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Myeloproliferative neoplasms (MPN) are associated with an increased risk of arterial and venous thrombosis. Pegylated-interferon alpha (IFN) and hydroxyurea (HU) are commonly used to treat MPN, but their effect on hemostasis has not yet been studied. The aim of our study was to determine whether IFN and HU impact the biological hemostatic profile of MPN patients by studying markers of endothelial, platelet, and coagulation activation. A total of 85 patients (50 polycythemia vera and 35 essential thrombocythemia) were included: 28 treated with IFN, 35 with HU, and 22 with no cytoreductive drug (non-treated, NT). Von Willebrand factor, shear-induced platelet aggregation, factor VIII coagulant activity (FVIII:C), fibrinogen, and thrombin generation with and without exogenous thrombomodulin were significantly higher in IFN-treated patients compared to NT patients, while protein S anticoagulant activity was lower. In 10 patients in whom IFN therapy was discontinued, these hemostatic biomarkers returned to the values observed in NT patients, strongly suggesting an impact of IFN therapy on endothelial and coagulation activation. Overall, our study shows that treatment with IFN is associated with significant and reversible effects on the biological hemostatic profile of MPN patients. Whether they could be associated with an increased thrombotic risk remains to be determined in further randomized clinical studies.