Evaluation of novel factor Xa inhibitors from Oxya chinensis sinuosa with anti-platelet aggregation activity

Abstract

The edible grasshopper Oxya chinensis sinuosa is consumed worldwide for its various medicinal effects. The purpose of this study was to investigate potential bioactive antithrombotic and antiplatelet compounds from O. chinensis sinuosa. Five N-acetyldopamine dimers (1-5) were isolated from O. chinensis sinuosa and compounds 1 and 2 were identified as new chemicals with chiral centers at H-2 and H-3 of the benzo-1,4-dioxane structure. Compounds 1-4 were found to have both FXa and platelet aggregation inhibitory activities. These compounds inhibited the catalytic activity of FXa toward its synthetic substrate, S-2222, by noncompetitive inhibition, and inhibited platelet aggregation induced by ADP and U46619. Furthermore, compounds 1-4 showed enhanced antithrombotic effects, which were assessed using in vivo models of pulmonary embolism and arterial thrombosis. The isolated compounds also showed anticoagulant effects in mice. However, compounds 1-4 did not prolong bleeding time in mice, as shown by tail clipping. N-Acetyldopamine dimers, including two new stereoisomers 1 and 2, are novel antithrombotic compounds showing both FXa inhibition and antiplatelet aggregation activity with a low bleeding risk. Collectively, these results suggest that compounds 1-4 could serve as candidates and provide scaffolds for development of new antithrombotic drugs.

Figure 1

Structure of active compounds 1–5 isolated from O. chinensis sinuosa.

Figure 2

Key HMBC () correlations of compounds 1 and 2.

Figure 3

The CD spectra of compounds 1 and 2.

Figure 4

Effects of compounds 1–4 on clotting and bleeding time. (A) One hour after administration (intravenous injection) of compound 1 (white box), 2 (light gray box), 3 (dark gray box), or 4 (black box), blood was collected from the mice and platelet-poor plasma (PPP) was obtained by centrifugation at 2,000 × g for 10 min at room temperature to test ex vivo activated partial thromboplastin time (aPTT). (B) Each group received a daily intravenous injection of the indicated compound for four consecutive days. Fifteen minutes after the last administration, blood samples were collected and in vivo aPTT was measured. (C) Fifteen minutes after administration of each compound or aspirin (4.5 or 9 mg/kg for 30 min) or the vehicle, tail tips (3 mm long) were cut from each mouse, and the remaining tail was immediately immersed into saline at 37 °C. Accumulated bleeding times (including periods of re-bleeding) were recorded. D = 0.2% DMSO used as the vehicle control. Data are presented as means ± SD of three independent experiments. *p < 0.05 vs. vehicle alone, analyzed by one-way ANOVA, followed by Tukey multiple comparison testing.

Figure 5

Effects of compounds 1–4 on agonist-induced platelet aggregation in vitro. Platelet-rich plasma (PRP) was preincubated for 5 min with different concentrations of compounds 1–4, or vehicle. Platelet aggregation was initiated with ADP (A, 10 µM), U46619 (B, 6 µM), or thrombin (C, 3 U/mL). D = 0.2% DMSO used as the vehicle control. Data are presented as means ± SD of three independent experiments. ^* p < 0.05 vs. vehicle alone, analyzed by one-way ANOVA, followed by Tukey multiple comparison testing.

Figure 6

Effects of compounds 1–4 on inhibition and production of FXa. (A–D) Michaelis-Menten and Lineweaver-Burk plots (inset) for compounds 1 (A), 2 (B), 3 (C), and 4 (D) in PBS at pH 8.34 and 37 °C, demonstrating a noncompetitive inhibition of each compound. The plots represent means of three independent measurements. (E) Human umbilical vein endothelial cells (HUVECs) were pre-incubated with the indicated concentration of each compound for 10 min. HUVECs stimulated with tumor necrosis factor-α (TNF-α, 10 ng/mL for 6 h) were incubated with FVIIa (10 nM) and FX (175 nM) in the absence or presence of anti-TF IgG (25 µg/mL). FXa production was determined as described in the “ Supplementary Materials and Methods” section. D = 0.2% DMSO used as the vehicle control. (F) Inhibition of FXa by each compound (5 μM) with or without TFPI (2 nM) was monitored using a chromogenic assay. ^* p < 0.05 vs. TNF-α alone (E) or each compound (F) alone, analyzed via one-way ANOVA, followed by Tukey multiple comparison testing.

Figure 7

Effects of compounds 1–4 on arterial and pulmonary thrombosis. (A) Time to large thrombi formation by compounds 1–4. Tirofiban (Tiro, 0.2 or 0.4 mg/kg) was used as a positive control. (B) Thrombi were scored for size 60 min after FeCl₃-treatment as described in “Supplementary Materials and Methods.” (C) After each compound was injected intravenously, a mixture of collagen (C, 500 µg/kg) and epinephrine (E, 50 µg/kg) was injected into the tail vein of mice to induce acute thrombosis 6 h later. Afterward, mice (20 mice per group) were carefully examined for 15 min to determine whether the mouse was paralyzed, dead, or had recovered from the acute thrombosis challenge. D = 0.2% DMSO used as the vehicle control. *p < 0.05 vs. DMSO, analyzed via one-way ANOVA, followed by Tukey multiple comparison testing.