Membrane Potential-Dependent Inhibition of Platelet Adhesion to Endothelial Cells by Epoxyeicosatrienoic Acids

Abstract

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 Ϫ58Ϯ9 mV. Different EET regioisomers hyperpolarized platelets down to Ϫ69Ϯ2 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 (BK Ca 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. Key Words: epoxyeicosatrienoic acids Ⅲ platelet adhesion Ⅲ membrane potential Ⅲ potassium channels Ⅲ EDHF I ntact endothelial cells continuously release autacoids such as nitric oxide (NO), prostacyclin (PGI 2 ), or adenosine and an endothelial-derived hyperpolarizing factor (EDHF), thereby controlling vascular tone and platelet activity. 1,2 Endothelial dysfunction and the associated activation of platelets are synergistic factors in the development of cardiovascular disorders. Both may precede atherosclerosis 3,4 and are associated with an enhanced risk of adverse cardiovascular events. 5 Little is known about the role of EDHF in the control of platelet function, although this factor may be less susceptible to mediators that deteriorate endothelial function such as reactive oxygen species.In several vascular beds, EDHF seems to be identical with epoxyeicosatrienoic acids (EETs), 6 which are products of cytochrome P450 enzymatic metabolism of arachidonic acid. 7 There are data indicating that EETs are released into the lumen of isolated vessels 8,9 or from endothelial cells in culture, 10 -12 so they could influence not only the adjacent smooth muscle cells but also circulating blood constituents like platelets. Although in 1986, years before these compounds have been postulated to represent an EDHF in the vasculature, Fitzpatrick et al observed inhibition of platelet aggregation by EETs, 13 it was not investigated whether the platelet-endothelium interaction was affected or whether platelet membrane potential has a role in this.In general, EETs could influence platelets by activation of calcium-activated potassium channels or by effects that are independent of the membrane potential, similar as described for endothelial cells. 14 Platelets not only contain voltageoperated potassium channels (K v channels) 15 but also calcium-activated potassium channels (K Ca channels), so they are potential targets of EETs. 16 In this study, we investigated whether EETs hyperpolarize platelets via K Ca channels and whether this has an effect on platelet activation parameters and platelet adhesion to the endothelium. MethodsFor a detailed Methods section, please see ht...

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“…It has been revealed that O 2 -reduces NO concentration by combining to it and form peroxynitrite (ONOO -). This combination has been speculated as a mechanism by which O 2 -diminishes the feedback effect of NO on Ca +2 and therefore, increases Ca +2 concentration [40,41]. Although, ONOO -is a pathogenic molecule in biological systems, its effect on PLT hyperactivity has not been clearly identified.…”

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