Phospholipase Cγ2 Signaling Cascade Contribute to the Antiplatelet Effect of Notoginsenoside Fc

Figures

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  FIGURE 1. Chemical structure of Notoginsenoside Fc.

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  FIGURE 2. Antiplatelet effect of Fc. (A) After incubating WP with Fc (400 μM) for indicated times (3, 5, 10 min), platelet aggregation was induced by thrombin. To determine the dose-dependent antiaggregatory effect of Fc, WP were incubated with indicated concentrations (50, 100, 200, 400, 800 μM) of Fc for 5 min. Platelet aggregation was induced by either thrombin (B), collagen (C), or ADP (D). Tracing graphs represent the percentage of platelet aggregation variation after treated with stimulators. Values are mean ± SEM (n = 3 for A; n = 3∼4 for B and C; n = 3∼5 for D).

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  FIGURE 3. Suppression of platelet PLCγ2 cascade by Fc. (A) After incubating WP with different concentrations of Fc, platelet PLCγ2 and PKC activity were evaluated according to the phosphorylation of PLCγ2 and P47. β-actin was used as a loading control. Platelet content of IP3, DAG and TXB2 were measured by ELISA kits. WP was incubated with the indicated concentrations of Fc, and platelet IP3 (B), DAG (C), and TXB2 (D) concentrations were assessed. Values are mean ± SEM (n = 3 for A; n = 5 for B; n = 4 for C,D). ∗P < 0.05 versus control.

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  FIGURE 4. Prevention of thrombin induced activation of PLCγ2 cascade by Fc. After incubating with different concentrations of Fc, WP was treated with thrombin for 3 min. (A) The phosphorylation of PLCγ2 and P47 in platelet were measured with western blotting. β-actin was used as a loading control. Platelet IP3 (B), DAG (C) and TXB2 (D) concentrations were assessed by corresponding ELISA kits. (E) Variation in [Ca2+] was assessed by detecting the fluorescence intensity emitted from intracellular Fluo-4. The images are from the peak point of each experiment. Tracings were from representative results in three independent experiments. Values are mean ± SEM (n = 6 for A; n = 4 for B,C; n = 3 for D). ∗P < 0.05 versus control; #P < 0.05 versus thrombin only.

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  FIGURE 5. Involvement of PLCγ2 cascade in the antiplatelet effect of Fc. WP were treated with either increasing concentrations of m-3M3FBS (100, 200, 400 μM) or m-3M3FBS (400 μM) followed Fc (800 μM). (A) Platelet aggregation was assessed by a 4-channed aggregometer. (B) The phosphorylation of PLCγ2 and P47 of platelet were measured with western blotting. β-actin was used as a loading control. Platelet IP3 (C), DAG (D), and TXB2 (E) concentrations were assessed by ELISA kits. (F) [Ca2+] was assessed by the fluorescence intensity from intracellular Fluo-4. The images are from the peak point of each experiment. Tracings were from representative results in three independent experiments. Values are mean ± SEM (n = 6∼11 for A; n = 3∼4 for B–E). ∗P < 0.05 versus control; #P < 0.05 versus 400 μM m-3M3FBS only.

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  FIGURE 6. In vivo anti-thrombotic effect of Fc. Rats were i.p. injected with saline, Fc or clopidogrel. 2 h later, a filter paper saturated with 20% FeCl3 was applied to the isolated femoral artery for 20 min. Then the injured arteries were isolated for observation under microscope and measurement of thrombus protein content. (A) The bar graph indicates the protein contents in FeCl3 induced thrombus of each group. (B) The representative graphs showed the artery condition after injured by FeCl3. Values are mean ± SEM (n = 6 for control and Fc group; n = 3 for clopidogrel group). ∗P < 0.05 versus control.

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  FIGURE 7. Mechanism of Fc inhibits platelet aggregation. Thrombin, collagen and ADP activate PLCγ2 signaling cascade through PAR, GPVI and P2Y1 receptors, respectively. PLCγ2 promotes the hydrolyzation of PIP2 to DAG and IP3 in platelets. The increase in platelet DAG and IP3 subsequently up-regulate PKC activity, TXA2 content and [Ca2+]. m-3M3FBS triggers activation of PLCγ2 and downstream signaling directly. Fc can abolish platelet aggregation induced by thrombin, collagen, ADP, as well as m-3M3FBS, through preventing the activation of PLCγ2 signaling. Arrows indicate the activation of target molecules or stimulation of production. Blunt line means target inhibition. The difference in color demonstrates different stimulators triggered effects.