Antiplatelet and Antithrombotic Effects of Epimedium koreanum Nakai

Abstract

Background and Objective. Epimedium koreanum Nakai is a medicinal plant known for its health beneficial effects on impotence, arrhythmia, oxidation, aging, osteoporosis, and cardiovascular diseases. However, there is no report available that shows its effects on platelet functions. Here, we elucidated antiplatelet and antithrombotic effects of ethyl acetate fraction of E. koreanum. Methodology. We analyzed the antiplatelet properties using standard in vitro and in vivo techniques, such as light transmission aggregometry, scanning electron microscopy, intracellular calcium mobilization measurement, dense granule secretion, and flow cytometry to assess integrin α _IIb β ₃ activation, clot retraction, and Western blot, on washed platelets. The antithrombotic effects of E. koreanum were assessed by arteriovenous- (AV-) shunt model in rats, and its effects on hemostasis were analyzed by tail bleeding assay in mice. Key Results. E. koreanum inhibited platelet aggregation in agonist-stimulated human and rat washed platelets, and it also reduced calcium mobilization, ATP secretion, and TXB2 formation. Fibrinogen binding, fibronectin adhesion, and clot retraction by attenuated integrin α _IIb β ₃-mediated inside-out and outside-in signaling were also decreased. Reduced phosphorylation of extracellular signal-regulated kinases (ERK), Akt, PLCγ2, and Src was observed. Moreover, the fraction inhibited thrombosis. HPLC results revealed that the fraction predominantly contained icariin. Conclusion and Implications. E. koreanum inhibited platelet aggregation and thrombus formation by attenuating calcium mobilization, ATP secretion, TXB2 formation, and integrin α _IIb β ₃ activation. Therefore, it may be considered as a potential candidate to treat and prevent platelet-related cardiovascular disorders.

Figure 1

E. koreanum inhibits agonist-induced human and rat platelet aggregation. Collagen-, ADP-, or thrombin-stimulated washed platelets ((a) and (d) from rats (3 × 10⁸ cells/mL); (b) from humans (5 × 10⁸ cells/mL)) pretreated with vehicle or various concentrations of E. koreanum ethyl acetate fraction (EAF) in the presence of 1 mM CaCl₂. (c, e) Rat PRP (5 × 10⁸ cells/mL) was incubated with vehicle or various concentrations of extract in the presence of 10-mM CaCl₂ for 1 min and then stimulated with ADP (25 μM) for 5 min. (d) Representative SEM images (5000×) of collagen (2.5 μg/mL)-stimulated rat platelets pre-treated with vehicle or various concentrations of EAF ((A) resting, (B) vehicle, (C) 50 μg/mL, (D) 100 μg/mL, and (E) 200 μg/mL). The scale bar represents 5 µm. The graphs show mean ± SD values from at least four independent experiments. ∗p < 0.05 and ∗∗∗p < 0.001 versus control.

Figure 2

E. koreanum inhibits (Ca²⁺)_i mobilization and reduces ATP release and TxB2 production. (a) Fura 2/AM-loaded rat platelets (3 × 10⁸ cells/mL) pre-treated with vehicle or different concentration of EAF and stimulated with collagen (2.5 μg/mL) for 3 min. Assessment of ATP concentration (b) and thromboxane-B2 production (c) was done in a supernatant of stimulated washed platelets (3 × 10⁸ cells/mL) suspension pre-treated with vehicle or various EAF concentrations and stimulated with collagen for 5 min on a luminometer or TxB2 ELISA kit, respectively. Results are represented as mean ± SD values from at least four independent experiments. ∗p < 0.05 and ∗∗∗p < 0.001 versus control. R: resting; C: control.

Figure 3

E. koreanum inhibits integrin α_IIbβ₃-mediated inside-out and outside-in signaling. ((a) and (b)) Flow cytometric measurements of fibrinogen binding in rat platelets (3 × 10⁸ cells/mL) treated with vehicle or different concentrations of E. koreanum fraction (EAF), or EGTA ((A) resting, (B) vehicle, (C) 50 μg/mL, (D) 100 μg/mL, (E) 200 μg/mL, and (F) 10 μM EGTA)), and stimulated with collagen (b–f). (c) Results of fibronectin adhesion assay, which was performed using an assay kit according to the manufacturer's instructions and by following the procedure described in the methods section. (d) In vitro effect of E. koreanum on clot retraction for 2 h at room temperature after thrombin addition and photographed at 15 min intervals. Representative images of clot retraction at 90 min after thrombin addition with or without EAF. Y-27632 (ROCK inhibitor) was used as a control. Lanes 1–5 correspond to lanes 1–5 in (e). (f) Clot retraction kinetics were measured by ImageJ software, and the clot surface areas were plotted as a percentage of retraction. Bar graphs summarizing the inhibitory effect of EAF on fibrinogen binding to integrin α_IIbβ₃ (B), fibronectin adhesion (C), clot retraction (E), and kinetics of clot retraction (F). Results are shown as mean ± SD values from at least four independent experiments. ∗p < 0.05,^∗∗p < 0.01, and ∗∗∗p < 0.001 versus control. R: resting; C: control; GR: GR155053; Y-2: Y-27632.

Figure 4

E. koreanum attenuates phosphorylation levels of ERK, Akt (a), Src, and PLCγ2 (b). Immunoblotting was conducted to analyze the phosphorylation of signaling molecules extracted from the lysates of collagen-stimulated washed rat platelets (3 × 10⁸ cells/mL) that were pretreated with vehicle or E. koreanum fraction (EAF). Representative immunoblot images and data (mean ± SD) from at least four independent experiments are shown. Lanes 1–5 of blot images correspond to each bar graph, respectively. ∗p < 0.05 and ∗∗∗p < 0.001 versus the agonist-treated group.

Figure 5

E. koreanum inhibits thrombus formation and modulates hemostasis. (a) In vivo evaluation of anti-thrombotic activity and determination of thrombus weight in AV-shunt model of rats that were orally administered with saline, ethyl acetate fraction of E. koreanum (EAF; 100–300 mg/kg), or ASA (50 mg/kg). (b) Results of tail bleeding assay for homeostasis measurement in mice administered with EAF (300 mg/kg), ASA (50 mg/kg), or saline (n = 5 in each group). Graph shows mean ± SD values from at least five independent experiments performed. ∗p < 0.05 and ∗∗∗p < 0.001 versus control.

Figure 6

Chemical constituents of E. koreanum ethyl acetate fraction. (a) Chromatograms of the standard solution. (b) The HPLC chromatogram of ethyl acetate fraction of E. koreanum (EAF) was detected at 280 nm UV.

Figure 7

A schematic summary of the inhibitory effects of E. koreanum on intracellular platelet signaling pathway.