AimIn healthy adults, there are sufficient amounts of antithrombin in the blood to regulate thrombin. However, the effects of high concentrations of antithrombin on dose‐dependent anticoagulation and platelet function have not been reported. In this study, we assessed platelet function and blood coagulation following high‐dose antithrombin supplementation in vitro.MethodsBlood samples were collected from 10 healthy volunteers, and samples with different antithrombin concentrations were prepared by adding an antithrombin agent (Neuart). Blood coagulation was assessed by the Thrombus‐Formation Analysis System (T‐TAS) and Rotational Thromboelastometry (ROTEM) using whole blood samples.ResultsThe data obtained by the platelet chip, exclusively representing platelet function, revealed that the onset of thrombus formation was significantly delayed in a dose‐dependent manner (100%–200%, P = 0.021; 100%–500%, P = 0.011; 200%–500%, P = 0.047). In measurements using the atheroma chip, which enables assessment of blood coagulation, the thrombus formation ability was found to be reduced (100%–200%, P = 0.022; 100%–500%, P = 0.05). In the ROTEM measurements, clotting time was prolonged in a dose‐dependent manner (100%–200%: P = 0.203, 200%–500%: P = 0.005, 500%–1000%: P = 0.022), except when comparing with 100% and 200%. Although antithrombin is reportedly saturated in healthy blood, its anticoagulant ability appears to be enhanced depending on its concentration. Furthermore, data obtained from the platelet chip showed that antithrombin might reduce platelet function.ConclusionsAntithrombin suppressed platelet function and blood coagulation in a dose‐dependent manner.