Celastrol is an active ingredient of the traditional Chinese medicinal plant, Tripterygium wilfordii Hook F, which is known especially for its anti-inflammatory effects. However, on the cellular and molecular levels, celastrol's mechanism of action is only poorly understood. Because platelets contribute to inflammatory events, this study investigates the effects of celastrol on platelet function using flow cytometry, aggregometry, and adhesion assays. In in vitro experiments with human platelets, celastrol inhibits adenosine-5-diphosphate (ADP)-induced expression of the platelet activation marker P-selectin and glycoprotein IIb/IIIa activation with 50% inhibition values of 1.62 and 1.86 microM, respectively. Celastrol also inhibits thrombin-stimulated and phorbol 12-myristate 13-acetate-stimulated P-selectin expression on platelets. Furthermore, ADP-stimulated platelet adhesion on fibrinogen is partially prevented by 5 microM celastrol. In platelet aggregometry, celastrol (0.05-0.5 mM) inhibits ADP-induced aggregation of platelet-rich plasma. Moreover, 12 male C57BL/6J mice were randomly grouped to receive intraperitoneal treatment with either celastrol (2 mg x kg x day) or vehicle. After 4 weeks of the respective treatment, celastrol inhibited 2 and 20 microM ADP-stimulated platelet fibrinogen binding by 34.5% (P < 0.01) and 28.9% (P < 0.05), respectively, compared with controls. In conclusion, these results indicate that celastrol exerts inhibitory effects on platelets. This new finding contributes to the understanding of antithrombotic and also anti-inflammatory effects of celastrol.