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
Este estudio investigó la base estructural de la unión de RGD-hirudina a la trombina mediante la expresión de RGD-hirudina recombinante y seis variantes mutantes de punto único (Y3A, S50A, Q53A, D55A, E57A, I59A) en Pichia pastoris, evaluando luego la actividad antitrombínica y la cinética de unión mediante resonancia de plasmón superficial. Las seis mutantes mostraron valores de KD superiores a los de RGD-hirudina, lo que indica una menor afinidad de unión en relación con la molécula parental; RGD-hirudina también se describe como inhibidora competitiva de la unión de fibrinógeno a la GPIIb/IIIa plaquetaria, inhibiendo así la agregación plaquetaria mientras conserva la actividad anticoagulante. Los autores concluyen que estos hallazgos contribuyen a la comprensión de la interacción entre RGD-hirudina y la trombina. Para el ámbito de la ASH, esto proporciona información a nivel molecular sobre una proteína derivada de la sanguijuela con doble actividad anticoagulante y antiplaquetaria. Salvedad: se trata de una investigación bioquímica in vitro que utiliza proteína recombinante expresada en levadura, no saliva nativa de la sanguijuela ni datos clínicos de hirudoterapia.
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: 18 de junio de 2026