Allosteric modulation of BPTI interaction with human alpha- and zeta-thrombin.
Research article published in European journal of biochemistry (1999)
Abstract
In this study, thrombin interaction with the basic pancreatic trypsin inhibitor (BPTI) was investigated in the presence of different allosteric modulators of thrombin, that is the C-terminal hirudin peptide 54-65 (Hir54-65), a recombinant thrombomodulin form (TMEGF4-6) and Na+. BPTI binding to alpha-thrombin is positively linked to Na+. Under low sodium concentration (5 mM Na+) the BPTI affinity for alpha-thrombin was roughly threefold lower than in the presence of 150 mM sodium (Ki = 320 microM vs. 100 microM). The hirudin fragment, which binds to the fibrinogen recognition site (FRS) of thrombin, induced a progressive and saturable decrease (3.6-fold) of alpha-thrombin affinity for BPTI, whereas the thrombomodulin peptide, which binds to a more extended region of FRS, caused a 5.5-fold increase of the enzyme affinity for the inhibitor. The opposite effect exerted by Hir54-65 and TMEGF4-6 was also observed for BPTI interaction with zeta-thrombin, in which the amidic bond between W148 and T149 is cleaved. However, in this case the effect by Hir54-65 and TMEGF4-6, although qualitatively similar to that observed with alpha-thrombin, had a smaller magnitude. Thrombin hydrolysis of Protein C was also differently affected by Hir54-65 and TMEGF4-6 peptides. While the latter enhanced the Protein C activation, the former caused a reduction of both alpha- and zeta-thrombin kcat/K(m)' for Protein C cleavage. These results showed that (a) Na+ facilitates BPTI interaction with thrombin; (b) Hir54-65 and TMEGF4-6, though sharing in part the same binding site at the thrombin FRS, can affect in opposite way thrombin's interaction with BPTI and Protein C; (c) such findings along with the results obtained with zeta-thrombin might be explained by admitting that the thermodynamic linkage between FRS and the critical W60-loop is also controlled by ligation and/or conformational state of the W148 insertion loop.
Abstract sourced from PubMed (NCBI) for the cited record. See the original publication for the authoritative version.
Zusammenfassung
In this study, thrombin interaction with the basic pancreatic trypsin inhibitor (BPTI) was investigated in the presence of different allosteric modulators of thrombin, that is the C-terminal hirudin peptide 54-65 (Hir54-65), a recombinant thrombomodulin form (TMEGF4-6) and Na+. BPTI binding to...
Warum dies für die Hirudotherapie relevant ist
Diese In-vitro-kinetische Studie untersuchte, wie allosterische Modulatoren – einschließlich des C-terminalen Hirudin-Peptids Hir54-65, eines rekombinanten Thrombomodulin-Fragments (TMEGF4-6) und Na+ – die Bindung von BPTI an menschliches Alpha- und Zeta-Thrombin beeinflussen. Das Hirudin-Fragment, das die Fibrinogen-Erkennungsstelle von Thrombin bindet, verringerte die Affinität von Alpha-Thrombin für BPTI um das 3,6-fache, während TMEGF4-6 sie um das 5,5-fache erhöhte; beide modulierten außerdem die Protein-C-Aktivierung in gegensätzlicher Weise. Diese Studie ist für die Hirudotherapie-Forschung direkt relevant, da sie die molekulare Wechselwirkung eines definierten Hirudin-abgeleiteten Peptids mit Thrombin charakterisiert und das Verständnis dafür vertieft, wie Komponenten des Blutegelsekretoms diese zentrale Gerinnungsprotease allosterisch regulieren. Die Einschränkung besteht darin, dass die Arbeit rein biochemisch/in vitro ist, ein synthetisches Hirudin-Fragment anstelle von Gesamthirudin oder rohem Blutegelsekret verwendet und keine therapeutische Anwendung der Hirudotherapie in vivo adressiert.
Zitation
Allosteric modulation of BPTI interaction with human alpha- and zeta-thrombin.
De Cristofaro et al. · European journal of biochemistry, 1999
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