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

Hirudin 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.

In 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.

Saturation 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.

The 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^-1 and shortened the thrombin time (TT) from 42.00 s to 30.94 s, whereas mutation of 26 K to W increased the ΔG to 24.70 kcal·mol^-1 and prolonged TT to 51.92 s. In addition, the aromatic effect of 20Y, combined with sulfation, synergistically enhanced binding. Based on these findings, the newly designed WPHVC_V1 showed a ΔG of 37.24 kcal·mol^-1 and, at 0.1 mg/ml, increased TT/APTT/PT from 41.72/14.38/15.86 s (WPHV_C) to 62.08/23.38/22.22 s. In in vivo studies, WPHVC_V1 achieved tail thrombus inhibition in the mouse tail by reducing the length of the thrombus from 3.562 cm in CK to 1.853 cm (1.729 cm for sodium heparin and 2.530 cm for WPHV_C), completely inhibited thrombus formation in a carotid artery model and reduced tail bleeding time by 35.2 s compared with heparin sodium. Safety evaluations revealed that WPHVC_V1 did not cause hemolysis, had no significant effect on blood pressure or cause pathological changes in major organs.

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