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.