Esculetin, a Coumarin Derivative, Prevents Thrombosis: Inhibitory Signaling on PLCγ2–PKC–AKT Activation in Human Platelets

Figures

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  Figure 1. Esculetin inhibits agonists-induced platelet aggregation in washed human platelets. (A) Chemical structure of esculetin (C9H6O4). (B) Washed human platelets (3.6 × 108 cells/mL) were preincubated with the solvent control (0.1% DMSO) or esculetin (10–100 μM) and subsequently treated with 1 μg/mL collagen, 0.01 U/mL thrombin, 1 μM U46619, and 60 μM AA to stimulate platelet aggregation. The aggregation curves in human platelets were monitored using lumi-aggregometer (Payton Associates, Scarborough, ON, Canada). ΔT/min = change in light transmission per min. (C) Concentration–response histograms of esculetin demonstrating its inhibitory activity for platelet aggregation (%). All data are presented as mean ± standard error of the mean (n = 4). * p < 0.05 and *** p < 0.001 vs. DMSO-treated group.

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  Figure 2. Effect of esculetin on surface P-selectin expression, ATP release, cytotoxicity, and LDH release in human platelets. Washed platelets (3.6 × 108 cells/mL) were preincubated with 0.1% DMSO, esculetin (50 and 80 µM), or FITC–P-selectin (2 µg/mL); collagen (1 μg/mL) was then added to trigger either (A) surface P-selectin expression or (B) ATP release (AU: arbitrary unit). The right panel of each figure shows the respective statistical data in (A,B). For other experiments, (C) washed platelets were pretreated with 0.1% DMSO or esculetin (100 μM) for 10 min and subsequently double washed by Tyrode’s solution; collagen (1 μg/mL) was added to activate platelet aggregation. ΔT/min = change in light transmission per min. (D) Washed platelets were preincubated with 0.1% DMSO or esculetin (50, 80, and 100 µM) for 20 min, and a 10-µL suspension of the supernatant was deposited on a Fuji Dri-Chem slide (LDH-PIII). Data are presented as mean ± standard error of the mean (n = 4). Profiles in (C) are representative of four independent experiments. *** p < 0.001 vs. resting control; # p < 0.05 and ## p < 0.01 vs. DMSO-treated group.

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  Figure 3. Effects of esculetin on Akt, PLCγ2, and PKC activation in human platelets. Washed platelets (1.2 × 109 cells/mL) were preincubated with 0.1% DMSO or esculetin (50 and 80 µM) and subsequently added collagen (1 μg/mL) to induce (A) Akt phosphorylation, (B) PLCγ2, and (C) PKC activation (p47 [pleckstrin] phosphorylation). Platelets were prepared and their suspension were analyzed to determine levels of protein phosphorylation. Data are presented as mean ± standard error of the mean (n = 4). *** p < 0.001 vs. the resting control; # p < 0.05, ## p < 0.01, and ### p < 0.001 vs. DMSO-treated group.

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  Figure 4. Effects of esculetin on ERK1/2, p38 MAPK, and JNK1/2 phosphorylation in collagen-activated human platelets. Washed platelets (1.2 × 109 cells/mL) were preincubated with 50 or 80 μM esculetin or 0.1% DMSO and subsequently treated with 1 μg/mL collagen to induce platelet activation. Platelets were collected and subcellular extracts were analyzed to determine the phosphorylation of (A) ERK1/2 (B) p38 MAPK, and (C) JNK1/2. Data are expressed as mean ± standard error of the mean (n = 4). ** p < 0.01 and *** p < 0.001 vs. the resting control.

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  Figure 5. Esculetin inhibits OH· formation in platelet suspensions and the closure time in human whole blood, which are estimated by the respective electron spin resonance (ESR) and PFA-100 analyses. (A) Washed human platelets was incubated with (a) Tyrode’s solution only (resting control); or with (b) 0.1% DMSO, esculetin at (c) 50 µM or (d) 80 µM, collagen (1 µg/mL) was then added for the ESR experiments, as described in the Section 4 (n = 4). Asterisk (*) indicates OH· formation. (B) Platelet plug formation induced by shear stress in whole blood was determined via recording the closure time in collagen-ADP (C-ADP)- and collagen-EPI (C-EPI)-coated membranes, as described in the Section 4. Data are presented as mean ± standard error of the mean (n = 6); p < 0.05 vs. DMSO-treated group.

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  Figure 6. The in vivo activities of esculetin in thrombotic platelet plug formation in mesenteric venules, tail bleeding time, and acute pulmonary thromboembolism, in experimental mice. (A) Mice were intravenously administered 0.1% DMSO or esculetin (2.5 or 5.0 μg/kg) (all in 50 μL), and the mesenteric venules were irradiated to induce microthrombus formation (occlusion time), as described in the Section 4. Microscopic images (×400 magnification) of DMSO-treated controls and esculetin (2.5 and 5.0 μg/kg)-treated groups were recorded at 5 and 150 s after irradiation, and the platelet plug formation represents by arrows. (B) The bleeding time was measured through the transection of mice tails after 30 min of intraperitoneal administration of either 0.1% DMSO or 2.5/5.0 μg/kg esculetin. (C) For the study of acute pulmonary thrombosis, 0.1% DMSO or esculetin at various doses (2.5 and 5.0 μg/kg) was administered intraperitoneally to mice, and ADP (0.7 mg/g) was then injected through the tail veins. Data are presented as mean ± standard error of the mean (n = 8); *** p < 0.001 vs. 0.1% DMSO-treated group.