Digging of Proline-Rich Peptides with Thrombin-Inhibitory Activity from Type V Collagen of Shortbill Spearfish ( <i>Tetrapturus angustirostris</i> ) Skin: A Combined <i>In Vitro</i> and <i>In Silico</i> Study

Type V collagen is a promising source to derive proline-rich peptides. High content of type V collagen in shortbill spearfish skin was utilized to dig proline-rich peptides with thrombin-inhibitory activity through <i>in vitro</i> and <i>in silico</i> strategies. Among identified peptides, Pro-Gly at 20 mg/mL prolonged fibrin polymerization formation rate by 41.6-fold, comparable to that of heparin. Enzyme kinetics analysis revealed that Pro-Gly inhibited the amidolytic activity of thrombin through a mixed-type inhibition mechanism (IC₅₀ = 0.61 mg/mL). Strong inhibitory effect of Pro-Gly was attributed to the preferential binding to the thrombin-substrate complex, facilitating spontaneous hydrophobic interactions and stable noncovalent complex formation. Molecular docking further demonstrated that Pro-Gly formed a hydrogen-bond contact with catalytic residues, enhancing the hydrophobic microenvironment and stabilizing the thrombin-peptide complex. Our work indicated that shortbill spearfish skin is a promising source of type V collagen for natural thrombin inhibitors, providing a scientific basis for the valorization of fish byproducts and the development of functional peptides.