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. 2024 Feb 8;25(4):2098.
doi: 10.3390/ijms25042098.

Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models

Wei-Chieh Huang  1 , Lan-Hsin Shu  2 , Yu-Ju Kuo  1 , Kevin Shu-Leung Lai  3 , Chih-Wei Hsia  4 , Ting-Lin Yen  5 , Chih-Hsuan Hsia  6 , Thanasekaran Jayakumar  7 , Chih-Hao Yang  8 , Joen-Rong Sheu  1   8
Affiliations

Eugenol Suppresses Platelet Activation and Mitigates Pulmonary Thromboembolism in Humans and Murine Models

Wei-Chieh Huang et al. Int J Mol Sci. .

Abstract

Platelets assume a pivotal role in the pathogenesis of cardiovascular diseases (CVDs), emphasizing their significance in disease progression. Consequently, addressing CVDs necessitates a targeted approach focused on mitigating platelet activation. Eugenol, predominantly derived from clove oil, is recognized for its antibacterial, anticancer, and anti-inflammatory properties, rendering it a valuable medicinal agent. This investigation delves into the intricate mechanisms through which eugenol influences human platelets. At a low concentration of 2 μM, eugenol demonstrates inhibition of collagen and arachidonic acid (AA)-induced platelet aggregation. Notably, thrombin and U46619 remain unaffected by eugenol. Its modulatory effects extend to ATP release, P-selectin expression, and intracellular calcium levels ([Ca2+]i). Eugenol significantly inhibits various signaling cascades, including phospholipase Cγ2 (PLCγ2)/protein kinase C (PKC), phosphoinositide 3-kinase/Akt/glycogen synthase kinase-3β, mitogen-activated protein kinases, and cytosolic phospholipase A2 (cPLA2)/thromboxane A2 (TxA2) formation induced by collagen. Eugenol selectively inhibited cPLA2/TxA2 phosphorylation induced by AA, not affecting p38 MAPK. In ADP-treated mice, eugenol reduced occluded lung vessels by platelet thrombi without extending bleeding time. In conclusion, eugenol exerts a potent inhibitory effect on platelet activation, achieved through the inhibition of the PLCγ2-PKC and cPLA2-TxA2 cascade, consequently suppressing platelet aggregation. These findings underscore the potential therapeutic applications of eugenol in CVDs.

