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. 2017 Jun;21(6):1217-1227.
doi: 10.1111/jcmm.13055. Epub 2016 Dec 20.

Inhibition of Platelet Aggregation and Thrombosis by Indole Alkaloids Isolated From the Edible Insect Protaetia Brevitarsis Seulensis (Kolbe)

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Inhibition of Platelet Aggregation and Thrombosis by Indole Alkaloids Isolated From the Edible Insect Protaetia Brevitarsis Seulensis (Kolbe)

JungIn Lee et al. J Cell Mol Med. .
Free PMC article

Abstract

Protaetia brevitarsis seulensis (Kolbe) has been temporarily registered as a food material by the Ministry of Food and Drug Safety of Korea (MFDS). The current study aimed to discover small antithrombotic molecules from this edible insect. Five indole alkaloids, 5-hydroxyindolin-2-one (1), (1R,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (2), (1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (3), (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (4) and L-tryptophan (5), were isolated from the insect. Among them, compounds 1 and 2 prolonged aPTT and PT and impaired thrombin and FXa generation on HUVEC surface. Moreover, these compounds inhibited platelet aggregation. Antithrombotic effects of compounds 1 and 2 were further confirmed in pre-clinical models of pulmonary embolism and arterial thrombosis. Collectively, these results demonstrated that compounds 1 and 2 could be effective antithrombotic agents and serve as new scaffolds for the development of antithrombotic drug.

Keywords: Protaetia brevitarsis seulensis (Kolbe); coagulation cascade; fibrinolysis; indole alkaloids.

Figures

Figure 1
Figure 1
Effects of compounds 1 and 2 on platelet aggregation. (A, B) The effect of each compound on human platelet aggregation induced by 2 μM U46619 (A) or 1 μg/ml collagen (B). (C) Each compound in DMSO was injected intravenously at the indicated concentration. The effects of each compound on mouse platelet aggregation induced by 2 μM U46619 (U) were monitored ex vivo. D = 0.2% DMSO used as the vehicle control. *P < 0.05 versus U46619 (A, C) or collagen (B) alone.
Figure 2
Figure 2
Effects of compounds 1 and 2 on PKC activation and intracellular calcium mobilization. (A) Platelet‐rich plasma (PRP) was incubated with DMSO or each compound (50 μM) at 37°C for 10 min. and was stimulated with U46619 (2 μM, left) or thrombin (0.05 U/ml, right) for another 1 min. Phospho‐MARCKS in the platelet lysates was detected using Western blotting (cropped images from full‐length gels). (B, C) Fura‐2‐loaded human platelets were incubated with DMSO (D), compound 1 (white box) and compound 2 (black box) at 37°C for 10 min. in the presence of 1 mM extracellular Ca2+, followed by the addition of U46619 (B, 2 μM) or thrombin (C, 0.05 U/ml) to trigger the increase in [Ca2+]i. D = 0.2% DMSO used as the vehicle control. The data represent the means ± S.E.M. of three independent experiments performed in triplicate. *P < 0.05 versus U46619 (B) or Th (C) alone.
Figure 3
Figure 3
Effects of compounds 1 and 2 on the inactivation and production of thrombin and factor Xa. (A) Inhibition of thrombin (Th) by compound 1 (white box) and compound 2 (black box) was measured using a chromogenic assay, as described in the ‘Materials and methods’ section. (B) The inhibition of factor Xa (FXa) by each compound was also monitored using a chromogenic assay, as described in the ‘Materials and methods’ section. Argatroban (A) or rivaroxaban (B) was used as a positive control. (C) The HUVEC monolayer was pre‐incubated with FVa (100 pM) and FXa (1 nM) for 10 min. and then with the indicated concentrations of each compound. Prothrombin was added at a final concentration of 1 μM and prothrombin activation was determined after 30 min., as described in the ‘Materials and methods’ section. (D) HUVECs were pre‐incubated with the indicated concentrations of each compound for 10 min. TNF‐α (10 ng/ml for 6 hrs)‐stimulated HUVECs were incubated with FVIIa (10 nM) and FX (175 nM) in the absence or presence of anti‐TF IgG (25 μg/ml); the FXa production was determined as described in the ‘Materials and methods’ section. D = 0.2% DMSO used as the vehicle control. *P < 0.05 versus 0.0001 μM each compound, or argatroban (A) or 0.0001 μM each compound, or 0.0001 nM rivaroxaban (B), DMSO (C) or TNF‐α alone (D).
Figure 4
Figure 4
Effects of compounds 1 and 2 on the secretion of PAI‐1 and t‐PA. (A) HUVECs were cultured with compound 1 (white box) and compound 2 (black box) in the absence or presence of TNF‐α (10 ng/ml) for 18 hrs and the PAI‐1 concentrations in the culture media were determined as described in the ‘Materials and methods’ section. (B) HUVECs were cultured with each compound in the absence or presence of TNF‐α (10 ng/ml) for 18 hrs and the t‐PA concentrations in the culture media were determined as described in the ‘Materials and methods’ section. (C) The PAI‐1/t‐PA ratio in TNF‐α activated HUVECs from (A) and (B). D = 0.2% DMSO used as the vehicle control. *P < 0.05 versus TNF‐α or D alone; n.s., not significant.
Figure 5
Figure 5
Effects of compounds 1 and 2 on arterial thrombosis and on acute thrombosis. (A) Time to large thrombus formation by compound 1 (white box) and compound 2 (black box). Tirofiban (Tiro) was used as a positive control. (B) The size score of the thrombus at 60 min. after FeCl3 treatment as described in ‘Materials and methods’. (C) After each compound was injected intravenously, a mixture of collagen (C, 500 μg/kg) plus epinephrine (E, 50 μg/kg) was injected into the tail vein of mice to induce acute thrombosis 6 hrs later. Then, mice (20 mice per group) were carefully examined for 15 min. to determine whether the mouse was paralysed, dead or recovered from the acute thrombotic challenge. D = 0.2% DMSO used as the vehicle control. *P < 0.05 versus DMSO.

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