Natural Coumarin Derivative Esculetin Regulates Platelet Activation via Modulating NF-κB Signaling in Cyclic Nucleotide-Independent Manner

Esculetin, a natural coumarin derivative, shows exciting biological activities in a variety of cell and animal models. Our recent study demonstrated that esculetin exhibits antiplatelet effects by obstructing the phospholipase C γ2/protein kinase C cascade, hydroxyl radical formation, and Akt activation. In this study, we further examined the involvement of cyclic 3′-5′adenosine monophosphate/, vasodilator-stimulated phosphoprotein (VASP), integrin α_IIbβ₃, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), since cyclic nucleotides reduce the phosphorylation of VASP and activate NF-κB, subsequently inducing α_IIbβ₃ activation that significantly involves the platelet inhibitory pathways. We found that esculetin (50 and 80 µM) did not significantly affect fibrinogen-induced aggregation of elastase-treated platelets; however, it markedly blocked integrin α_IIbβ₃ activation by interrupting the binding of fluorescein isothiocyanate-labeled PAC-1. In addition, neither ODQ nor SQ22536 significantly reversed esculetin-mediated antiplatelet activity stimulated by collagen. Nitroglycerin and prostaglandin E₁ significantly increased VASP phosphorylation, but esculetin had no effect in this reaction, the values being almost identical with those of normal platelets. Furthermore, esculetin, at its maximum concentration of 80 μM significantly reduced the phosphorylation of IκBα and p65 and reversed IκBα degradation in collagen-induced platelets. These results suggest that the NF-κB-dependent α_IIbβ₃ inhibition of esculetin might represent a novel feedback inhibitory mechanism to regulate platelet functions.