Novel Therapeutic Agent against Platelet Activation In Vitro and Arterial Thrombosis In Vivo by Morin Hydrate

Figures

• 

  Figure 1. Inhibitory activity of morin hydrate in platelet aggregation stimulated by various agonists in washed human platelets. (A) Chemical structure of morin hydrate; (B) washed human platelets (3.6 × 108 cells/mL) were preincubated with the solvent control (0.1% dimethyl sulfoxide, DMSO) or morin hydrate (20, 50, and 80 μM) and subsequently treated with 1 μg/mL of collagen, 0.01 U/mL of thrombin, 1 μM of U46619, and 60 μM of arachidonic acid (AA) to stimulate platelet aggregation; (C) concentration–response histograms of morin hydrate in inhibition of platelet aggregation (%). All data are presented as means ± standard errors of means (n = 4).

• 

  Figure 2. Effects of morin hydrate on adenosine triphosphate (ATP) release, relative intracellular Ca2+ ([Ca2+]i) mobilization, and surface P-selectin expression in human platelets. Washed platelets (3.6 × 108 cells/mL) were preincubated with the solvent control (0.1% DMSO), morin hydrate (40 and 80 µM), or fluorescein isothiocyanate (FITC)–P-selectin (2 µg/mL); collagen (1 μg/mL) was then added to trigger either: (A) ATP release (AU; arbitrary unit), (B) relative [Ca2+]i mobilization, or (C) the fluorescence profiles of (a) FITC only as a resting control; washed platelets were preincubated with the (b) solvent control (0.1% DMSO) or morin hydrate (c, 40 µM; d, 80 µM) for 3 min, followed by the addition of FITC–P-selectin and subsequently treated with collagen to induce platelet activation. The corresponding statistical data are shown on the right panel of each figure (A–C). Data are presented as means ± standard errors of means (n = 4). *** p < 0.001 compared with the (A) DMSO-treated group or (B,C) resting control; ### p < 0.001 compared with the DMSO-treated group.

• 

  Figure 3. Effects of morin hydrate on cytotoxicity, lactate dehydrogenase (LDH) release, and PLCγ2, PKC, and Akt activation in human platelets. (A) Washed platelets (3.6 × 108 cells/mL) were preincubated with the solvent control (0.1% DMSO) or morin hydrate (100 μM) for 10 min and consequently washed twice with Tyrode’s solution; collagen (1 μg/mL) was then added to trigger platelet aggregation; (B) washed platelets were preincubated with the solvent control (0.1% DMSO) or morin hydrate (40, 80, and 100 µM) for 20 min and a 10-µL aliquot of the supernatant was deposited on a Fuji Dri-Chem slide LDH-PIII, as described in Materials and Methods. (C–E) For the other experiments, washed platelets were preincubated with the solvent control (0.1% DMSO) or morin hydrate (40 and 80 µM) and subsequently treated with collagen (1 μg/mL) to induce (C) PLCγ2 and (D) PKC activation (p47, pleckstrin phosphorylation) and (E) Akt phosphorylation. Data are presented as means ± standard errors of means (n = 4). *** p < 0.001 compared with the resting control; # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the DMSO-treated group.

• 

  Figure 4. Morin hydrate inhibits p38 MAPK, ERK2, and JNK1 phosphorylation in collagen-activated platelets. Washed platelets were preincubated with the solvent control (0.1% DMSO) or morin hydrate (40 and 80 µM) and subsequently treated with collagen (1 μg/mL) to trigger (A) p38 MAPK, (B) ERK2, and (C) JNK1 activation. Data are presented as means ± standard errors of means (n = 4). * p < 0.05 and *** p < 0.001 compared with the resting control; # p < 0.05 and ### p < 0.001 compared with the DMSO-treated group.

• 

  Figure 5. Activities of morin hydrate on OH· formation in human platelet suspensions or the Fenton reaction solution. (A) Washed platelet suspensions or (B) the Fenton reaction solution was preincubated with (a) Tyrode’s solution (resting control), (b) 0.1% DMSO, or morin hydrate at (c) 40 µM or (d) 80 µM. Collagen (1 µg/mL) was then added for the Electron Spin Resonance (ESR) experiments, as described in Materials and Methods. Profiles are representative of four independent experiments, and an asterisk (*) indicates OH· formation.

• 

  Figure 6. In vivo activities of morin hydrate on thrombotic platelet plug formation in the mesenteric venules and bleeding time in the tail veins of mice. (A) Mice were given an intravenous bolus of the solvent control (0.1% DMSO) or morin hydrate (5 and 10 mg/kg) and the mesenteric venules were irradiated to induce microthrombus formation (occlusion time); (B) microscopic images (400× magnification) of 0.1% DMSO-treated controls (a and b); the 5 mg/kg (c and d) and 10 mg/kg (e and f) morin hydrate–treated groups were recorded at 5 s (a, c, and e) and 150 s (b, d, and f) after irradiation. The photographs are representative of six similar experiments. The black arrows indicate platelet plug formation; (C) bleeding time was measured through transection of the tail in mice after 30 min of administration of 5 or 10 mg/kg of morin hydrate intraperitoneally. Data are presented as means ± standard errors of means (A–C, n = 6). *** p < 0.001 compared with the 0.1% DMSO-treated group.