Demonstration of exosite I-dependent interactions of thrombin with human factor V and factor Va involving the factor Va heavy chain: analysis by affinity chromatography employing a novel method for active-site-selective immobilization of serine proteinases.
Biochemistry
; 37(38): 13143-52, 1998 Sep 22.
Article
en En
| MEDLINE
| ID: mdl-9748321
ABSTRACT
In an essential step of blood coagulation, factor V is proteolytically processed by thrombin to generate the activated protein cofactor, factor Va, and to release the activation fragments E and C1. For the identification and characterization of sites of thrombin binding to factor V and its activation products, a new method was developed for immobilizing thrombin and other serine proteinases specifically (>/=92%) through their active sites and used in affinity chromatography studies of the interactions. Interactions of factor V with exosite I of thrombin were shown to regulate the factor V activation pathway from the 93% +/- 12% inhibition of the rate of activation correlated with specific binding of hirudin54-65 to this exosite. Chromatography of factor V on active-site-immobilized thrombin showed only a weak interaction, while the factor Va heterodimer bound specifically and with apparently higher affinity, in an interaction that was prevented by hirudin54-65. The heavy chain of subunit-dissociated factor Va bound to immobilized thrombin, while the light-chain subunit and fragment E had no detectable affinity. These results demonstrate a previously undescribed, exosite I-dependent interaction of thrombin with factor Va that occurs through the factor Va heavy chain. They support the further conclusion that similar exosite I-dependent binding of thrombin to the heavy-chain region of factor V contributes to recognition of factor V as a specific thrombin substrate and thereby regulates proteolytic activation of the protein cofactor.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Factor V
/
Serina Endopeptidasas
/
Trombina
/
Factor Va
/
Enzimas Inmovilizadas
Tipo de estudio:
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Biochemistry
Año:
1998
Tipo del documento:
Article
País de afiliación:
Estados Unidos