Construction of safer hirudin-derived peptides with enhanced anticoagulant properties based on C-terminal active residue adaptation and modification.

introductionHirudin exerts anticoagulant effects by inhibiting the binding and catalytic activity of thrombin to fibrinogen. However, its rigid N-terminal region irreversibly occupies the thrombin catalytic center, raising concerns about potential bleeding.

objectivesIn this study, a novel lead compound, WPHVC_V1, which is based on the competitive binding mechanism of the hirudin variant WPHV_C, was developed and validated for in vitro and in vivo activity and safety.

methodsSaturation mutagenesis, molecular dynamics simulations and mutant protein activity assays were used to elucidate the competitive anticoagulant mechanism between WPHV_C and thrombin. Next, a recombinantly expressed tyrosylprotein sulfotransferase was used to modify and confirm the sulfation site on the C-terminal tyrosine of hirudin. Finally, a multisite aromatic amino acid mutation strategy was implemented to design and synthesize the lead anticoagulant, WPHVC_V1.

resultsThe acidic amino acid cluster in WPHV_C formed strong electrostatic interactions with the positively charged thrombin exosite I, blocking fibrinogen binding. The introduction of aromatic amino acids further stabilized the complex through π-π stacking and π-cation interactions. For example, mutation of 13E to A decreased the free energy of dissociation (ΔG) from 19.27 to 10.93 kcal·mol

conclusionThese findings provide an initial foundation and sequence reference for the development of safe and effective anticoagulant drugs with potential for clinical translation.