Sanguinarine Attenuates Collagen-Induced Platelet Activation and Thrombus Formation

Figures

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  Figure 1. Chemical structure of sanguinarine.

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  Figure 2. Sanguinarine inhibits the formation of thrombus in vivo and in vitro without prolonging bleeding time. (a) Summary data of ferric chloride (FeCl3)-induced thrombus formation. Thrombus formation in vivo was assessed by occlusion time(s) after FeCl3 -induced injury of the carotid artery, which was measured with a doppler flow probe. The occlusion time of the sanguinarine-treated (5 mg/kg or 10 mg/kg, i.g.) were compared with the control (saline + 5% DMSO i.g.) group. The results were expressed as mean ± SEM (n = 7) and were analyzed using one-way ANOVA. (b) The effect of sanguinarine on bleeding time. Results are shown as mean ± SEM (n = 15), and data were analyzed using an unpaired t test. (c) Sanguinarine reduced thrombosis in vitro. Washed human platelets were labeled with CellTrace Calcein Green and then incubated with DMSO (control) or sanguinarine (1, 2.5 and 5 μM) for 10 min. The mixture of labeled platelets and washed red blood cells (with a hematocrit of about 40%) and CaCl2/MgCl2 (75 mM/37.5 mM) was perfused through Bioflux plates at a wall shear rate of 2000 s−1 for 5 min. Representative images of surface coverage were revealed. (d) Summary data of area covered by…

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  Figure 3. Effects of sanguinarine on collagen and collagen-related peptide-induced human platelet aggregation of washed human platelets. (a) Washed human platelets (3 × 108/mL) were preincubated for 10 min with different concentrations of sanguinarine (1, 2.5, 5 µM) or DMSO (control), followed by stimulation with different agonists. (b) Data were shown as mean ± SEM (n = 5) and examined using one-way ANOVA analysis.

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  Figure 4. Sanguinarine decreases collagen-induced human platelet granule release and integrin αIIbβ3 activation. (a) Washed human platelets were prepared as shown in Figure 3. ATP secretion was documented and computed following the instructions in the Materials and Methods Section. (b) Data were shown as mean ± SEM (n = 5) and examined using one-way ANOVA analysis. (c–f) First, washed human platelets (5 × 107/mL) were preincubated for 10 min at 37 °C with sanguinarine (1, 2.5, 5 µM) or DMSO (control) in the presence of FITC-conjugated P-selectin and FITC-conjugated PAC-1 antibodies. Afterward, the platelets were stimulated with collagen (2 µg/mL) and incubated for another 5 min. P-selectin expression (c,d) and PAC-1 binding (e,f) were identified by flow cytometry. Results and data were expressed as mean ± SEM (n = 5).

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  Figure 5. Sanguinarine has an inhibitory effect on collagen-induced phosphorylation of the signaling cascade. (a,c) Washed human platelets were treated initially with sanguinarine (1, 2.5, 5 µM) or DMSO (control) at 37 °C for 10 min, then stimulated with collagen (1 µg/mL) for 5 min, and later underwent lysis with lysis buffer, followed by Western blotting using specific antibodies. (b,d) The Syk/PLCγ2 and PI3K/Akt/GSK3β phosphorylation ratios were measured. The blot was a representative of three independent experiments that showed statistical significance at * p < 0.05 and ** p < 0.01 compared with the control group (n = 5).

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  Figure 6. Sanguinarine dose-dependently attenuates intracellular Ca2+ concentration ([Ca2+]i) in human platelets. (a) Intracellular Ca2+ flux was measured by flow cytometry. Washed human platelets (5 × 107/mL) were labeled with 1 μM Fluo-3AM at 37 °C for 30 min, then incubated with sanguinarine (1, 2.5 and 5 μM) or DMSO (control) for 10 min. After collecting a baseline for 20 s, collagen (2 µg/mL) and 2 mM extracellular calcium were added to the FACS tube, and the change in fluorescence intensity was recorded on BD Biosciences flow cytometer and analyzed using Flowjo software. The fitting curve was plotted using Sigma Plot software. (b) Values of [Ca2+]i elevation upon collagen stimulation shown in (a) were plotted as mean ± SEM from five independent experiments.

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  Figure 7. Sanguinarine dose-dependently represses human platelet spreading on the fibrinogen-coated surface and inhibits integrin αIIbβ3-mediated outside-in signaling. (a) A representative image of platelets spreading on the coated BSA or fibrinogen cover glasses. (b) The figure expressed the quantity of the single adherent platelet area as shown in the right panel, and the average coverage of the spreading platelets in five independent experiments. (c,d) Typical figure of phosphorylation of β3/Src of lysed spreading platelets for five independent experiments. The data had statistical significance at * p < 0.05 when compared to the control group.

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  Figure 8. Schematic of sanguinarine antiplatelet activity. Sanguinarine attenuates collagen-induced platelet activation (including α and dense granule release) and thrombus formation by targeting the GPVI pathway (inhibiting the phosphorylation of Syk-PLCγ2 and PI3K-Akt-GSK3β), intracellular Ca2+ mobilization and integrin αIIbβ3-mediated “outside-in” signal transduction (inhibiting the phosphorylation of src). (Inhibit ; Induce )