Background <p>The mechanism of thrombotic complications in patients with aortic stenosis (AS) is unclear so far. Our aim was to evaluate the levels of phosphatidylserine (PS) exposed on blood cells, endothelial cells (ECs), and extracellular vesicles (EVs) and its procoagulant activity (PCA) in mild to severe AS patients.</p> Methods <p>Exposed PS on blood cells, ECs and EVs were analyzed by flow cytometry. PCA was evaluated by clotting time (CT), intrinsic factor Xa (FXa), extrinsic FXa, thrombin and fibrin formation assays. We also evaluated the inhibitory effects of lactadherin (Lact) on PCA in AS patients.</p> Results <p>Our results demonstrated that patients with AS had significantly higher percentages of positive phosphatidylserine (PS⁺) red blood cells (RBCs), platelets (PLTs), white blood cells (WBCs) and ECs compared to controls. Total EVs with PS⁺, platelet EVs (PEVs), endothelial-derived EVs (EEVs) and positive tissue factor EVs (TF⁺EVs) levels were significantly higher in mild to severe AS. In addition, we further confirmed that PS⁺ blood cells, ECs and EVs significantly contributed to shortened CT and dramatically increased FXa, thrombin and final fibrin generation in mild to severe AS compared to controls. Furthermore, lactadherin significantly inhibited the PCA of PS exposure on blood cells, ECs and EVs in AS patients, whereas anti-TF had no this effect.</p> Conclusion <p>Our study revealed a previously unrecognized association between exposed PS levels on blood cells, ECs and EVs and PCA in AS. Lactadherin promises to be a new therapy by blocking PS to prevent thrombosis in AS patients.</p>