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

Background/Objectives: Platelets are anucleate cells that play a crucial role in primary hemostasis and arterial thrombosis, contributing to cardiovascular diseases. Eugenol, a bioactive phenolic compound, exhibits vasodilatory, antibacterial, and anticancer properties and inhibits platelet aggregation induced by collagen and arachidonic acid (AA). AA is a critical lipid component of the platelet membrane and a precursor for potent signaling molecules that mediate platelet activation. However, the precise mechanisms through which eugenol modulates AA-stimulated platelet activation remain unclear. Methods: Human platelets were pretreated with eugenol and subsequently stimulated with AA. Platelet aggregation, ATP release, intracellular calcium mobilization, and P-selectin expression were measured. JNK, p38 MAPK, ERK, and vasodilator-stimulated phosphoprotein (VASP) phosphorylation were analyzed by Western blotting. SP600125, SQ22536, and ODQ were used to examine the involvement of JNK and cyclic nucleotide signaling pathways. Antithrombotic effects were further evaluated in a mouse mesenteric thrombosis model. Results: Eugenol significantly suppressed AA-induced platelet aggregation, ATP release, calcium mobilization, and P-selectin expression, selectively reducing JNK phosphorylation without affecting p38 MAPK or ERK. SP600125 produced similar inhibitory effects. Neither SQ22536 nor ODQ reversed eugenol’s inhibitory effects on platelet aggregation. Furthermore, eugenol did not alter VASP phosphorylation at Ser157 or Ser239. In vivo, real-time intravital imaging showed that eugenol and SP600125 delayed thrombus formation and prolonged occlusion time. Conclusions: These findings suggest that eugenol exerts inhibitory effects that may involve modulation of JNK phosphorylation, independent of cyclic nucleotide/VASP pathways in AA-induced platelet activation, highlighting its potential as an antithrombotic agent.