Anti-Thrombotic Effect of Hemp Seed Peptides in Zebrafish by Dual Regulation of vegfr1 Signaling and Caspase-3-Mediated Apoptosis

Abstract

This study aimed to investigate the antithrombotic activity of hemp seed peptides derived from trypsin hydrolysates and elucidate their molecular mechanisms using a ponatinib-induced zebrafish (Danio rerio) thrombosis model. Through ultrafiltration and LC–MS/MS analysis, we identified 1741 peptide sequences (< 3 kDa), with 67% distributed in the 0.5–1.5 kDa range. In vivo experiments demonstrated that 125 μg/mL hemp seed peptides significantly ameliorated ponatinib-induced thrombosis by increasing 16.55% cardiac erythrocyte staining intensity, enhancing 54.8% blood flow velocity, and restoring 11.2% intersegmental blood vessel diameters. In addition, the observed anticoagulant effect may be partially attributed to upregulated expression levels of vascular endothelial growth factor receptor 1 (vegfr1) and down-regulated expression levels of cysteinyl aspartate-specific proteinase-3 (caspase-3) at the mRNA level. These findings propose hemp seed peptides as novel dietary antithrombotic agents with potential applications in functional food development.