The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1
Research article published in European journal of biochemistry (2001)
Abstract
The role of thrombin anion-binding exosite-I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site-directed mutagenesis. Basic residues in anion-binding exosite-I (Arg35, Arg36, Arg67, Arg73, Arg75, Arg77A, Lys81, Lys109, Lys110 and Lys149E) were substituted with glutamines and the resultant recombinant mutant thrombins were used to determine kinetic parameters for the cleavage of a peptide (PAR38-60) based on the PAR1 extracellular domain. Compared with wild-type thrombin, replacement of Arg67 and Arg73 had a dramatic effect on the cleavage of PAR38-60 (k(cat)/K(m) = 1.8 x 10(6) and 4.6 x 10(6) vs 9.2 x 10(7) M(-1).s(-1)), whereas the remaining mutations of the anion-binding exosite-I of thrombin had a less pronounced effect, with k(cat)/K(m) values ranging from 3.3 x 10(7) M(-1). s(-1) (R77(a)Q) to 5.8 x 10(7) M(-1).s(-1) (K109Q). The ability of thrombin mutants to activate platelets paralleled that of PAR38-60 cleavage, whereas their ability to clot fibrinogen differed profoundly, as did their susceptibility to hirudin inhibition. Results are interpreted with respect to known interactions of thrombin with thrombomodulin, hirudin, rhodniin and heparin cofactor II. We conclude that the basic residues of anion-binding exosite-I contribute significantly to enhancing the rate of complex formation in two ways; the first (general) ensures electrostatic steering of ligands with complementary electrostatic fields, the second (specific) involves a combination of molecular contacts within the complex that is unique for each ligand.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
The role of thrombin anion-binding exosite-I in the recognition and cleavage of the extracellular domain of the seven transmembrane domain thrombin receptor (PAR1) was determined using site-directed mutagenesis.
Por qué esto importa para la hirudoterapia
Este estudio utilizó mutagénesis dirigida de los residuos básicos del exositio I de unión a aniones de la trombina (ABE-I) para diseccionar sus funciones en el reconocimiento y escisión del receptor 1 activado por proteasa (PAR1). Las mutaciones de Arg67 y Arg73 redujeron de forma más drástica la eficiencia de escisión del péptido PAR1 (descenso de kcat/Km desde 9,2×10^7 hasta 1,8×10^6 y 4,6×10^6 M−1s−1, respectivamente), mientras que los efectos sobre la coagulación del fibrinógeno y la susceptibilidad a la inhibición por hirudina variaron profundamente entre los mutantes. Los autores interpretan estos hallazgos en el contexto de las interacciones conocidas de la trombina con la hirudina, la trombomodulina, la rodniina y el cofactor II de heparina, concluyendo que los residuos del ABE-I contribuyen tanto mediante direccionamiento electrostático general como mediante contactos específicos de ligando. Esto es relevante para el ámbito de ASH, ya que profundiza en la comprensión mecanística de cómo la hirudina se une al exositio I de la trombina. Sin embargo, el estudio se centra en la biología molecular de la trombina mediante mutagénesis; la hirudina se analiza como ligando comparador en lugar de ser el objeto principal de investigación.
Citación
The dual role of thrombin's anion-binding exosite-I in the recognition and cleavage of the protease-activated receptor 1
Myles T et al. · European journal of biochemistry, 2001
Contexto clínico relacionado
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026