Purification and Characterization of Novel Collagen Peptides from Oncorhynchus mykiss Inhibiting Platelet Aggregation, and the Underlying Mechanism via Molecular Docking.

Rainbow trout is produced in large quantities worldwide, generating substantial processing by-products, among which collagen-rich skin and bone require high-value utilization. Rainbow trout collagen peptides potentially exhibit antiplatelet activity based on structural similarity to reported antiplatelet fish collagen peptides, while their active peptide sequences and underlying mechanisms require investigation. In this study, rainbow trout skin collagen was hydrolyzed by neutral protease, yielding a hydrolysate with 70.52% inhibition of platelet aggregation at 4 mg/mL. The fraction with high antiplatelet activity, N2-2, was obtained via separation by ODS-C18 and Sephadex LH-20 chromatography, which was enriched in Gly, Pro, and Hyp residues and characterized by mild hydrophilicity and a moderate molecular weight. Peptide sequences in the N2-2 were identified by HPLC-MS/MS, followed by molecular docking-based screening, and the four peptides selected were synthesized and evaluated for antiplatelet activity in vitro. Peptides MTGP and OOGGHG exhibited higher antiplatelet activity, with IC50 values of 0.52 mM and 0.54 mM, respectively. Molecular docking indicated that MTGP and OOGGHG stably bind to the P2Y12 receptor via hydrogen bonding with key residues (Lys280, Arg256, and Asn191), with hydrophobic interactions contributing to complex stability. This study adds support to the feasibility of natural antiplatelet functional foods from rainbow trout collagen.