IntroductionPlatelet activation is thought to be a key event in acute vascular thrombosis. Therefore, prevention of enhanced platelet activation is a major target of therapeutic strategies fighting cardiovascular and cerebrovascular diseases. [1][2][3] An important stimulus for physiologic platelet activation and thrombus formation is the contact of platelets with components of the subendothelial matrix, like collagen. 4 Although Marcus et al have shown as early as 1977 that platelets have the ability to release superoxide anions (O 2 Ϫ ), 5 it was only recently proposed that platelets stimulated by collagen produce reactive oxygen species (ROS) such as hydrogen peroxide, 6 hydroxyl radicals, 7 or O 2 Ϫ . 7,8 While O 2 Ϫ , a highly reactive radical, damages cells in high concentrations by reacting with proteins, lipids, and DNA, in low concentrations its continuous production, with similarity to second messengers, has been suggested to indirectly affect signal transduction processes. 9,10 Platelet agonists other than collagen, such as thrombin or ADP, do not seem to induce ROS formation during aggregation. 8 This difference raises the question whether O 2 Ϫ formation could serve a modulating function when thrombus formation is induced by collagen.The cellular source of platelet O 2 Ϫ is unclear. Growing evidence supports the assumption that platelet activation by collagen is specifically due to binding to the glycoprotein VI (GPVI)-receptor, 11,12 resulting in a cascade of tyrosine phosphorylation events ultimately leading to activation of phospholipase C␥ (PLC␥), 13 which is known to strongly activate protein kinase C (PKC) through production of diacylglycerol. 4 Recently, evidence for the existence of a neutrophil-type reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidase in platelets that can be activated by PKC and is involved in O 2 Ϫ formation has been presented, [14][15][16] similar as in other O 2 Ϫ -generating systems, like the vascular endothelium. In endothelial cells, an NAD(P)H oxidase is the main source of O 2 Ϫ . 17 As O 2 Ϫ readily reacts with NO, this has been suggested to result in attenuated 18,19 and the role of O 2 Ϫ in the regulation of vascular tone has become a major focus of interest. 17 Moreover, antioxidants like N-acetylcysteine (NAC) have been shown to exert direct antiaggregatory effects. 20 Although these findings raise the possibility that platelet-derived O 2 Ϫ is involved in regulating platelet activation, evidence for a role of platelet-derived O 2 Ϫ in platelet function is rare. In a canine model of coronary arterial thrombosis, thrombus formation was regulated by intraplatelet redox state. 21 Leo and colleagues have shown that platelets subjected to anoxia/ reoxygenation are more reactive, due to an enhanced O 2 Ϫ generation. 14 However, so far it remains unclear whether an enhanced O 2 Ϫ production occurs also during direct platelet activation, such as with collagen, and how this could affect thrombus growth. Whereas Supported by a grant from the Friedrich-...

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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...