Identification of berbamine hydrochloride as a novel coagulation factor VII inhibitor by natural product library screening using zebrafish as a model.

Current antithrombotic drugs cause gastrointestinal and intracranial hemorrhage and interact with dietary components and other medications. Thus, there is a need for alternative antithrombotic drugs that have fewer side effects. We screened 547 natural products (NPs) for lethality and found 321 NPs were non-lethal to zebrafish larvae. Laser-induced venous thrombosis on larvae incubated with 321 NPs identified 76 NPs with prolonged time to occlusion of the vessel (TTO). Out of these, 43 were already identified platelet inhibitors and the remaining 33 NPs were tested using laser-induced arterial, ferric chloride-induced, and phenylhydrazine-induced thrombosis assays. Of the 33 NPs tested, 4 prolonged TTO in the laser-induced arterial thrombosis assay, 3 in the ferric chloride-induced thrombosis assay, and 5 in the phenylhydrazine-induced thrombosis assay. The remaining 24 NPs were tested with kPT and kPTT, the coagulation assays that measure extrinsic and intrinsic pathways, respectively. We found that acetosyringone and aphyllic acid affected the intrinsic coagulation pathway, while berbamine hydrochloride affected the extrinsic coagulation pathway, and the other 15 compounds did not affect intrinsic or extrinsic pathways in our assays, suggesting alternative mechanisms. Since berbamine hydrochloride did not inhibit the intrinsic pathway, we posit that it should inhibit factor VII. Molecular docking of berbamine hydrochloride on human factor VII revealed hydrogen bonding with active site amino acids and is consistent with berbamine hydrochloride’s activity. In conclusion, NP screening identified many novel antithrombotic compounds, including a novel inhibitor for factor VII. Future chemical modifications of these compounds may result in novel antithrombotic drugs.