A cytochrome P450 2C8/9-derived EDHF inhibits platelet adhesion to endothelial cells in vitro

Abstract number: P0195

Krötz^* F., Riexinger† T., Sohn‡ H. Y., Bürkle§ M. A., Gloe† T., Pohl† U.

‡Cardiology Department, Germany; †Germany; §Institute of Anaesthesiology, Germany ^*Institute of Physiology, Germany;

The endothelium produces a factor that induces hyperpolarization of vascular smooth muscle cells resulting in vasodilatation (Endothelium-derived hyperpolarizing factor, EDHF). Whether EDHF also influences blood constituents like platelets is unclear. We investigated, whether cultured endothelial cells produce an EDHF that influences platelet membrane potential and function. Flow cytometric measurement of platelet membrane potential using the fluorescent dye DiBac4(3) showed a resting potential of washed platelets of -58 ± 9 mV. Supernatants of cultured human umbilical vein endothelial cells (HUVEC) grown in the presence of b-naphtoflavone (3 mmol L^-1) and nifedipine (100 nmol L^-1) hyperpolarized platelets, when stimulated with bradykinin under inhibition of NO-synthase and cyclooxygenase. This effect was inhibited by blocking endothelial CYP2C8/9 using sulfaphenazole (30 mmol L^-1) and by inhibition of platelet calcium-activated potassium channels of intermediate (IKCa-channels) and large conductance (BKCa-channels) by charybdotoxin (Cbtx) or iberiotoxin (Ibtx), respectively. CYP2C8/9-mRNA was detected in cultured HUVEC. Stable overexpression of CYP2C9 in EA.hy926 cells resulted in release of a factor that, alike different stereoisomers of the CYP2C8/9-products, epoxyeicosatrienoic acids (EETs), hyperpolarized platelets, which was inhibited by Cbtx and Ibtx. EETs and the supernatants of HUVEC and of CYP2C9 overexpressing EA.hy926 cells inhibited platelet adhesion to HUVEC in an I/BKCa-channel-dependent manner, which was related to inhibition of ADP-induced surface expression of platelet P-selectin and the platelet ? IIb-integrin. We show that cultured endothelial cells release a factor that hyperpolarizes platelets, which has similar properties as EETs. The release of this endothelial factor could be of importance for inhibition of platelet activation.