Molecular dynamic and pharmacological studies on protein‐engineered hirudin variants of Hirudinaria manillensis and Hirudo medicinalis

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.

Anti-thrombotic Effects Mediated by a Novel Dual-Target Peptide Inhibiting Both Platelet Aggregation and Thrombin Activity without Causing Bleeding

“…Thrombin is a central regulator in the coagulation cascade and inflammation process. In the process of venous thrombogenesis, several thrombin inhibitors with pharmacological profiles different from the conventional anticoagulant heparin are currently in clinical use for thromboembolic indications 37 . One of them is the direct thrombin inhibitor recombinant hirudin 37 .…”

“…In the process of venous thrombogenesis, several thrombin inhibitors with pharmacological profiles different from the conventional anticoagulant heparin are currently in clinical use for thromboembolic indications 37 . One of them is the direct thrombin inhibitor recombinant hirudin 37 . In the artery, platelet aggregation may play a crucial role in thrombosis.…”

“…37 One of them is the direct thrombin inhibitor recombinant hirudin. 37 In the artery, platelet aggregation may play a crucial role in thrombosis. Platelet aggregation inhibitors, which Fig.…”

“…37 One of them is the direct thrombin inhibitor recombinant hirudin. 37 In the artery, platelet aggregation may play a crucial role in thrombosis. Platelet aggregation inhibitors, which interfere in the interactions between fibrinogen and its receptor, the GP IIb/IIIa complex, show extreme structural diversity but they share the common functional feature of (RGD) tripeptide segment.…”

Protein-Engineered Hirudins with Enhanced Anticoagulant Activities Developed by a Molecular Integrative Approach

“…An integrative analytical approach was employed in a study published in the British Journal of Pharmacology by Yan Sun and et al 6 to clarify the anticoagulant effects of C-terminusmodified hirudins, HV1 and hirudin variant 2 of Hirudinaria manillensis (HM2). In umbrella sampling of the changes in the Gibbs free energy in the molecular system when the Cterminus of hirudin was pulled away from human thrombin, it was found that the Asp61 mutation of HM2 to Ala decreased the binding free energy of the HM2 C-terminus to thrombin, with the Coulombic potential energy playing a substantial role (unpublished data), whereas the Glu60Asp (E60D) and Ile62Asp (I62D) double mutation enhanced the binding free energy.…”