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rdf:type | |
lifeskim:mentions | |
pubmed:issue |
32
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pubmed:dateCreated |
1997-9-5
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pubmed:abstractText |
Exosite I of the blood clotting proteinase, thrombin, mediates interactions of the enzyme with certain inhibitors, physiological substrates and regulatory proteins. Specific binding of a fluorescein-labeled derivative of the COOH-terminal dodecapeptide of hirudin ([5F] Hir54-65) to exosite I was used to probe changes in the function of the regulatory site accompanying inactivation of thrombin by its physiological serpin inhibitor, antithrombin. Fluorescence-monitored equilibrium binding studies showed that [5F]Hir54-65 and Hir54-65 bound to human alpha-thrombin with dissociation constants of 26 +/- 2 nM and 38 +/- 5 nM, respectively, while the affinity of the peptides for the stable thrombin-antithrombin complex was undetectable (>/=200-fold weaker). Kinetic studies showed that the loss of binding sites for [5F]Hir54-65 occurred with the same time-course as the loss of thrombin catalytic activity. Binding of [5F] Hir54-65 and Hir54-65 to thrombin was correlated quantitatively with partial inhibition of the rate of the thrombin-antithrombin reaction, maximally decreasing the bimolecular rate constants 1.7- and 2.1-fold, respectively. These results support a mechanism in which thrombin and the thrombin-Hir54-65 complex can associate with antithrombin and undergo formation of the covalent thrombin-antithrombin complex at modestly different rates, with inactivation of exosite I leading to dissociation of the peptide occurring subsequent to the rate-limiting inactivation of thrombin. This mechanism may function physiologically in localizing the activity of thrombin by allowing inactivation of thrombin that is bound in exosite I-mediated complexes with regulatory proteins, such as thrombomodulin and fibrin, without prior dissociation of these complexes. Concomitant with inactivation of thrombin, the thrombin-antithrombin complex may be irreversibly released due to exosite I inactivation.
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pubmed:grant | |
pubmed:language |
eng
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pubmed:journal | |
pubmed:citationSubset |
IM
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pubmed:chemical | |
pubmed:status |
MEDLINE
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pubmed:month |
Aug
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pubmed:issn |
0021-9258
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pubmed:author | |
pubmed:issnType |
Print
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pubmed:day |
8
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pubmed:volume |
272
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pubmed:owner |
NLM
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pubmed:authorsComplete |
Y
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pubmed:pagination |
19837-45
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pubmed:dateRevised |
2007-11-14
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pubmed:meshHeading |
pubmed-meshheading:9242645-Antithrombin III,
pubmed-meshheading:9242645-Binding, Competitive,
pubmed-meshheading:9242645-Binding Sites,
pubmed-meshheading:9242645-Catalysis,
pubmed-meshheading:9242645-Hirudins,
pubmed-meshheading:9242645-Humans,
pubmed-meshheading:9242645-Kinetics,
pubmed-meshheading:9242645-Macromolecular Substances,
pubmed-meshheading:9242645-Spectrometry, Fluorescence,
pubmed-meshheading:9242645-Thrombin
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pubmed:year |
1997
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pubmed:articleTitle |
Inactivation of thrombin by antithrombin is accompanied by inactivation of regulatory exosite I.
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pubmed:affiliation |
Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA. paul.bock@mcmail.vanderbilt.edu
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pubmed:publicationType |
Journal Article,
Research Support, U.S. Gov't, P.H.S.,
Research Support, Non-U.S. Gov't
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