Involvement of monocyte-derived extracellular vesicle-associated tissue factor activity in convallatoxin-induced hypercoagulability

Convallatoxin (CNT) is a natural cardiac glycoside extracted from lily of the valley ( Convallaria majalis ). Although it is empirically known to cause blood coagulation disorders, the underlying mechanism remains unclear. CNT exerts cytotoxicity and increases tissue factor (TF) expression in endothelial cells. However, the direct action of CNT on blood coagulation remains unclear. Therefore, herein, we investigated the effects of CNT on whole blood coagulation system and TF expression in monocytes.Blood samples were collected from healthy volunteers to measure plasma thrombin-antithrombin complex (TAT) concentration using ELISA and to perform rotational thromboelastometry (ROTEM) and whole-blood extracellular vesicle (EV)-associated TF (EV-TF) analysis. The effects of CNT were also investigated using the monocytic human cell line THP-1. Quantitative real-time PCR and western blotting were performed, and PD98059, a mitogen-activated protein kinase (MAPK) inhibitor, was used to elucidate the action mechanism of CNT-mediated TF production.CNT treatment increased EV-TF activity, shortened the whole blood clotting time in rotational thromboelastometry analysis, and increased TAT levels, which is an index of thrombin generation. Furthermore, CNT increased TF mRNA expression in THP-1 cells and EV-TF activity in the cell culture supernatant. Therefore, CNT may induce a hypercoagulable state with thrombin generation, in which elevated EV-TF activity derived from monocytes might be involved. These procoagulant effects of CNT were reversed by PD98059, suggesting that CNT-induced TF production in monocytes might be mediated by the MAPK pathway.The findings of the present study have further clarified the procoagulant properties of CNT.