Figure 1.
Effects of chlorogenic acid on P-selectin expression and GPIIb/IIIa activation.
Washed platelets were preincubated with vehicle (DMSO 0.2%) or chlorogenic acid (0.1 to 1 mmol/L) for 3 min. Then, samples were treated for 6 min with ADP 8 µmol/L. P-selectin expression (A) and GPIIb/IIIa activation (B) were determined by flow cytometry. Results were expressed as mean ± SEM, n = 6. **p<0.01 and ***p<0.001. The results presented are from 6 separate volunteers.
Figure 2.
Effects of chlorogenic acid on platelet ATP secretion (A) and aggregation (B).
For A) ADP 8 µmol/L (thromboxane A2 dependent) and collagen 1.5 µg/mL induced platelet ATP secretion. For B) platelet aggregation induced by ADP 8 µmol/L and collagen 1.5 µg/mL and each aggregation curve is representative of multiple traces obtained from six separate platelet donors. Results were expressed as % inhibition (mean ± SEM, n = 6).
Figure 3.
Effects of chlorogenic acid on collagen-induced platelet adhesion and aggregation under arterial flow conditions.
Citrate-anticoagulated blood was pre-incubated with vehicle (DMSO 0.2%) or chlorogenic acid (0.1 to 1 mmol/L) for 15 min and then was perfused over plaque-coated surfaces for 10 min at room temperature at a shear rate of 10 dyne/cm2. A) Timelapse of 10 min at 10 dyne/cm2, at 2 min intervals. B) Surface covered by platelets expressed as the percentage of the total surface observed, values are mean ± SEM; n = 6. ***p<0.001. The results presented are from 6 separate volunteers.
Figure 4.
Chlorogenic acid reduces leukocyte rolling and firm adhesion over collagen-bound platelet monolayers.
Velocities of leukocyte rolling and firm adhesion on platelet monolayer surface in presence of chlorogenic acid. Results were expressed as mean ± SEM of n = 6. The significant results (*p<0.05, **p<0.01 and ***p<0.001) are between vehicle group (DMSO 0.2%) and chlorogenic acid dose. The results presented are from 6 separate volunteers.
Figure 5.
Effect of chlorogenic acid on intraplatelet levels of cAMP formation in human platelets.
Platelets were incubated with PGE1 (0.02 mmol/L, positive control) or chlorogenic acid (0.1 to 1 mmol/L) for measurement of cAMP formations as described in “Materials and Methods.” Results were expressed as mean ± SEM of n = 6. The significant results (*p<0.05 and ***p<0.001) are between resting group, and PGE1 and chlorogenic acid dose. The results presented are from 6 separate volunteers.
Figure 6.
Effects of chlorogenic acid on platelet sP-selectin, sCD40L, CCL5 and IL-1β release.
Washed platelets were pretreated with vehicle (DMSO 0.2%), ASA (0.3 mmol/L) or chlorogenic acid (0.1 to 1 mmol/L) for 15 min at 37°C and then stimulated by thrombin (2 U/mL). The graph depicts the mean ± SEM of n = 6 experiments. *p<0.05 and *** p<0.001. The results presented are from 6 separate volunteers.
Figure 7.
Effects of SQ22536 and ZM241385 on antiplatelet activity of chlorogenic acid.
For platelet aggregation washed platelets suspension was incubated with ADP, chlorogenic acid plus ADP or pretreated with SQ22536 (A) or ZM241385 (B) for 3 min, followed by addition of chlorogenic acid (CA) and ADP. The graph depicts the mean ± SEM of n = 6 experiments. ** p<0.01. The results presented are from 6 separate volunteers.
Figure 8.
Effects of SQ22536 on mechanism of antiplatelet action of chlorogenic acid.
For sP-selectin (A) and sCD40L (B) washed platelets were incubated with thrombin, chlorogenic acid plus thrombin or pretreated with SQ22536 for 3 min, followed by the addition of chlorogenic acid and thrombin. The graph depicts the mean ± SEM of n = 6 experiments. * p<0.05, ** p<0.01 and *** p<0.001. The results presented are from 6 separate volunteers.
Figure 9.
Effect of chlorogenic acid on PKA activation.
Platelets were collected and subcellular extracts were analyzed for phospho-PKA as described in Materials and Methods. Data are presented as the mean ± SEM of n = 6 experiments. **p<0.01 and *** p<0.001. The results presented are from 6 separate volunteers.
Figure 10.
Chlorogenic acid inhibited arterial thrombosis formation.
A) Representative images of thrombus formation after laser irradiation in the vehicle group (DMSO 0.2%, n = 6); ASA (200 mg/kg, n = 6) or chlorogenic acid (200 mg/kg, n = 6) to 60 min. B) Time course changes of thrombus growth rate. I, intima; M, media and A, adventitia.
Figure 11.
Effect of chlorogenic acid on bleeding time.
Same amount of vehicle (DMSO 0.2%, n = 6, intraperitoneally), chlorogenic acid (200 mg/kg, n = 6, intraperitoneally) or ASA (200 mg/kg, n = 6, intraperitoneally) as in thrombosis model were given. Each dot represents the bleeding time measured in each individual mouse (n = 6), mean is also shown.
Figure 12.
Predicted binding conformation of the chlorogenic acid inside A2A receptor binding pocket.
A) Alignment of docked structures of chlorogenic acid on X-ray reference structure of A2A receptor binding pocket. B) Pocket of A2A receptor occupied by chlorogenic acid.