Sinomenium acutum Modulates Platelet Aggregation and Thrombus Formation by Regulating the Glycoprotein VI-Mediated Signalosome in Mice

Figures

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  Figure 1. The suppressing effect of Sinomenium acutum (SA) on platelet aggregation and adenosine triphosphate (ATP) secretion activated by different agonists. Platelets were preincubated with the different characteristics of the solutions of SA (50, 100, and 200 µg/mL) for 10 min at 37 °C. Subsequently, they were induced by 1 µg/mL collagen (A), 0.5 µg/mL collagen-related peptide (CRP) (B), 0.05 U/mL thrombin (C), 3 µM U46619 (D), and 2.5 µM adenosine diphosphate (ADP) (E). In ATP secretion, cleansed platelets’ surfaces were prepared with the various concentrations of SA (50, 100, and 200 µg/mL) for 10 min at 37 °C. After the addition of the luciferin/luciferase reagent, platelets were activated with various agonists, then ATP secretion was assessed with a luminometer. PRP was preincubated with either 0.01% DMSO, or one of three concentrations of SA (50, 100, or 200 µg/mL) for 10 min at 37 °C. They were then stimulated with 1 µg/mL collagen (F) or 0.5 µg/mL CRP (G). The data are shown as means ± standard deviations (n = 4). *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

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  Figure 2. The preventive effect of Sinomenium acutum (SA) on P-selectin exposure, αIIbβ3 integrin stimulation, and Ca2+ mobilization. The binding of anti-P-selectin (A) and anti-activated αIIbβ3 (JON/A) antibodies (B) to platelets was measured as the ratio of the geometric mean fluorescence intensity value of antibodies to that of the control immunoglobulin G. The quantitative data are presented as means ± standard deviations (n = 3). In a Ca2+ mobilization assay, mouse platelets were incubated with a calcium-sensitive dye for 30 min at 37 °C in the dark, and then platelets were induced using 0.5 µg/mL collagen-related peptide (CRP) for 10 min. Intracellular Ca2+ secretion and flow (C) were evaluated and quantified with the area under the curve (arbitrary units). The quantitative data are demonstrated as means ± standard deviations (n = 4). *, p < 0.05; **, p < 0.01; and ***, p < 0.001.

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  Figure 3. Sinomenium acutum (SA) extract regulates the phosphorylation of spleen tyrosine kinase (Syk), phospholipase Cγ2 (PLCγ2), phosphatidylinositol 3-kinase (PI3K), AKT, and ERK after collagen-related peptide (CRP) stimulation. Mouse platelets were pretreated with various concentrations of SA (50, 100, and 200 µg/mL) and stimulated with 0.5 µg/mL CRP. Equal amounts (30 µg) of cell lysate protein were immunoblotted to determine specific Syk, PLCγ2, PI3K, AKT, and ERK phosphorylation inhibition. The results are shown as blots and graphs. The data are presented as means ± standard deviations (n = 3). #, p < 0.05 and ###, p < 0.001 versus the vehicle control (unstimulated) after Student’s t-test. *, p < 0.05; **, p < 0.01; and ***, p < 0.001 versus the vehicle control (stimulated) after analysis of variance and Tukey’s test.

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  Figure 4. Sinomenium acutum (SA) delays FeCl3-induced arterial thrombus formation without causing bleeding. FeCl3-activated arterial thrombus formation was carried out, as shown in Methods. After oral administration of 0.5% low-viscosity carboxymethylcellulose (CMC) and/or SA (50 and 100 mg/kg, body weight) or acetylsalicylic acid (ASA; 100 mg/kg, body weight) once a day for seven days (A,B), 10% FeCl3 was injected into the mouse carotid artery for two minutes, and blood flow traces were controlled until stable occlusion was achieved. In the bleeding time assay, the tails of mice were removed, and the bleeding time (C) was monitored, as demonstrated in Methods. (D) Blood loss during the bleeding time assay was measured by assessing the absorbance at 575 nm for hemoglobin (Hb). The horizontal bars illustrate the median occlusion time (n = 10). #, p < 0.05, ##, p < 0.01 and ###, p < 0.001 versus the vehicle control after Student’s t-test.

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  Figure 5. Ultra-high-performance liquid chromatography–tandem mass spectrometry examination of Sinomenium acutum. (A) Base peak chromatogram of Sinomenium acutum in positive and negative ion mode. (B) Elicit ion chromatogram of Sinomenium acutum. 1, higenamine; 2, acutumidine; 3, acutumine; 4, sinomenine; 5, gelsemine; 6, magnoflorine; 7, scopoletin; 8, columbamine or jatrorrhizine; 9, palmatine; 10, syringin; 11, eleutheroside E.