Molecular dynamic and pharmacological studies on protein-engineered hirudin variants of Hirudinaria manillensis and Hirudo medicinalis
Basic science / preclinical published in British journal of pharmacology (2022)
Abstract
BACKGROUND AND PURPOSE: Hirudin variants are the most powerful thrombin inhibitors discovered to date, with a lower risk of bleeding than heparin. For anticoagulation, the C-termini of hirudin variants bind to the exocite I of thrombin. Anticoagulant effects of gene-recombinant hirudin are weaker than natural hirudin for the reason of lacking tyrosine O-sulfation at C-terminus. EXPERIMENTAL APPROACH: An integrative pharmacological study was carried out using molecular dynamic, molecular biological and in vivo and in vitro experiments to elucidate the anticoagulant effects of protein-engineered hirudins. KEY RESULTS: Molecular dynamic analysis showed that modifications of the C-termini of hirudin variant 1 of Hirudo medicinalis (HV1) and hirudin variant 2 of Hirudinaria manillensis (HM2) changed the binding energy of the C-termini to human thrombin. The study indicated that Asp61 of HM2 that corresponds to sulfated Tyr63 of HV1 is critical for inhibiting thrombin activities. Further, the anticoagulant effects of HV1 and HM2 were improved when the amino acid residues adjacent to Asp61 were mutated to Asp. These improvements were prolongation of the activated partial thromboplastin time, prothrombin time and thrombin time of human blood, and decreased Ki and IC50 values. In the in vivo experiments, mutations at C-termini of HV1 and HM2 significantly changed partial thromboplastin time, prothrombin and thrombin time CONCLUSION AND IMPLICATIONS: The study indicated that the anticoagulant effects of gene-engineered HM2 are stronger than gene-engineered HV1 and HM2-E60D-I62D has the strongest effects and could be an antithrombotic with better therapeutic effects.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Summary
Hirudin variants are the most powerful thrombin inhibitors discovered to date, with a lower risk of bleeding than heparin. For anticoagulation, the C-termini of hirudin variants bind to the exocite I of thrombin.
Why This Matters for Hirudotherapy
This study examined how protein engineering and specific amino acid mutations at the C-terminus of hirudin variants from Hirudo medicinalis and Hirudinaria manillensis affect their binding to human thrombin and overall anticoagulant activity in vitro and in vivo. For hirudotherapy and the clinical application of the leech salivary secretome, these findings are highly relevant because they provide mechanistic insights into overcoming the reduced efficacy of recombinant hirudin caused by the lack of natural tyrosine O-sulfation. By identifying specific mutations that enhance thrombin inhibition and prolong blood clotting times, the research highlights pathways to optimizing naturally derived leech anticoagulants for therapeutic use. As an honest caveat, this is a preclinical investigation utilizing molecular dynamics and laboratory animal models; it focuses entirely on recombinant protein engineering rather than live leech therapy, and the clinical safety or efficacy of these engineered variants in actual human patients remains unestablished.
Citation
Molecular dynamic and pharmacological studies on protein-engineered hirudin variants of Hirudinaria manillensis and Hirudo medicinalis.
Sun Y et al. · British journal of pharmacology, 2022
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