Keywords: MAPK; PI3K/Akt/GSK-3β; PLCγ2–PKC; cPLA2/TxA2; eugenol; human platelets; pulmonary thrombosis.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
(A) The molecular structure of eugenol, with the molecular formula C10H12O2, is illustrated. Washed human platelets (3.6 × 108 cells/mL) were preincubated with either a solvent control (0.1% DMSO) or varying concentrations of eugenol (0.5 to 100 μM). Subsequently, platelets were exposed to different agonists, including (B) collagen (1 μg/mL), (C) arachidonic acid (AA; 60 μM), (D) thrombin (0.02 U/mL), or (E) U46619 (1 μM), to induce platelet aggregation. Concentration-response histograms for eugenol highlight its inhibitory effects on platelet aggregation triggered by various agonists (%). (F) Cytotoxicity assessment involved preincubating platelets with 0.1% DMSO, 10 μM, or 20 μM eugenol for 10 min, followed by two washes with Tyrode’s solution. Subsequently, platelets were stimulated with collagen (1 μg/mL). Statistical significance levels of * p < 0.05 and *** p < 0.001 indicate differences compared to the 0.1% DMSO-treated group. The presented data in (B–F) represent the mean ± standard error of the mean (n = 4).
Figure 2
Figure 2
Eugenol’s inhibitory effects on ATP release, relative [Ca2+]i level, and surface P-selectin expression in human platelets were investigated. Washed platelets (3.6 × 108 cells/mL) were preincubated with either 0.1% DMSO or eugenol (2 and 4 µM), followed by collagen (1 μg/mL) stimulation to elicit the following responses: (A) ATP release, quantified in arbitrary units (AU); (B) relative [Ca2+]i level; and (C) surface P-selectin expression (a, Tyrode’s solution; b, 0.1% DMSO + collagen group; c, 2 µM eugenol + collagen group; d, 4 µM eugenol + collagen group). Detailed experimental methodologies are provided in Section 4. Statistical significance in (A,B) is denoted by * p < 0.05 and *** p < 0.001 compared to the 0.1% DMSO-treated group. In (C), *** p < 0.001 indicates deviations from the resting control (Tyrode’s solution), while ### p < 0.001 signifies differences compared to the 0.1% DMSO-treated group. The data are presented as the mean ± standard error of the mean (n = 4).
Figure 3
Figure 3
The impact of eugenol on the activation of cytosolic phospholipase A2 (cPLA2), phospholipase Cγ2 (PLCγ2), and protein kinase C (PKC) in platelets was investigated. Washed platelets were preincubated with either 0.1% DMSO or eugenol (2 and 4 µM) and subsequently stimulated with collagen (1 µg/mL) to induce the following responses: phosphorylation of (A) cPLA2, (B) PLCγ2, and (C) activation of PKC, as indicated by p-p47 phosphorylation. Data are presented as the mean ± standard error of the mean (n = 4). Significant differences are denoted by *** p < 0.001 in comparison to resting platelets exposed to Tyrode’s solution. Furthermore, ### p < 0.001 is used to indicate disparities compared to the group treated with 0.1% DMSO.
Figure 4
Figure 4
Eugenol’s inhibitory effects on the activation of cytosolic phospholipase A2 (cPLA2) and phospholipase Cγ2 (PLCγ2) were visualized using confocal laser microscopy. Washed platelets were pre-incubated with either 0.1% DMSO or eugenol (4 µM) and subsequently exposed to collagen (1 μg/mL) for confocal microscopic evaluation at 1000× magnification. This assessment specifically focused on the visualization of phosphorylated (A) cPLA2 and (B) PLCγ2, represented by green fluorescence, along with α-tubulin indicated by red fluorescence. The presented images are representative of four independent experiments. The red boxes serve to highlight one of the numerous cells that have been phosphorylated and are further magnified within white boxes. The scale bar is 10 μm.
Figure 5
Figure 5
Illustrates the regulatory impact of eugenol on the phosphoinositide 3-kinase (PI3K)/Akt/glycogen synthase kinase-3β (GSK3β) and mitogen-activated protein kinases (MAPKs) pathways. Platelets were preincubated with either 0.1% DMSO or eugenol (2 and 4 µM) and subsequently exposed to collagen (1 μg/mL). This allowed for immunoblotting analysis of key components within the (A) PI3K, (B) Akt, (C) GSK3β, (D) p38 MAPK, (E) ERK, and (F) JNK pathways. The data are presented as the mean ± standard error of the mean (n = 4). Statistical significance is denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the results observed in resting platelets (Tyrode’s solution); and # p < 0.05, ## p < 0.01, and ### p < 0.001 compared with the results observed in the 0.1% DMSO group.
Figure 6
Figure 6
The study investigated the effects of eugenol on cytosolic phospholipase A2 (cPLA2) and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, as well as thromboxane B2 (TxB2) formation in human platelets. Platelets were preincubated with either 0.1% DMSO, eugenol (4 µM) or with SB203580 (20 µM) and subsequently exposed to arachidonic acid (AA; 60 µM). Immunoblotting analysis was conducted to assess the levels of (A) cPLA2 and (B) p38 MAPK proteins. (C) In another set of experiments, platelets were preincubated with Tyrode’s solution alone, or with either 0.1% DMSO or eugenol (4 µM), followed by exposure to collagen (1 μg/mL) and AA (60 µM) to quantify TxB2 formation. The data are presented as the mean ± standard error of the mean (n = 4). Statistical significance is indicated as * p < 0.05 and *** p < 0.001 compared to the results observed in resting platelets (Tyrode’s solution); # p < 0.05, ## p < 0.01 and ### p < 0.001 compared to the results observed in the 0.1% DMSO group.
Figure 7
Figure 7
The study evaluated the efficacy of eugenol in mitigating thromboembolism in the lungs of mice. (A) Acute pulmonary thrombosis was induced by intraperitoneally administering either 0.1% DMSO, eugenol (15 mg/kg), or aspirin (15 mg/kg) to mice, followed by the injection of ADP (700 mg/kg) into the tail vein. Histological examination of lung tissue sections stained with hematoxylin–eosin focused on alveoli (stars), blood vessels (arrows), and bronchioles (arrowheads), with a scale bar indicating 200 μm. The mortality rate (%) of ADP-induced pulmonary thromboembolism in mice (n= 12) was presented in (B). (C) In a separate investigation, bleeding time was determined by transecting mouse tails after a 30-min interval following intraperitoneal administration of either normal saline (NS), 0.1% DMSO, eugenol (15 mg/kg), or aspirin (15 mg/kg). The data are presented as the mean ± standard error of the mean (n = 12). Statistical significance is denoted as *** p < 0.001 compared to the results observed in the 0.1% DMSO group.
Figure 8
Figure 8
A theoretical framework provides insights into the complex mechanisms through which eugenol exerts inhibitory effects on human platelet activation. Eugenol’s impact involves targeting pivotal signaling cascades, specifically cPLA2/TxA2 and PLCγ2/PKC, followed by the activation of PI3K-Akt-GSK3β and MAPKs pathways. This orchestrated modulation leads to a precise control of reducing intracellular calcium ([Ca2+]i) levels, ultimately resulting in the suppression of platelet aggregation. In the diagram, red suppress arrows represent inhibition, orange double-head arrows signify mutual influence, and black arrows denote standard signaling pathways.
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