washed platelets (3.6 × 108 cells/mL) were preincubated with the solvent control (0.1% DMSO) or CBN (60 and 80 μM), followed by the addition of collagen (1 μg/mL) or arachidonic acid (AA; 60 μM) to trigger (a) surface P-selectin expression (resting control, red line; collagen-activated, black line; CBN 60 μM, blue line; CBN 80 μM, green line), (b) ATP release (AU; arbitrary unit), and (c) relative [Ca2+]i mobilization, as described in the Materials and methods section. The corresponding statistical data are presented in the right panel of each figure. Data are presented as means ± SEM (n = 4). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the resting control; ##p < 0.01 and ###p < 0.001, compared with the 0.1% DMSO-treated group

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  Fig. 3. Regulatory effects of columbianadin (CBN) on PLCγ2/PKC and Akt phosphorylation in platelets. Washed platelets were preincubated with a solvent control (0.1% DMSO) or CBN (60 and 80 μM) and subsequently treated with collagen (1 μg/mL), PDBu (150 nM) or arachidonic acid (AA; 60 μM) to trigger either (a) PLCγ2, (b) PKC (p47) activation or (c) platelet aggregation, and (d) Akt phosphorylation. Profiles in (c) are representative of four independent experiments. Data are presented as the means ± SEM (n = 4). **p < 0.01 and ***p < 0.001, compared with the resting platelets; #p < 0.05, ##p < 0.01, and ###p < 0.001, compared with the 0.1% DMSO-treated group

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  Fig. 4. Effect of columbianadin (CBN) on ERK1/2, JNK1/2, and p38 MAPK phosphorylation in collagen-activated platelets. Washed platelets were preincubated with a solvent control (0.1% DMSO) or CBN (60 and 80 μM) and subsequently treated with collagen (1 μg/mL) for immunoblotting the (a) ERK1/2, (b) JNK1/2, and (c) p38 MAPK phosphorylation. Data are presented as the means ± SEM (n = 4). **p < 0.01 and ***p < 0.001, compared with the resting platelets; ##p < 0.01 and ###p < 0.001, compared with the 0.1% DMSO-treated group

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  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

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  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…

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  Fig. 7. Effects of columbianadin (CBN) on integrin β3, Src, and FAK phosphorylation in platelets exposed to a fibrinogen-coated surface and on vascular thrombosis in the mesenteric venules of mice. (a) For immunoprecipitation study, washed platelets were preincubated with the solvent control (0.1% DMSO) or CBN (80 μM) and allowed to spread on immobilized fibrinogen (100 μg/mL). The platelets were lysed and Protein G Mag Sepharose Xtra beads were added with the anti-integrin β3 mAb (1 μg/mL) for immunoblotting. (b, c) Washed human platelets were preincubated with the solvent control (0.1% DMSO) or CBN (60 and 80 μM) and subsequently activated by immobilized fibrinogen (100 μg/mL) for determining the levels of (b) Src and (c) FAK phosphorylation. (d) For animal study, mice were administered an intravenous bolus of the solvent control (0.1% DMSO) or CBN (5 and 10 mg/kg), and the mesenteric venules were irradiated to induce microthrombus formation (occlusion time). Microscopic images (400× magnification) of 0.1% DMSO-treated controls and the 5 and 10 mg/kg CBN-treated groups were recorded at 5 and 150 s after irradiation, respectively. The photographs are representative of eight similar…