Eugenol Exhibits Cyclic Nucleotide/VASP-Independent Antiplatelet Activity Associated with Inhibition of Arachidonic Acid-Induced JNK Phosphorylation and Reduces Thrombus Formation as Visualized by Real-Time Intravital Imaging

Figure 1. Effects of eugenol on AA-induced platelet aggregation, ATP release, calcium mobilization, and surface P-selectin expression. Washed human platelets (3.6 × 108 cells/mL) were pretreated with 0.1% DMSO or various concentrations of eugenol (1–5 μM) for 3 min. Subsequently, AA (60 μM) was added to induce (A) platelet aggregation, (B) ATP release, and (C) relative [Ca2+]i level. Statistical analyses are presented in the right panels of each subfigure. Data are presented as means ± standard error of the mean (n = 4). * p < 0.05, and *** p < 0.001 indicate differences between the 0.1% DMSO and eugenol groups.

Figure 2. Eugenol suppresses AA-induced P-selectin exposure on the platelet surface. Washed human platelets were treated with 0.1% DMSO or eugenol (5 μM) for 3 min, followed by stimulation with AA (60 μM) to induce platelet activation. (A) Flow cytometric analysis of surface P-selectin expression, expressed as mean fluorescence intensity (MFI), in the following groups: (a) resting platelets; (b) AA-stimulated platelets treated with 0.1% DMSO; and (c) AA-stimulated platelets treated with eugenol. (B) Representative confocal laser fluorescence microscopy images showing surface P-selectin expression in platelets stained with FITC-conjugated anti-P-selectin antibody (green). Data are presented as means ± standard error of the mean (n = 4). *** p < 0.001 compared with resting platelets; ### p < 0.001 compared with 0.1% DMSO group. The scale bar is 5 μm.

Figure 3. Effects of eugenol on MAPK phosphorylation in platelets. Washed human platelets (3.6 × 108 cells/mL) were incubated with either 0.1% DMSO or eugenol (5 µM) for 3 min and subsequently exposed to AA (60 μM). The phosphorylation levels of MAPK family members, including (A) p38 MAPK, (B) ERK, and (C) JNK, were examined by immunoblotting. Data are presented as means ± standard error of the mean (n = 4). *** p < 0.001 compared with resting platelets; ### p < 0.001 compared with 0.1% DMSO group.

Figure 4. Eugenol-induced inhibition of MAPK activation on confocal laser microscopy. Washed platelets (3.6 × 108 cells/mL) were preincubated with 0.1% DMSO or eugenol (5 µM) for 3 min and subsequently exposed to AA (60 μM) for confocal microscopic evaluation at 1000× magnification, focusing on visualization of (A) phosphorylated p38 MAPK, (B) ERK, and (C) JNK through green fluorescence and α-tubulin through red fluorescence. Images represent four independent experiments. The scale bar is 5 μm.

Figure 5. SP600125-induced regulation of ATP release, calcium mobilization, and surface P-selectin expression in AA-induced platelet activation. Washed human platelets (3.6 × 108 cells/mL) were preincubated with 0.1% DMSO or SP600125 (20 μM) for 3 min. Subsequently, AA (60 μM) was added to trigger (A) ATP release, (B) calcium mobilization, and (C) Surface P-selectin expression, presented as MFI: (a) resting; (b) 0.1% DMSO + AA; (c) SP600125 + AA. (D) Representative confocal laser fluorescence microscopy images of platelets stained with FITC-conjugated anti-P-selectin antibody (green fluorescence) to visualize surface P-selectin expression. Data are expressed as means ± standard error of the mean (n = 4). In (A,B), *** p < 0.001 indicates differences between the SP600125- and 0.1% DMSO-treated groups. In (C,D), ** p < 0.01 and *** p < 0.001 indicate differences between the resting and 0.1% DMSO-treated groups, and ## p < 0.01 and ### p < 0.001 indicate differences between the SP600125- and 0.1% DMSO-treated groups. The scale bar is 5 μm.

Figure 6. Cyclic nucleotide/VASP signaling is not involved in the antiplatelet action of eugenol. (A) Platelet aggregation was assessed in washed human platelets (3.6 × 108 cells/mL) after a 3 min treatment with NTG (5 µM), PGE1 (10 nM), or eugenol (5 µM) in the absence or presence of the guanylate cyclase inhibitor ODQ (10 µM) or the adenylyl cyclase inhibitor SQ22536 (10 µM). Aggregation was initiated by AA (60 µM). In parallel experiments, platelets were treated with 0.1% DMSO, NTG (5 µM), PGE1 (10 nM), or eugenol (5 µM), and the phosphorylation of (B) VASP Ser239 and (C) VASP Ser157 was determined by immunoblot analysis. Immunofluorescence staining was further performed to detect phosphorylated (D) VASP Ser239 and (E) VASP Ser157 in green, with α-tubulin counterstained in red. Data from four independent experiments are shown for (A,D,E). Bar graphs are expressed as the mean ± standard error of the mean (n = 4). ** p < 0.01 and *** p < 0.001 compared with the 0.1% DMSO-treated group. Scale bar = 5 µm.

Figure 7. Efficacy of eugenol in platelet plug occlusion in mesenteric vessels. Mice received intraperitoneal administration of either a solvent control (0.1% DMSO) or eugenol at doses of 6 and 15 mg/kg, and mesenteric vessels were subjected to fluorescein irradiation to induce thrombus formation. In panel (A), real-time microscopic images were captured at intervals from 0 to 150 s post-irradiation at 400× magnification. Yellow dashed lines and white arrows indicate the blood vessel wall and thrombus formation. In panel (B), occlusion time was quantified as the primary measure, and data are presented as means ± standard error of the mean (n = 12).

Figure 8. Efficacy of SP600125 in platelet plug occlusion in mesenteric vessels. Mice received intraperitoneal administration of either a solvent control (0.1% DMSO) or SP600125 at doses of 20 mg/kg, and mesenteric vessels were subjected to fluorescein irradiation to induce thrombus formation. In panel (A), real-time microscopic images were captured at intervals from 0 to 150 s post-irradiation at 400× magnification. Yellow dashed lines and white arrows indicate the blood vessel wall and thrombus formation. In panel (B), occlusion time was quantified as the primary measure, and data are presented as means ± standard error of the mean (n = 12).

Figure 9. Potential mechanisms through which eugenol inhibits AA-induced platelet activation. Eugenol inhibits AA-induced platelet activation, which may involve modulation of JNK phosphorylation. Under therapeutically relevant conditions, eugenol does not affect NO/cGMP or PGE1/cAMP signaling, as indicated by unchanged VASP phosphorylation levels. GTP: guanosine triphosphate, NO: nitric oxide, PKA: protein kinase A (cAMP-dependent), PKG: protein kinase G (cGMP-dependent), sGC: soluble guanylate cyclase, sAC: soluble adenylate cyclase.