Saccharides from Arctium lappa L. root reduce platelet activation and thrombus formation in a laser injury thrombosis mouse model

Figure 1. ALR-S reduces platelet aggregation and ATP release in a concentration-dependent manner. (A and B) Representative (A) aggregation and (B) ATP release traces of washed platelets from healthy donors, in response to collagen, thrombin or ADP. Aggregation and ATP release were assessed with a Chrono-log aggregometer under stirring. The y-axis represents the relative percentage change of light transmission, when the light transmission of Tyrode's buffer (without platelet) is defined as 100%, and washed platelets (resting) were adjusted to 0% before the aggregation was started. (C and D) Quantification of (C) platelet aggregation and (D) ATP release. *P<0.05, **P<0.01 and ***P<0.001 vs. control (vehicle). ALR-S, saccharides from Arctium lappa L. root.

Figure 2. ALR-S attenuates platelet αIIbβ3 activation and CD62P expression. Representative histograms and summaries of αIIbβ3 activation and CD62P expression induced by (A) CRP (2 µg/ml), (B) thrombin (0.025 U/ml) and (C) ADP (10 µM) are presented. Dashed lines are used to gate a subpopulation of activated platelets. *P<0.05, **P<0.01 and ***P<0.001 vs. vehicle group. MFI, mean fluorescence intensity; ALR-S, saccharides from Arctium lappa L. root; CRP, collagen-related peptide.

Figure 3. ALR-S inhibits platelet spreading and delays clot retraction. (A) Representative images and (B) summary of platelet spreading. Original magnification, x100. Scale bars, 2 µm. (C) Representative results and (D) summaries of clot retraction. After incubation with ALR-S or vehicle, platelet-rich plasma (3x108/ml) was stimulated with thrombin (0.4 U/ml) and images were captured at designated time points. **P<0.01 and ***P<0.001 vs. vehicle group. ALR-S, saccharides from Arctium lappa L. root.

Figure 4. ALR-S decreases ROS generation during platelet activation. (A) Platelet intracellular ROS level was determined using flow cytometry. Platelets loaded with H2DCFDA (50 µmol/l) were incubated with ALR-S or vehicle for 10 min and then stimulated with CRP (2 µg/ml), thrombin (0.025 U/ml), or ADP (10 µM) for 5 min. *P<0.05 and **P<0.01 vs. control. (B) Western blotting was performed to analyze the effects of ALR-S on phosphorylated ERK1/2, p38 and HSP27 during platelet activation in an aggregometer. (C) Representative aggregation traces of washed platelets in response to collagen, thrombin or ADP. Washed platelets were preincubated with ALR-S (0, 20, 60 and 200 µg/ml), respectively. H2O2 (20 µM) was added before aggregation was started, and traces were recorded by a Chrono-log aggregometer under stirring. ALR-S, saccharides from Arctium lappa L. root; p38, p38 mitogen-activated protein kinase; HSP27, heat shock protein 27; Col, collagen; Thr, thrombin; MFI, mean fluorescence intensity; ROS, reactive oxygen species; CRP, collagen-related peptide.

Figure 5. ALR-S inhibits platelet adhesion onto fibrillar collagen under shear and thrombosis formation in vivo. (A) Representative images and (B) coverage area quantification of platelet adhesion on fibrillar collagen in a microfluidic perfusion system. Original magnification, x20. Scale bar, 100 µm. (C) Representative images of mouse cremaster arteriole thrombosis induced by laser injury (white circle), and thrombi (green) observed using anti-GPVI-labeled platelets. Original magnification, x40. Scale bars, 50 µm. (D) Time course of relative thrombi fluorescence intensity at laser-injured site. The fluorescence intensity of injured site after 8 sec was normalized to 1. (E) Summary of the time for thrombus reaching maximal fluorescence intensity after laser injury. **P<0.01 and ***P<0.001 vs. control (vehicle). ALR-S, saccharides from Arctium lappa L. root; GPVI, glycoprotein VI.