OC 17.3 - Identification of a novel factor VII inhibitor by natural product library screening using zebrafish as a model.

Background: 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.

Aims: To screen natural products (NP) using zebrafish and identify novel antithrombotics.

Methods: Non-toxic NPs were selected by 4 dpf larval survival after 24 hours of incubation with these products. These NP-treated larvae were screened for anti-thrombotic activity using a laser-induced venous thrombosis (LIVT) assay that measures multiple hemostatic pathways including coagulation, platelet function, endothelial and red cell involvement, and stasis. NPs were then classified as platelet inhibitors, compounds affecting endothelial and RBCs by using arterial laser thrombosis assay, FeCl3-induced thrombosis, and phenylhydrazine-induced thrombosis assays respectively, kPT and kPTT assays, were used to measure extrinsic pathway (EP) and intrinsic pathway (IP), respectively. One-way ANOVA was used for statistical significance.

Results: We screened 547 NPs for toxicity and found 321 NPs were non-toxic to larvae. LIVT on larvae incubated with 321 NPs identified 74 NPs prolonged time to occlusion of vessel (TTO). Out of these 42 were already identified platelet inhibitors and remaining 32 NPs were tested using laser-induced arterial, FeCl3-Induced, and phenylhydrazine-induced thrombosis assays. We found prolonged TTO in 4, 4, and 6 among 32 NPs, respectively. The remaining 18 NPs were tested with kPT and kPTT. We found acetosyringone, and aphyllic acid affected IP while berbamine-hydrochloride (BH) affected EP and other 15 compounds affected unknown pathways. Since BH did not inhibit IP we posit it should inhibit factor VII. Molecular docking of BH on human factor VII revealed hydrogen bonding with an active site amino acid that explains BH activity.

Conclusion(s): NP screening identified many novel antithrombotic compounds including an inhibitor for factor VII. Future chemical modifications of this compound may result in a novel antithrombotic drug.