The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa
Research article published in Archives of biochemistry and biophysics (1992)
Abstract
Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa. Prolonged incubation of ATS with factor Xa leads to the highly specific hydrolysis of the peptide bond between residues Arg34 and Val35, implicating this peptide bond as the putative reactive site. We report here the preparation of pure, cleaved (modified) recombinant ATS (rATS) and utilize this material to provide additional proof that the cleaved peptide bond is in fact the reactive site. Modified rATS retains strong inhibitory potency against factor Xa as evidenced by a dissociation constant of 166.3 +/- 9.6 pM; four-fold greater than that of native inhibitor, 43.4 +/- 1.4 pM. Incubation of pure, modified rATS with catalytic amounts of factor Xa results in resynthesis of the hydrolyzed peptide bond, achieving an equilibrium near unity between native and modified inhibitors. Specific removal of the newly formed carboxy-terminal Arg residue from modified rATS by carboxypeptidase B treatment obviates its conversion to native inhibitor coincident with the complete loss of inhibitory activity. These results establish that rATS inhibits factor Xa according to a standard mechanism of serine protease inhibitors and support the contention that the Arg34-Val35 peptide bond constitutes the reactive site.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
Antistasin (ATS) is a 119-amino acid, leech-derived protein which exhibits selective, tight-binding inhibition of blood coagulation factor Xa.
Por qué esto importa para la hirudoterapia
La antistasina es una proteína de 119 aminoácidos, derivada de la sanguijuela, que inhibe de manera selectiva y con alta afinidad el factor Xa de la coagulación sanguínea. Este estudio empleó antistasina recombinante purificada para demostrar que el enlace peptídico Arg34–Val35 constituye el sitio reactivo, mostrando que el inhibidor modificado (escindido) conserva una potente actividad anti-Xa (Kd ~166 pM) y puede resintetizar su enlace hidrolizado en presencia de factor Xa catalítico. Este trabajo es directamente relevante para el secretoma de la sanguijuela y el dominio de ASH, ya que caracteriza el mecanismo de una proteína anticoagulante específica derivada de la sanguijuela. La advertencia honesta es que se trata de un estudio puramente bioquímico in vitro con proteína recombinante; no se incluyen sanguijuelas vivas, saliva cruda, ni datos clínicos o in vivo.
Citación
The hydrolysis and resynthesis of a single reactive site peptide bond in recombinant antistasin by coagulation factor Xa
Dunwiddie CT et al. · Archives of biochemistry and biophysics, 1992
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026