Pimpinellin Inhibits Collagen-induced Platelet Aggregation and Activation Through Inhibiting Granule Secretion and PI3K/Akt Pathway

Figures

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  FIGURE 1. Effects of pimpinellin on platelet aggregation and LDH release. (A) The washed human platelets (3 × 108/ml) were incubated with different concentrations of pimpinellin (10 and 20 μM) or the vehicle group for 5 min. Stimulated platelet aggregation with collagen (1 and 2 μg/ml), Thrombin (0.04 and 0.08 U/ml), ADP (3 and 5 μM). (B) Fixed and observed untreated platelets or platelets treated with vehicle or pimpinellin (10 and 20 μM) and collagen 2μg/mL. Values are the average of three independent experiments ± S.E.M. (C) To assess the cytotoxicity, platelets were pre-incubated with 0.1% DMSO (vehicle), or pimpinellin (10 and 20 μM) for 10 min, and 20 μl of the supernatant from the aliquots was precipitated on an LDH-assay kit. Data are presented as the mean ± SEM (n = 9). One-way ANOVA followed by Bonferroni post-hoc analysis of the data. (**p < 0.01, ***p < 0.001 compared with the vehicle group, #p < 0.05 compared with the PIN 10 μM group, NS means no significance.).

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  FIGURE 2. Pimpinellin inhibited platelet ATP release, P-selectin expression and PAC-1. (A) Washed platelets were treated with pimpinellin (10 and 20 μM) at 37°C for 5 min and ATP release was measured after stimulation with collagen (1 μg/ml) using a Lumi-Aggregometer. One-way ANOVA followed by Bonferroni post-hoc analysis of the data. (*p < 0.05, **p < 0.01; compared to the vehicle group). (B,C) Washed platelets (3.0 × 108/ml) were incubated with vehicle or pimpinellin (20 μM), Then CRP (1 μg/ml) was added to stimulate the expression of P-selection (B) and PAC-1 (C). (resting control, black line; CRP-activated, blue line; pimpinellin 20 μM, red line; Tirofiban, brown line). Detection of P-selection, PAC-1 expression by flow cytometry (n = 3). Bar graphs show mean ± SEM (n = 3). *p < 0.05 and **p < 0.01 compared to the vehicle group.

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  FIGURE 3. The effect of pimpinellin on clot retraction and platelet spreading on fibrinogen surfaces. (A) Human platelet-rich plasma were treated with pimpinellin (10, 20 and 40 μM) at 37°C for 5 min. The clot retraction was triggered by adding thrombin (0.4 U/ml) and proceeding at 37°C. The clot retraction was photographed after 30 min. (B) Human washed platelets (1.0 × 107/ml) were pretreated with pimpinellin (10 and 20 μM) or vehicle at 37°C for 5 min, and then allowed to adhere to the fibrinogen-coated glass slide. Platelets were spread on a slide coated with fibrinogen at 37°C for 60 min, after which spreading was stopped by fixing in 4% paraformaldehyde. The platelet images were obtained by fluorescent microscopy. Representative images were obtained from three similar experiments. Data are presented as the mean ± SEM (n = 3). *p < 0.05 and **p < 0.01 compared to the vehicle group.

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  FIGURE 4. Effects of pimpinellin on cAMP levels, and TxB2 production. (A) We pre-incubated washed human platelets (3 × 108/ml) with vehicle, different concentrations of pimpinellin, or aspirin (100 μM) for 10 min, after which the platelets were stimulated with collagen (1 μg/ml). TxB2 production levels were evaluated by measuring the OD values. Results were expressed as TxB2 production (ng/ml). (B) Washed human platelets (3 × 108/ml) were pre-incubated with the vehicle, different concentrations of pimpinellin or PGE1 (50 ng/ml) for 10 min, after which they were stimulated with collagen. After 5 min, the supernatant was obtained by centrifugation. The effect of pimpinellin on cAMP levels was determined by detecting OD value. Data are mean ± SEM values (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 compared to the vehicle group.

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  FIGURE 5. Effect of pimpinellin on collagen-induced signal transduction. Washed platelets were stimulated with 2 μg/ml collagen (3.0 × 108/ml) and then lysed with cell lysis buffer. Syk, PLCγ2, SLP76, JNK, ERK, P-38, p85, Akt, GSK3β, and PKC phosphorylation levels were detected with relevant antibodies. Band density was analyzed using the ImageJ software. Data are presented as the mean ± SEM (n = 3). *p < 0.05 and **p < 0.01 compared to the vehicle group; NS means no significance.

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  FIGURE 6. Effects of pimpinellin on platelet aggregation in the presence of ADP or LY294002. (A) Washed platelets (3 × 108/ml) were pretreated with 20 μM pimpinellin, 1 μM ADP or both for 10 min after which they were stimulated with collagen (1 μg/ml). Platelet aggregation rates were calculated in at least three independent experiments and the results are shown as mean ± SEM (*p < 0.05 compared with the PIN 20+Col group, PIN 20 means pimpinellin 20 μM, COL means collagen). (B) Lysates from each group were analyzed by immunoblotting to determine the Akt phosphorylation levels. (C) Washed platelets (3 × 108/ml) were pre-incubated with 10 μM pimpinellin, 2 μM LY294002 or both for 10 min, after which they were stimulated with 2 μg/ml collagen. (*p < 0.05 and **p < 0.01 compared to the vehicle group, PIN 10 means pimpinellin 10 μM). (D) Lysates in each group were analyzed by immunoblotting to determine the Akt phosphorylation levels. Band density was calculated by the ImageJ software. Bar graphs represent the mean ± SEM (n = 3).

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  FIGURE 7. The effects of pimpinellin on coagulation in mice. (A,B) The effect of pimpinellin on the coagulation system was assessed by the APTT and PT. (*p < 0.05, **p < 0.01, NS means p > 0.05). (C) Representative experiments of platelet aggregation induced by collagen (2 μg/ml) in control mice and mice treated with pimpinellin or aspirin. Pimpinellin produced a inhibition of collagen-induced ex vivo aggregation in platelets. Statistics of aggregation rates (n = 3). Data are presented as mean ± SEM. *p < 0.05 compared to the vehicle group. (D) The tail tip was excised and soaked at 37°C for the bleeding test. Symbols represent the time to stop bleeding in individual animals and error bars represent the mean ± SEM (n = 10).