FIGURE LEGENDS
Figure 1. Inhibitory effect of quercitrin on platelet aggregation and ATP secretion following stimulation with numerous agonists. Washed platelets were preincubated with various concentrations of quercitrin (10, 20, and 30 µM) for 5 minutes at 37°C and then stimulated with 0.1 µg/ml CRP (A), 3 µM U46619 (B), 1 µg/ml Collagen (C), 0.025 U/ml thrombin (D), and 2.5 µM ADP (E). Similarly, the effect of quercitrin on PRP was analyzed following stimulation with 1 µg/ml Collagen (F). Aggregation was measured for 5 minutes at 37°C under constant stirring (1,000 rpm) conditions in a platelet aggregometer (Chrono-Log). In the ADP-induced aggregation assay, 30 µg/ml of human FG was added to the platelet suspension before ADP stimulation. The representative aggregation traces were obtained from three independent experiments. (i) Platelet aggregation and quantitative graphs. Data represent the mean ± SD (n = 3). In the ADP secretion, washed platelets were preincubated with various concentrations of quercitrin (10, 20, and 30 µM) for 5 minutes at 37°C before adding a luciferin/luciferase reagent. After the luciferin/luciferase reagent added, platelets were stimulated with 0.1 µg/ml CRP (A), 3µM U46619 (B), 1 µg/ml Collagen (C), and 0.025 U/ml thrombin (D). (ii) ATP secretion was measured using a luminometer. Data represent the mean ± SD (n = 3). *:P<0.05, **:P<0.01, and ***:P<0.001 versus vehicle control after ANOVA and Dunnett’s test.
Figure 2. Quercitrin modulates αIIbβ3 integrin activation and P-selectin exposure during platelet activation. The inhibitory effect of quercitrin on CRP or U46619 induced αIIbβ3 integrin activation (A and C) and P-selectin exposure (B and D) was measured by flow cytometry. Washed human platelets were pre-treated with various concentration of quercitrin (10, 20, and 30 µM) for 5 minutes at 37°C and stimulated with 0.1 µg/ml CRP (A and B) or 3 µM U46619 (C and D). Binding of anti-activated αIIbβ3 (JON/A) and anti-P-selectin antibodies to platelets was calculated by the ratio of the geometric mean fluorescence intensity (MFI) value of antibodies to that of control IgG. Data represent mean ± SD (n = 3). *:P<0.05, **:P<0.01, and ***:P<0.001 versus vehicle control after ANOVA and Dunnett’s test.
Figure 3. Quercitrin regulates Ca2+mobilization during platelet activation. Human platelets were resuspended in HEPES-Tyrode buffer without 1 mM CaCl2and preincubated with various concentrations of quercitrin (10, 20, and 30 µM) for 5 minutes at 37°C and incubated with a calcium-sensitive dye for 30 minutes at 37°C in the dark. After treatment with a Ca2+ dye, platelets were stimulated with 0.1 µg/ml CRP (A) or 3 µM U46619 (B) for 10 minutes and 2 mM CaCl2 was then added. Intracellular Ca2+ release (i) and influx (ii) were measured and quantified by the AUC (arbitrary units). Quantitative data represent the mean ± SD (n = 3). *:P<0.05 and **:P<0.01 versus vehicle control after ANOVA and Dunnett’s test.
Figure 4. Quercitrin modulates intracellular and extracellular H2O2 following agonist stimulation.Vehicle control (0.02% DMSO) and quercitrin (10, 20, and 30 µM) were incubated with H2DCFDA and stimulated with 0.1 µg/ml CRP (A) or 3 µM U46619 (B). DCF fluorescence was measured using flow cytometry and is quantified as mean ± SD (n = 3). *:P<0.05 versus vehicle control after ANOVA and Dunnett’s test. Extracellular H2O2 levels were measured in CRP-stimulated platelets using the Amplex Red assay (C). The fluorescence signal was measured by a microplate reader and is quantified as mean ± SD (n = 3). **:P<0.01 and ***:P<0.001 versus vehicle control after ANOVA and Dunnett’s test. ###:P<0.001 between resting and stimulated control group.
Figure 5. The inhibitory effect of quercitrin on platelet spreading on immobilized fibrinogen. Human platelets, 8 x 106 platelets in 0.4 ml, were pre-treated with various concentrations of quercitrin (10, 20, and 30 µM) or vehicle control (0.02% DMSO) and incubated on FG-coated surfaces for 2 hours at 37°C. Adherent and spread platelets were stained with rhodamine-conjugated phalloidin. (A) Representative images. Bar = 10 µm. (B) Number of adherents (but not spread, gray bars) and fully spread (white bars). Platelet spreading was analyzed by the surface area (C) which was measured by the number of pixels divided by the number of platelets (D) in the field. *:P<0.05, **:P<0.01, and ***:P<0.001 versus vehicle control after ANOVA and Dunnett’s test. Data represent mean ± SD (n = 5).
Figure 6. Quercitrin plays a crucial role in the GPVI-mediated signalosome. Human platelets were pre-treated with various concentrations of quercitrin (10, 20, and 30 µM), and stimulated with 0.1 µg/ml CRP (A, E, and I) or 3 µM U46619 (C) under stirring conditions (1,000 rpm) in an aggregometer. An equal amount of cell lysate protein (30 µg) was immunoblotted, followed by densitometry (arbitrary unit (AU)). Representative blots (A, C, E, and I). Quantitative graphs (B, D, F, and J). (G-H) Human platelets were stimulated by 0.1 µg/ml CRP for 5 minutes under stirring conditions. The lysates were immunoprecipitated with control IgG or antibodies against GPVI or 4G10, followed by immunoblotting and densitometry (n = 3). Data represent the mean ± SD (n = 3-4). ***:P<0.01 versus vehicle control (unstimulated) after Student’s t -test, and #:P<0.05, ##:P<0.01 and ###:P<0.001 versus vehicle control (stimulated) after ANOVA and Turkey’s test.
Figure 7. Role of Quercitrin in thrombus formation and tail bleeding time. Blood drawn from vehicle or quercitrin (50 or 100 mg/kg, BW) treated mice were perfused over collagen-coated surfaces at a wall shear rate of 1000 s-1 for 1 minute through the chamber. Adherent thrombi were stained with rhodamine-conjugated phalloidin and analyzed as described in Method. (A) Representative images. Bar = 10 µm. (B) Surface coverage and (C) thrombus volume were measured and presented as mean ± SD (n = 3). (D) FeCl3-induced arterial thrombus formation was performed as described in Methods. Mouse carotid artery was isolated and treated with 10% FeCl3 for 2 minutes, and blood flow traces were monitored until stable occlusion took place using a blood flowmeter. (E) Tails of vehicle or quercitrin treated mice were amputated, and bleeding time was monitored as described in Methods. Horizontal bars represent the median of occlusion and bleeding times for each group of animals (n = 10). *:P<0.05 and ***:P<0.001 versus vehicle control after ANOVA and Dunnett’s test.