Abstract-Platelets participate not only in thrombus formation but also in the regulation of vessel tone, the development of atherosclerosis, angiogenesis, and in neointima formation after vessel wall injury. It is not surprising, therefore, that the platelet activation cascade (including receptor-mediated tethering to the endothelium, rolling, firm adhesion, aggregation, and thrombus formation) is tightly regulated. In addition to already well-defined platelet regulatory factors, such as nitric oxide (NO), prostacyclin (PGI 2 ), and adenosine, reactive oxygen species (ROS) participate in the regulation of platelet activation. Although exogenously derived ROS are known to affect the regulation of platelet activation, recent data suggest that the platelets themselves generate ROS. Intracellular ROS signaling in activated platelets could be of significant relevance after transient platelet contact with the vessel wall, during the recruitment of additional platelets, and in thrombus formation. This review discusses the potential cellular and enzymatic sources of ROS in platelets, their molecular mechanisms of action in platelet activation, and summarizes in vitro and in vivo evidence for their physiological and potential therapeutic relevance. Key Words: platelets Ⅲ reactive oxygen species Ⅲ NAD(P)H-oxidase Ⅲ aggregation Ⅲ adhesion Ⅲ thrombus formation P latelet interaction with the vessel wall serves numerous physiological and pathophysiological functions. This is reflected by the fact that platelets release growth factors, 1 lipid mediators, 2,3 and cytokines. 4 Consequently, the regulation of platelet activity plays a role not only for thrombus formation and the regulation of vascular tone 5 but also for the vascular pathophysiology of angiogenesis and inflammation. Moreover, platelets participate in the development of atherothrombotic disease by promoting atherosclerotic lesion 6 and neointima formation. 7 Not surprisingly, the activation of platelets is regulated and modulated by numerous factors, blood-borne and cell-derived. Most of these factors are relatively well-characterized. 8 However, in recent time, several publications have suggested that reactive oxygen species (ROS) represent a new modulator of platelet activity. It has been known for some time that ROS exert critical regulatory functions within the vascular wall and it is therefore plausible that platelets represent a relevant target for their action.Within the vessel wall (where endothelial cells, vascular smooth muscle cells, and fibroblasts express a variety of ROS-generating enzymes), there is a constant, low-quantity flux of ROS. It is already established that enhanced ROS release from the vascular wall can indirectly affect platelet activity by scavenging nitric oxide (NO), thereby decreasing the antiplatelet properties of the endothelium. 9 In addition to their exposure to ROS derived from the vascular wall, platelets themselves also can generate ROS, and there is some evidence for a more direct role of ROS in the control of platelet activi...

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...

Modification of cellular functions by overexpression of genes is increasingly practised for research of signalling pathways, but restricted by limitations of low efficiency. We investigated whether the novel technique of magnetofection (MF) could enhance gene transfer to cultured primary endothelial cells. MF of human umbilical vein endothelial cells (HUVEC) increased transfection efficiency of a luciferase reporter gene up to 360-fold compared to various conventional transfection systems. In contrast, there was only an up to 1.6-fold increase in toxicity caused by MF suggesting that the advantages of MF outbalanced the increase in toxicity. MF efficiently increased transfection efficiency using several commercially available cationic lipid transfection reagents and polyethyleneimine (PEI). Using PEI, even confluent HUVEC could be efficiently transfected to express luciferase activity. Using a green fluorescent protein vector maximum percentages of transfected cells amounted up to 38.7% while PEI without MF resulted in only 1.3% transfected cells. Likewise, in porcine aortic endothelial cells MF increased expression of a luciferase or a β-galactosidase reporter, reaching an efficiency of 37.5% of cells. MF is an effective tool for pDNA transfection of endothelial cells allowing high efficiencies. It may be of great use for investigating protein function in cell culture experiments.

Superoxide anions impair nitric oxide-mediated responses and are involved in the development of hypertensive vascular hypertrophy. The regulation of their production in the vascular system is, however, poorly understood. We investigated whether changes in membrane potential that occur in hypertensive vessels modulate endothelial superoxide production. In cultured human umbilical vein endothelial cells, changes in membrane potential were induced by high potassium buffer, the non-selective potassium channel blocker tetrabutylammonium chloride (1 mM), and the non-selective cation ionophore gramicidin (1 M). Superoxide formation was significantly elevated to a similar degree by all three treatments (by ϳ60%, n ‫؍‬ 23, p < 0.01), whereas hyperpolarization by the K ATP channel activator Hoe234 (1 M) significantly decreased superoxide formation. Depolarization also induced an increased tyrosine phosphorylation of several not yet identified proteins (90 -110 kDa) and resulted in a significant increase in membrane association of the small G-protein Rac. Accordingly, the Rac inhibitor Clostridium difficile toxin B blocked the effects of depolarization on superoxide formation. The tyrosine kinase inhibitor genistein (30 M, n ‫؍‬ 15) abolished depolarization-induced superoxide formation and also prevented depolarization-induced Rac translocation associated with it. It is concluded that depolarization is an important stimulus of endothelial superoxide production, which involves a tyrosine phosphorylation-dependent translocation of the small G-protein Rac.