Structural basis of RGD-hirudin binding to thrombin: Tyr3 and five C-terminal residues are crucial for inhibiting thrombin activity
Basic science / structural biology published in BMC Struct Biol (2014)
Abstract
BACKGROUND: Hirudin is an anti-coagulation protein produced by the salivary glands of the medicinal leech Hirudomedicinalis. It is a powerful and specific thrombin inhibitor. The novel recombinant hirudin, RGD-hirudin, which contains an RGD motif, competitively inhibits the binding of fibrinogen to GPIIb/IIIa on platelets, thus inhibiting platelet aggregation while maintaining its anticoagulant activity. RESULTS: Recombinant RGD-hirudin and six mutant variants (Y3A, S50A, Q53A, D55A, E57A and I59A), designed based on molecular simulations, were expressed in Pichia pastoris. The proteins were refolded and purified to homogeneity as monomers by gel filtration and anion exchange chromatography. The anti-thrombin activity of the six mutants and RGD-hirudin was tested. Further, we evaluated the binding of the mutant variants and RGD-hirudin to thrombin using BIAcore surface plasmon resonance analysis (SPR). Kinetics and affinity constants showed that the KD values of all six mutant proteins were higher than that of RGD-hirudin. CONCLUSIONS: These findings contribute to a novel understanding of the interaction between RGD-hirudin and thrombin.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
Structural characterization of recombinant RGD-hirudin and six mutants binding thrombin via Biacore SPR. Tyr3 and five C-terminal residues identified as crucial for thrombin inhibition.
Por qué esto importa para la hirudoterapia
This study investigated the structural basis of RGD-hirudin binding to thrombin by expressing recombinant RGD-hirudin and six single-point mutant variants (Y3A, S50A, Q53A, D55A, E57A, I59A) in Pichia pastoris, then testing anti-thrombin activity and binding kinetics via surface plasmon resonance. All six mutants exhibited higher KD values than RGD-hirudin, indicating reduced binding affinity relative to the parent molecule; RGD-hirudin is also described as competitively inhibiting fibrinogen binding to platelet GPIIb/IIIa, thus inhibiting platelet aggregation while retaining anticoagulant activity. The authors conclude these findings contribute to understanding the interaction between RGD-hirudin and thrombin. For ASH's domain, this provides molecular-level insight into a leech-derived protein with dual anticoagulant and anti-platelet activity. Caveat: This is in-vitro biochemical research using recombinant protein expressed in yeast, not native leech saliva or clinical hirudotherapy data.
Citación
Structural basis of RGD-hirudin binding to thrombin: Tyr3 and five C-terminal residues are crucial for inhibiting thrombin activity.
Huang Y et al. · BMC structural biology, 2014
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: June 18, 2026