Structure-function and refolding studies of the thrombin-specific inhibitor hirudin
Research article published in Haemostasis (1991)
Abstract
We have developed a novel expression and purification system that yields recombinant desulfo-hirudin (HV-1) with high specific activity (10,000 antithrombin units/mg) and an inhibition constant (Ki) for human alpha-thrombin of 0.2 pM. Reduced and denatured hirudin rapidly refolds to the native, fully active conformation at high concentration (greater than 50 mg/ml) by incubation at pH 10. Analytical gel filtration studies at neutral pH suggest that hirudin is a multimer. Initial binding of hirudin to thrombin appears to be followed by dissociation of the hirudin multimer to give a tight-binding 1:1 hirudin:thrombin complex. Thrombin inhibition studies showed that hirudin synthetic peptide fragments 42-65 and 51-65 [but not (Ala22)-6-28, containing two of the three disulfide bonds formed in native hirudin] were similarly effective in inhibiting thrombin cleavage of fibrinogen (IC50 = 4.9 and 6.0 microM, respectively, at a thrombin concentration of 1 microM). We conclude that hirudin has unusual structural and refolding properties and that its mechanism of inhibition involves noncovalent interaction with multiple sites on thrombin. The interaction of hirudin (specifically the region of Lys-47) with the basic specificity pocket of thrombin may contribute to the binding but is not essential for its inhibitory activity.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Resumen
Structure-function and refolding studies of the thrombin-specific inhibitor hirudin.
Por qué esto importa para la hirudoterapia
El resumen describe un sistema novedoso de expresión y purificación que produce desulfo-hirudina recombinante (HV-1) con alta actividad específica (10.000 unidades antitrombina/mg) y una Ki para alfa-trombina humana de 0,2 pM, junto con estudios de replegamiento, multimerización e inhibición de trombina por fragmentos peptídicos (42-65, 51-65). El trabajo caracteriza las propiedades estructurales y de replegamiento inusuales de la hirudina y su mecanismo multisitio no covalente de inhibición de la trombina. El resumen en sí no identifica la hirudina como derivada de sanguijuela, no menciona sanguijuelas ni invoca el secretoma de la sanguijuela; la relevancia para ASH o la hirudoterapia es por tanto solo inferible a partir del conocimiento general del origen de la hirudina. Se trata estrictamente de un estudio bioquímico in vitro utilizando proteína recombinante y fragmentos sintéticos.
Citación
Structure-function and refolding studies of the thrombin-specific inhibitor hirudin
Johnson PH et al. · Haemostasis, 1991
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Añadido a la biblioteca ASH: May 27, 2026 · Última actualización del sitio: 18 de junio de 2026