Membrane potential-dependent inhibition of platelet adhesion to endothelial cells by epoxyeicosatrienoic acids

Objective - Epoxyeicosatrienoic acids (EETs) are potent vasodilators produced by endothelial cells. In many vessels, they are an endothelium-derived hyperpolarizing factor ( EDHF). However, it is unknown whether they act as an EDHF on platelets and whether this has functional consequences. Methods and Results - Flow cytometric measurement of platelet membrane potential using the fluorescent dye DiBac(4) showed a resting potential of - 58 +/- 9 mV. Different EET regioisomers hyperpolarized platelets down to - 69 +/- 2 mV, which was prevented by the non-specific potassium channel inhibitor charybdotoxin and by use of a blocker of calcium-activated potassium channels of large conductance (BKCa channels), iberiotoxin. EETs inhibited platelet adhesion to endothelial cells under static and flow conditions. Exposure to EETs inhibited platelet P-selectin expression in response to ADP. Stable overexpression of cytochrome P450 2C9 in EA. hy926 cells ( EA. hy2C9 cells) resulted in release of EETs and a factor that hyperpolarized platelets and inhibited their adhesion to endothelial cells. These effects were again inhibited by charybdotoxin and iberiotoxin. Conclusions - EETs hyperpolarize platelets and inactivate them by inhibiting adhesion molecule expression and platelet adhesion to cultured endothelial cells in a membrane potential-dependent manner. They act as an EDHF on platelets and might be important mediators of the anti-adhesive properties of vascular endothelium.