OBJECTIVE:Epoxyeicosatrienoic acids (EETs) act as vasodilators activating high conductance calcium-operated potassium (K) channels (Kca1.1, also named BK, MAXI-K). We found expression of MAXI-K channel in platelets. The present study aimed at defining its functionality in platelet using an in vitro model of platelet thrombosis. DESIGN AND METHOD:We tested the effects of 5 μmol/L 11,12-EET and the pharmacological modulation of MAXI-K channel (agonists5 and 20 μmol/L BMS 191011, 5 μmol/L NS1619, 5 μmol/L NS11021). Platelet rich plasma was used to assess adhesion-induced thrombi formation under flow by microfluidics technology with collagen-coated microchips mimicking arterial blood flow. The kinetic of platelet responses to scalar doses of 0.3–10 μmol/L ADP, 0.05–2 μmol/L U46619 0.5–10 μg/mL collagen was determined by paired analysis using Born aggregometry. Flow-cytometry was used to analyse the expression of active fibrinogen receptor and P-selectin in stimulated platelets. The effects of 100 μmol/L aspirin and 1 μmol/L ticagrelor were also assessed. RESULTS:In vitro thrombi formation was halved (expressed as platelet-covered area) by pre-treatment with either 11,12-EET (−45 ± 11%, n = 5, P < 0.001 vs control, Mean ± SD), aspirin (−66 ± 8%, n = 4, P < 0.001) or ticagrelor (−55 ± 8%, n = 4, P < 0.001). Similar results were obtained using BMS 5 μmol/L (−54 ± 17%, n = 6, P < 0.001), NS1619 (−50 ± 19%, n = 9, P < 0.001), NS11021 (−60 ± 21%, n = 6, P < 0.001). The addition of 20 μmol/L BMS191011 prior to platelet aggregation (EC502.67 μmol/L, 95%CI0.97–7.29, n = 36) significantly shifted to the right the dose response-curve to ADP (EC500.91 μmol/L, 0.43–1.92, n = 36). Platelet aggregation was further blunted by the addition of aspirin to BMS191011 (EC506.18 μmol/L, 2.11–18.09, n = 36). U46619- and collagen-induced aggregation was not altered. ADP-induced activation of the fibrinogen receptor (−48 ± 14% to −62 ± 10%, n = 7, P < 0.05) and P-selectin expression (−37 ± 15% to −41 ± 13%, n = 7, P < 0.01) were blunted by the activators of MAXI-K channel. CONCLUSIONS:Activation of the MAXI-K channel by 11,12-EET and all the tested synthetic compounds is associated with reduced sensitivity to ADP and reduced thrombus generation through the inhibition of the amplificatory phase of platelet activation. The present results reveal new mechanisms of platelet activation and suggest that targeting MAXI-K might be of potential pharmacological interest for the prevention of atherothrombosis.