Xinmailong Modulates Platelet Function and Inhibits Thrombus Formation via the Platelet αIIbβ3-Mediated Signaling Pathway

Figures

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  Figure 1. The main active components of XML. (A) adenosine, (B) protocatechuic acid, (C) inosine, (D) pyroglutamate dipeptide.

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  Figure 2. HPLC fingerprint of XML extractum containing three marker compounds. Uracil (C4H4N2O2) (Michelson, 2010), hypoxanthine (C5H4N4O) (Varga-Szabo et al., 2008), inosine (C10H12N4O5) (Watson et al., 2001). (A) HPLC fingerprint of standards provided by the CFDA; (B) HPLC chromatogram of the XML extractum sample used in the present research. The fingerprint spectrum similarity between the standard and the sample is ≥0.99.

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  Figure 3. Inhibitory effect of XML on agonist-induced platelet aggregation in human PRP and washed platelets. (A) PRP (2.0 × 108/ml) and (B) washed platelets (2.0 × 108/ml) were pre-incubated with XML (0.1 mg/ml, 1 mg/ml, and 10 mg/ml) or vehicle prior to the addition of thrombin (0.04 U/ml), collagen (1 μg/ml), or AA (62.5 μM) to trigger platelet aggregation. Quantification of aggregation (%) is also shown in the lower panel. Data are expressed as the mean ± SEM of three experiments. *P < 0.05, **P < 0.01 vs. vehicle.

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  Figure 4. XML affects platelet activity in vivo in rats. Rats were pre-treated with vehicle, 50 mg/kg XML by tail vein injection for 30 min. Then, washed platelets (3.0 × 108/ml) were treated with thrombin (0.08 U/ml), collagen (1 μg/ml), or AA (100 μM) to trigger platelet aggregation. Quantification of aggregation (%) is shown in the lower panel. Data are expressed as the mean ± SEM of 10 experiments. **P <0.01 vs. vehicle.

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  Figure 5. Inhibitory effect of XML on agonist-induced ATP-release, P-, and PAC-1 expressions in washed human platelets. (A) Washed platelets (5.0 × 107/ml) were pre-treated with XML (0.1, 1, and 10 mg/ml) or vehicle before the addition of thrombin (0.04 U/ml), collagen (1 μg/ml), or AA (62.5 μM) in the ATP-release reaction experiment. Quantification of ATP release (nM) is shown in the right panel. (B, C) Washed human platelets (5.0 × 107/ml) were pre-incubated with XML (0.1, 1, and 10 mg/ml) at 37°C for 10 min in the presence of PE-conjugated P-selectin and Alexa Fluor–conjugated PAC-1 antibodies, stimulated with thrombin (0.04 U/ml), collagen (1 μg/ml), or AA (62.5 μM), and incubated for a further 5 min. P-selectin expression (%) and PAC-1 expression (%) were determined by flow cytometry. Quantification of P-selectin expression (%) (B) and PAC-1 expression (%) (C) are shown in the lower panel. The results are expressed as mean ± SEM of three experiments. *P <0.05, **P <0.01 vs. vehicle.

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  Figure 6. XML inhibits platelet adhesion on collagen-coated surfaces. (A) Human washed platelets (2.0 × 107/ml) were pre-treated with vehicle or XML (1 mg/ml, 10 mg/ml) for 10 min at 37°C, then allowed to adhere on collagen-coated slides. We selected representative images from three independent experiments. (B) The number of adherent platelets and (C) the average surface area of individual platelets was analyzed. (Mean ± SEM, n = 3, *P <0.05, **P <0.01 vs. vehicle).

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  Figure 7. Effect of XML on agonist-induced signal transduction in washed human platelets. Washed platelets (5.0 × 108/ml) were stimulated by (A) 0.08 U/ ml thrombin, (B) 1 μg/ml collagen, and (C) 100 μM AA for 120 s and subsequently lysed with cell lysis buffer. The levels of the phosphorylation of Syk, PLCγ2, Akt, GSK3β, JNK, ERK1/2, and p-38 were detected with the relevant antibodies. Total protein and GAPDH were used as loading controls. The band density was calculated with ImageJ. Values are expressed as the mean ± SEM (n = 3). *P <0.05, **P <0.01 vs. vehicle.

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  Figure 8. XML negatively regulated signaling. (A) Platelets were pretreated with either XML (0.1 mg/ml), PP2 (SFK inhibitor, 2 μM), or XML (0.1 mg/ml) and PP2 (2 μM) before collagen stimulation. (B) Platelets were pretreated with either XML (0.1 mg/ml), LY294002 (PI3K inhibitor, 2 μM), or XML (0.1 mg/ml) and LY294002 (2 μM) before collagen stimulation. Bar graphs show mean ± SEM of three experiments. *P <0.05, **P <0.01 vs. vehicle. # P <0.01 vs. PP2 or LY294002 group.

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  Figure 9. XML inhibits clot retraction. The clot retraction assay was performed, and images were taken 30 min later. Representative results were selected from three independent experiments.

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  Figure 10. XML inhibits platelet spreading on fibrinogen-coated surfaces. (A) Human washed platelets (2.0 × 107/ml) were pre-treated with vehicle or XML (1 mg/ml, 10 mg/ml) for 10 min at 37°C, then allowed to adhere to fibrinogen-coated slides. We selected representative images from three independent experiments. (B) The number of adherent platelets and (C) the average surface area of individual platelets was analyzed. (mean ± SEM, n = 3, *P <0.05, **P <0.01 vs. vehicle).

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  Figure 11. Effect of XML on hemorrhage and hemostasis. The mouse tails were transected to induce bleeding, and the time to bleeding cessation was recorded. Data are reported as means ± SEM (n = 6). N.S., no significant difference versus vehicle.

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  Figure 12. Effect of XML on prothrombin time (PT) and activated partial thromboplastin time (APTT). Results are expressed as means ± SEM (n = 10). N.S., no significant difference versus vehicle.