Curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbβ3 sign pathway

Abstract

Curdione, a sesquiterpene compound isolated from the essential oil of Curcuma aromatica Salisb. inhibits platelet aggregation, suggesting its significant anticoagulant and antithrombotic effects. However, the mechanisms have not been fully elucidated. We hypothesized that curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbβ3 signaling pathway. We performed in vitro assays to evaluate the effect of curdione on thrombin-induced expression levels of the AMPK signaling molecule and integrin αIIbβ3 signaling pathway components. Platelet proteins were extracted from washed human platelets, and the effects of curdione on thrombin-induced platelet aggregation were evaluated. The expression levels of the AMPK signaling molecule and integrin αIIbβ3 signaling pathway-related proteins were examined using western blot and RT-PCR. The binding of vinculin and talin were studied using immunoprecipitation, double immunofluorescence staining and microscale thermophoresis. Platelet aggregation analysis showed that 0.02 U/ml thrombin significantly induces platelet aggregation. Western blot and RT-PCR analysis revealed that AMPK inhibits the vinculin/talin-mediated integrin αIIbβ3 signaling pathway, and curdione downregulates the thrombin-induced expression of phosphorylated AMPK (P-AMPK) and P-integrin at both the protein and mRNA levels and downregulates vinculin and talin at the protein level. Furthermore, microscale thermophoresis experiments showed that curdione inhibits the binding of vinculin and talin. The results from the immunoprecipitation and double immunofluorescence staining were consistent with the results of the microscale thermophoresis experiments. Curdione inhibits thrombin-induced platelet aggregation via regulating the AMP-activated protein kinase-vinculin/talin-integrin αIIbβ3 signaling pathway, which suggests its therapeutic potential in ethnomedicinal applications as an anti-platelet and anti-thrombotic compound to prevent thrombotic diseases.