RvE1 regulates P2Y12signaling in a ChemR23-dependant manner. receptor, human ChemR23 (present on human platelets), addition of RvE1 (0.1nM-10.0nM) blocked ADP signals (IC50~1.61011M) in P2Y12-ChemR23-expressing cells compared to mock transfections. == Conclusions == These results demonstrate that RvE1s regulatory actions (i.e reducing ADP-stimulated P-selectin mobilization and actin polymerization) are hChemR23-dependent. Moreover, they document specific platelet actions of RvE1 selectively engaged with ADP-activated platelets that illuminate a new cellular mechanism and impact of omega-3 EPA that may contribute to both resolution of vascular inflammation and ADP-dependent platelet activation relevant in pathologic cardiovascular events. Keywords:Eicosapentaenoic acid (EPA), Resolvin E1, inflammation, platelet, adenosine diphosphate (ADP) Excessive inflammation is now recognized as a central component in several of the most prevalent human diseases in the developed world, including rheumatic diseases, diabetes, and cardiovascular diseases13. Identification of cellular and molecular mechanisms in resolution and inflammation as well as the roles of novel local chemical mediators demonstrate that resolution of acute inflammation is an active process46rather than passive as believed earlier2. It is becoming increasingly apparent that inflammatory diseases, such as periodontal disease7, atherosclerosis8and Henoch-Schonlein purpura9, arise due to a failure in mounting endogenous resolution programs10. Timely resolution of acute inflammation is usually actively regulated by a new genus of endogenous mediators collectively called specialized pro-resolving mediators (SPM). This genus includes several families of structurally unique, potent and local-acting mediators, namely lipoxins, resolvins and protectins, which are biosynthesized from polyunsaturated essential fatty acids, reviewed in11,12. Results from several clinical studies (recently reviewed in13) demonstrate that diets rich in marine fish omega-3 polyunsaturated fatty acids (PUFA) reduce the risk of coronary events13. The cellular mechanisms responsible for omega-3 fatty acid responses are of increasing general interest14. Along these lines, circulating omega-3 fatty acids (eicosapentaenoic acid, EPA, and docosahexaenoic acid, DHA) rapidly appear in resolving inflammatory exudates carried by edema proteinsin vivo,thus emphasizing the importance of circulating levels of omega-3 PUFA and their availability for conversion by exudates into resolvins AT 56 and AT 56 protectins15. Resolvins are enzymatic products of EPA and DHA biosynthesized during the resolution phase of acute inflammation, initially isolated in self-limited murine exudates6,16. They are agonists that exert both anti-inflammatory (i.e. limit further neutrophil infiltration) and pro-resolving (i.e. stimulate non-phlogistic activation of macrophages) actions11. Specifically, Resolvin E1 (RvE1), a member of the E series of resolvins, is usually biosynthesized from EPA and was first identifiedin vivoduring the resolution phase of inflammation from exudates6. The levels of RvE1 were monitored in plasma from healthy human volunteers taking fish oil ranged from 0.1 to 0.4 ng/ml.17This is commensurate with RvE1 bioactions evoked in the pM-nM range. With isolated human cells, RvE1 is usually produced by hypoxia-activated human endothelial cells that convert EPA to an 18R-hydroxy-containing intermediate via aspirin-acetylated COX-26. Also, 18R-hydroxy-eicosapentanoic acid is usually produced by microbial P45018. Once created, 18R-HEPE is usually rapidly transformed by activated leukocytes to a 5(6)-epoxide-18R-hydroxy containing intermediate where 5-LOX carries out consecutive actions19. RvE1 total stereochemistry is established (5S,12R,18R-trihydroxy-6Z,8E,10E,14Z,16E-eicosapentaenoic acid) and is a stereoselective agonist that interacts with at least two recognized G-protein-coupled receptors (GPCRs), ChemR2317and BLT120(reviewed in21). RvE1 possesses potent anti-inflammatory and pro-resolving actions in animal models of disease and acts on a wide range of cell types11including leukocytes and platelets in whole blood22. Unlike BLT1, which is present on platelets23, we recently recognized ChemR23 on megakaryocytes and the surface of human platelets22. RvE1 has stereoselective anti-aggregatory actions with human platelets,22and at concentrations as low as 1 nM RvE1 blocks adenosine diphosphate (ADP, 10M)-stimulated platelet aggregation and thromboxane generation22. The platelet actions of ADP are of particular interest because it is usually well appreciated as an important local mediator of hemostasis and thrombosis, as well as when aberrantly regulated platelets contribute to the pathogenesis of cardiovascular diseases24,25. On platelets, ADP specifically binds and activates two G-protein coupled receptors, P2Y1and P2Y12. Transduction of the ADP signal entails inhibition of adenyl cyclase (via P2Y12) and a concomitant transient increase of intracellular Ca2+(via P2Y1)25. Further downstream signaling results in robust shape changes, inside-out activation of platelet receptors, such as GPIIbIIIa, and granule secretion25. Current anti-platelet therapies targeted at blocking purinergic receptor signaling, specifically Mouse monoclonal to ABCG2 P2Y12antagonists, are the focal point of platelet therapeutics26and are among the most widely used pharmacotherapy in cardiovascular diseases27,28. Since we found that RvE1 has potent and stereoselective actions in blocking ADP-stimulated platelet aggregation,22we examined RvE1s actions in ADP-activated platelets. Here, we AT 56 statement that RvE1 regulates ADP-stimulated P-selectin surface mobilization and actin polymerization, and that RvE1 counterregulates P2Y12signaling in a receptor-dependant (ChemR23) manner in transfected cells. Together, these findings may have implications for dietary EPA supplementation for the maintenance of vascular homeostasis and in the resolution of acute inflammation. ==.