VAS2870 and VAS3947 attenuate platelet activation and thrombus formation via a NOX-independent pathway downstream of PKC

Figures

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  Figure 1. VAS1 mediated the inhibition of platelet aggregation in human platelets stimulated by various agonists. Washed platelets (3.6 × 108 cells/ml) were preincubated with DMSO (solvent control) or VAS1 (VAS2870, 1–10 μM) following the stimulation with (A) collagen (1 μg/ml), (B) convulxin (10 ng/ml), (C) thrombin (0.02 U/ml) and (D) U46619 (1 μM) to trigger platelet aggregation. Left panels indicate the tracing curve of platelet aggregation, and right panels indicate the statistical bar graphs of platelet aggregation (%). Data (A–D) are presented as means ± SEM (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the DMSO (solvent control) group. Comparisons were made by ANOVA.

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  Figure 2. Effects of VAS2 and ML171 on platelet aggregation induced by various agonists. Washed platelets (3.6 × 108 cells/ml) were pre-incubated with DMSO (solvent control), (A–D) VAS2 (VAS3947, 1–10 μM), or (E–H) ML171 (ML, 0.5–10 μM) following the stimulation of collagen (1 μg/ml), convulxin (10 ng/ml), thrombin (0.02 U/ml) and U46619 (1 μM) to trigger platelet aggregation. Profiles (A–H) are representative examples of three similar experiments.

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  Figure 3. Effects of selective and nonspecific NOX inhibitors on platelet aggregation. Washed platelets (3.6 × 108 cells/ml) were pre-incubated with DMSO (solvent control), (A) GKT136901 (GKT1, 20 μM), (B) GKT137831 (GKT2, 20 μM), (C) GSK2795039 (GSK, 20 μM), or (D,E) combination of ML171 (ML, 0.5 μM), GSK (20 μM), and GKT1 or GKT2 (20 μM) following stimulation with collagen (1 μg/ml), convulxin (10 ng/ml), thrombin (0.02 U/ml) and U46619 (1 μM) to trigger platelet aggregation. Profiles (A–E) are representative examples of three similar experiments.

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  Figure 4. Effects of VAS compounds on the PKC/NOX downstream signaling pathway. (A) Washed platelets (3.6 × 108 cells/ml) were pre-incubated with DMSO (solvent control), VAS compounds (2–10 μM), or Ro 31-8220 (2 μM) following stimulation with PDBu (150 nM) to trigger platelet aggregation. (B) The statistical analysis in (A). (C) After the reaction, platelet lysates were directly collected, and then subjected to Western blotting. Specific antibodies were used to detect PKC, IKKβ, and p38 MAPK. (D,E) Luciferase/luciferin and FITC-P-selectin antibody were used to detect ATP release and P-selectin using a microplate reader and flow cytometry, respectively. Data (B,D) are presented as means ± S.E.M. (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001, compared with the DMSO (solvent control) group. Data (C,E) are presented as means ± SEM (C, n = 4; E, n = 3). ***p < 0.001, compared with the resting group. ##p < 0.01 and ###p < 0.001, compared with the PDBu-treated (positive control) group. Comparisons were made by ANOVA.

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  Figure 5. Effects of VAS compounds on calcium mobilization and GPIIbIIIa activation. (A,B) Washed platelets were pre-incubated with DMSO (solvent control) and VAS1 (2–10 μM) before the addition of PDBu to trigger platelet activation. Fura-2 and FITC-PAC1 antibodies were used to detect calcium mobilization and GPIIbIIIa activation by a microplate reader and flow cytometry, respectively. (C) FITC-triflavin was used to observe the binding capacity of VAS compounds (10 μM) on the GPIIbIIIa receptor through flow cytometry. (D) LDH assay kits were used to determine the cytotoxicity of VAS compounds (10–100 μM) on platelets. The Triton-treated group as maximum toxicity (100%). Data (A) are presented as means ± SEM (n = 3). *p < 0.05 and ***p < 0.001, compared with the DMSO (solvent control) group. Data (B) are presented as means ± S.E.M. (n = 3). ***p < 0.001, compared with the resting group. #p < 0.05 and ##p < 0.01, compared with the PDBu-treated (positive control) group. Comparisons were made by ANOVA. Profiles (C) are representative examples of three similar experiments.

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  Figure 6. Effects of VAS compounds on platelet aggregation, thrombus formation, and hemostasis in mice. Washed mouse platelets (1 × 108 cells/ml) were preincubated with DMSO (solvent control) or VAS compounds (10–20 μM) following stimulation with 1 μg/ml collagen (A) and 0.02 U/ml thrombin (B) to trigger platelet aggregation. (C) Mice received an intravenous bolus of DMSO, VAS1 (3.7 mg/kg) or VAS2 (4.5 mg/kg), and their mesenteric venules were irradiated to induce microthrombus formation. The arrow indicates occlusion of the mesenteric venule. The scale bar indicates 30 μm. (D) Bleeding was induced by severing the tail at 3 mm from the tail tip, and the bleeding tail stump was immersed in saline. Subsequently, the bleeding time was continually recorded until no sign of bleeding was observed for at least 10 s. Each point in the scatter plots graph represents a mouse (n = 6). Profiles (A,B) are representative examples of three similar experiments. Data (C) are presented as the mean ± SEM (n = 6). *p < 0.05, compared with the DMSO group. Comparisons were made by ANOVA.

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  Figure 7. Schematic illustration of VAS compounds-mediated inhibition of platelet activation and thrombus formation.