Honokiol as a specific collagen receptor glycoprotein VI antagonist on human platelets: Functional ex vivo and in vivo studies

Figures

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  Figure 1. Concentration-dependent inhibitory effect of honokiol in agonist-stimulated platelet aggregation. Washed human platelets (3.6 × 108 cells/ml) were preincubated with honokiol (0.1–5 μM) (A, chemical structure) or a solvent control (0.5% DMSO), and subsequently treated with 1 μg/ml of collagen (○), 0.05 IU/ml of thrombin (◇), 1 μM U46619 (□), or 60 μM arachidonic acid (∇) to trigger platelet aggregation for 6 min (B–D). For the other experiment, platelet-rich plasmas were preincubated with honokiol (1–10 μM), subsequently treated with 20 μM ADP to trigger platelet aggregation for 6 min (C). Statistical data (D) are presented as the means ± SEM (n = 4).

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  Figure 2. Honokiol in the ATP-release reaction, relative [Ca2+]i mobilization in human platelets, and toxicity in zebrafish embryonic development. Washed platelets (3.6 × 108 cells/ml) were preincubated with honokiol (0.6 μM and 1 μM) or the solvent control (0.5% DMSO), after which 1 μg/ml of collagen was added to stimulate either the ATP-release reaction for 6 min (A,B) or to induce relative [Ca2+]i mobilization for 10 min (C,D). Data (B,D) are presented as the means ± SEM (n = 4). *p < 0.05 and **p < 0.01, compared with the control (resting) platelets; #p < 0.05 and ##p < 0.01, compared with the collagen-treated platelets. For the other experiments (E), wild-type zebrafish embryos at 6 postfertilization were exposed to a solvent control (a, 0.5% DMSO) or various concentrations of honokiol (b, 1 μM; c, 5 μM; d, 10 μM) (scale bar 100 μm). Statistical data (F) show the means ± SEM (n = 60).

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  Figure 3. Effects of honokiol on platelet aggregation and Lyn, phospholipase Cγ2, and protein kinase C substrate (p47) phosphorylation stimulated with convulxin in washed human platelets. Washed platelets (3.6 × 108 cells/ml) were preincubated with honokiol (1–10 μM) or the solvent control (0.5% DMSO), followed by the addition of 5 ng/ml of convulxin (A) or 1 μg/ml of aggretin (B) to trigger platelet activation. The profiles in A and B represent 4 independent experiments. Furthermore, washed platelets (1.2 × 109 cells/ml) were preincubated with or without honokiol (5 μM and 10 μM) or the solvent control (0.5% DMSO), followed by the addition of 5 ng/mL of convulxin to trigger platelet activation at the indicated times (0.5 to 5 min). The cells were collected, and the subcellular extracts were analyzed for the presence of phosphorylated (C,D) Lyn, (E,F) phospholipase Cγ2 (PLCγ2), and (G,H) p47. Based on the convulxin’s maximum stimulation time points, immunoblot analysis was performed with convulxin stimulation of 1 min for phosphorylated (D) Lyn and (F) PLCγ2, and 3 min for (H) p47. Data are presented as the means ± SEM (n = 4). *p < 0.05 and **p < 0.01, compared with the control (resting)…

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  Figure 4. Effects of honokiol on ERK2, JNK, p38 MAPK, and Akt phosphorylation stimulated with convulxin. Washed platelets (1.2 × 109 cells/ml) were preincubated with or without honokiol (5 μM and 10 μM) or the solvent control (0.5% DMSO), followed by the addition of 5 ng/ml of convulxin at the indicated times (1 to 5 min). Cells were collected, and the subcellular extracts were analyzed for the presence of phosphorylated (A,B) ERK2, (C,D) JNK, (E,F) p38 MAPK, and (G,H) Akt. Data are presented as the means ± SEM (n = 4). *p < 0.05, **p < 0.01 and ***p < 0.001, compared with the control (resting) platelets; #p < 0.05, ##p < 0.01 and ###p < 0.001, compared with the convulxin-treated platelets.

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  Figure 5. Association of honokiol with GP VI on human platelets. (A) The solid line represents the fluorescence profiles of the platelets in the presence of (a) FITC only (background) or (b) FITC-JAQ1 mAb (1 μg/ml); or preincubated with (c) convulxin (5 ng/ml) or (d) honokiol (10 μM), followed by the addition of FITC-JAQ1 mAb (1 μg/ml). Statistical graphs (B) show the means ± SEM (n = 4). ***p < 0.001, compared with the control (background) group; #p < 0.05 and ##p < 0.01, compared with the FITC-JAQ1 mAb group. (C) For the immunoprecipitation study, washed platelets (1.2 × 109 cells/ml) were preincubated with honokiol (10 μM) or the solvent control (0.5% DMSO) for 3 min, followed by the addition of convulxin (5 ng/ml) to trigger platelet activation. The proteins in the cellular extracts were then immunoprecipitated using the anti-Lyn pAb (1 μg/ml) or control IgG (1 μg/ml). The immunoprecipitates were then analyzed through immunoblotting using the anti-JAQ1 mAb and anti-Lyn pAb. The profile represents 4 independent experiments. (D) The direct binding of honokiol to GP VI was examined using surface plasmon resonance (SPR) technology, as described in the Methods section. In brief, the…

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  Figure 6. Honokiol on closure time determined through PFA-100 analysis and thrombotic platelet plug formation in the mesenteric venules of mice. (A) Shear-induced platelet plug formation in whole blood was determined according to closure time (CT), as described in the Methods section. The CT of CEPI for whole blood was recorded for the solvent control (0.5% DMSO), honokiol (10 μM), or CAPE (50 μM). Data are presented as the means ± SEM (n = 6). **p < 0.01 and ***p < 0.001, compared with the solvent control group. (B) Mice were administered an intravenous bolus of the solvent control (0.5% DMSO) or honokiol (0.5 or 1.0 mg/kg), and the mesenteric venules were irradiated to induce microthrombus formation (occlusion time). Data are presented as the means ± SEM (n = 6). **p < 0.01, compared with the 0.5% DMSO group. (C) Microscopic images (x400 magnification) of DMSO-treated controls (a,b) and honokiol (1.0 mg/kg)-treated groups (c,d) were recorded at 5 s (a,c) and 150 s (b,d) after irradiation. The photographs are representative examples of 6 similar experiments. The arrow indicates platelet plug formation.

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  Figure 7. Hypothetical scheme of the inhibitory signaling of honokiol in platelet activation. Honokiol binds to GP VI before interfering with the activation of convulxin-mediated signal events, such as Lyn phosphorylation and phospholipase Cγ2 (PLCγ2)-diacylglycerol (DAG)-protein kinase C (PKC)-p47, MAPKs, and Akt signaling pathways. It subsequently inhibits [Ca+2]i mobilization and ultimately inhibiting platelet activation. DTS, dense tubular system; IP3, inositol 1,4,5-trisphosphate; ITAM, immunoreceptor tyrosine-based activation motif.