In vivo screening of Thai plant extracts for antithrombocyte activity using zebrafish: discovery of novel antiplatelet compounds

Current antithrombotic therapies are associated with significant side effects, including gastrointestinal and intracranial bleeding, as well as interactions with diet and other medications. These limitations underscore the need for safer, more effective alternatives. Given the conservation of coagulation and platelet activation pathways between zebrafish and mammals, we investigated the utility of zebrafish as an in vivo model for screening plant-derived compounds with anti-platelet activity. Unlike conventional in vitro platelet aggregation assays, this approach allows simultaneous assessment of biological activity and systemic toxicity. A total of 41 plant tissue extracts were screened using a zebrafish-based thrombocyte aggregation assay, with five extracts showing significant inhibition of ADP-induced thrombocyte activation. These findings were validated using human whole blood impedance aggregation assays. Active extracts were then subjected to chromatographic fractionation, and select fractions retaining anti-platelet activity were further analyzed. From these, three compounds were isolated in sufficient quantity for structure elucidation using mass spectrometry and nuclear magnetic resonance spectroscopy. Structural analysis revealed three related compounds, two of which represent novel inhibitors of ADP-mediated platelet aggregation. This study highlights the zebrafish as a powerful platform for the in vivo screening of natural product libraries for antithrombotic agents and identifies two previously unreported plant-derived compounds with promising anti-platelet properties.