PMC

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.

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).

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.

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).