Luteolin inhibits GPVI-mediated platelet activation, oxidative stress, and thrombosis

Figures

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  FIGURE 1. Effect of luteolin on collagen- and convulxin-induced platelet aggregation, ATP release, P-selectin expression, and LDH release. Human washed platelets (WPs) were incubated with different concentrations of luteolin or DMSO at 37°C for 10 min after the addition of indicated agonists. (A–D) After incubation, the platelet aggregation and ATP release curves were depicted in real-time, and the inhibition rate curves were drawn (A, far right) to calculate the IC50 values using nonlinear regression. (B) Platelet aggregation curves under different stimulations. (C) The platelet aggregation rates were calculated from the curves in (B). (D) ATP release and inhibition rate IC50 values using nonlinear regression (E) Flow cytometric scatter plots were performed for CD62p and CD61. (F) Platelets were incubated at 37°C for 60 min with different doses of luteolin, DMSO, or Triton X-100. The LDH release was measured. All experimental results are expressed as mean ± SEM (n ≥ 3). Compared with the Vehicle(DMSO) group, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data were analyzed with one-way ANOVA followed by Dunnett’s test.

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  FIGURE 2. Effect of luteolin on GPVI-mediated oxidative stress in human platelets. The reactive oxygen species (ROS) assay probes DCFH-DA (5 μM), 2 μM Amplex Red, and 2 μM DHE were used to pre-stained human washed platelets (WPs). The WPs were incubated with different concentrations of luteolin, DMSO, and Syk-specific inhibitor (BAY61-3606) for 10 min at 37°C. (A) Collagen (10 μg/mL) and GPVI-specific activator convulxin (5 ng/mL) activation of human platelets. Human platelet ROS release was monitored in real-time at ex/em = 480/530 nm. RFU: relative fluorescence value. (B,C) Flow cytometry detection of platelet superoxide anion and H2O2 release. (D) Effect of luteolin and VAS2870 (10 μM) on collagen (10 μg/mL) and convulxin (5 ng/mL) -induced NOXs activity. (E–G) Platelet lysate supernatant MDA, SOD, and GPx activities were measured. (H) Effect of luteolin (25 μM) and BAY61-3606 (63 nM) on collagen- (10 μg/mL) and convulxin (5 ng/mL)-induced platelet ROS release. Absorbance values were detected at 450 nm after the reaction of platelet membrane protein lysate with NADPH (0.5 mM) and lucigenin (10 μM). Compared with the BAY61-3603 group, # p < 0.05, ### p < 0.001. Data were analyzed with…

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  FIGURE 3. Effect of luteolin on platelet static adhesion to the collagen surface. Adhesion of human washed platelets (WPs) on the surface of collagen (50 μg/mL) collagen-coated coverslips (n = 3) after incubation with different concentrations of luteolin or DMSO at 37°C for 10 min. Magnification ×40. The bar graphs compare the number and area of platelet adhesions. The experimental results are expressed as mean ± SEM, analyzed with two-way ANOVA followed by Dunnett’s test. Compared with the vehicle group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  FIGURE 4. Effect of luteolin on integrin αIIbβ3-mediated “inside-out” and “outside-in” activation in human platelet. (A) Washed platelets (WPs) were incubated with DMSO or luteolin at 37°C for 10 min CaCl2 (1 mM) and collagen (10 μg/mL) or convulxin (5 ng/mL) were added to stimulate the WPs. The expression of the PAC-1 protein on the platelet surface was analyzed using flow cytometry. n = 3. Statistical analysis of PAC-1 cells was calculated by the FlowJo software. (B) Convulxin (5 ng/mL) and collagen (10 μg/mL) induced platelets intracellular calcium release were recorded and traced using a Fluorescence microplate reader at ex/em = 480/530 nm (left panel). n = 3. (C) The WPs were spread on 25 μg/mL of fibrinogen-coated slides Magnification ×40. n = 3. (D) WPs were incubated with 1 mM CaCl2 with 0.1 U/mL thrombin at 37°C and photographed every 5 min to observe clot retraction, n = 3. Data are expressed as mean ± SEM, analyzed with ANOVA followed by Dunnett’s test. Compared with the vehicle group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  FIGURE 5. Binding of luteolin to GPVI and effect on convulxin-induced activation of ITAM and MAPK signaling pathways. (A) Molecular docking showing the schematic diagram of luteolin binding to GPVI (PDB code: 2GI7). Yellow: luteolin; green: GPVI; blue: interacting amino acid residues; yellow dashed lines: hydrogen bonds. (B) Inhibition of rhGPVI binding to immobilized collagen by increasing concentrations of luteolin. The binding of rhGPVI at 30 nM (black circles) to collagen was dose-dependently inhibited by luteolin with IC50 values of 5.49 µM using nonlinear regression. In contrast, luteolin had no chromogenic reaction on the 5 μg/mL (black triangles) collagen. n = 3. (C) SPR analysis of the interaction between luteolin and GPVI. (D, E) Effect of luteolin on convulxin-induced activation of platelet ITAM and MAPK signaling pathway molecule. n = 3. Results are expressed as mean ± SEM analyzed with one-way ANOVA followed by Dunnett’s test. Compared with the vehicle group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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  FIGURE 6. Effect of luteolin on thrombus formation and oxidative stress in vivo. (A,B) Effect of luteolin on ferric chloride-induced thrombosis in the mesenteric arteries of mice. Magnification ×4. n = 10. (C,D) Effects of luteolin on collagen and epinephrine injection induced pulmonary thrombosis in mice. H&E (C) and CD41 (D) immunohistochemical analyses were performed, and the photo-grams were magnified at ×10, with arrows indicating thrombus (n = 6). (E) Effect of luteolin suppresses on malonaldehyde (MDA) generation and superoxide dismutase (SOD) activity in plasma of pulmonary embolism mice (n = 6). Results are expressed as mean ± SEM. Differences between DMSO and luteolin groups were analyzed with unpaired t-tests to compare the differences between the two groups. One-way ANOVA followed by Dunnett’s test was applied to multiple comparisons. *p < 0.05, ***p < 0.001.

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  FIGURE 7. Effect of luteolin on hemostasis, coagulation, and platelet production in mice. (A) Mice were anesthetized 30 min after injection of DMSO or luteolin and aspirin. Effect of luteolin on tail bleeding time in mice. n = 10. (B) Coagulation analysis, n = 10. (C,D) Luteolin was injected intraperitoneally daily for 15 days. Changes in platelet count and volume in mice during administration, n = 10. (E) Immunohistochemistry of sternum bone marrow sections from luteolin-treated (35 μM/kg/day) or DMSO-treated mice. Mouse sternum was taken and prepared for immunohistochemical analysis. CD41 staining was positive for macrophages, as shown by the arrow. The photo-grams were magnified at ×40. The bar graph shows the numbers of megakaryocytes from six fields from 10 mice in each group. Results are shown as mean ± SEM, Differences between DMSO, aspirin, and luteolin groups were analyzed with unpaired t-tests to compare the differences between the two groups. One-way ANOVA followed by Dunnett’s test was applied to multiple comparisons. *p < 0.05.