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.
Summary
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...
Why This Matters for Hirudotherapy
This in vitro kinetic study investigated how allosteric modulators — including the C-terminal hirudin peptide Hir54-65, a recombinant thrombomodulin fragment (TMEGF4-6), and Na+ — influence the binding of BPTI to human alpha- and zeta-thrombin. The hirudin fragment, which binds thrombin's fibrinogen recognition site, reduced alpha-thrombin affinity for BPTI 3.6-fold, while TMEGF4-6 increased it 5.5-fold; both also oppositely modulated Protein C activation. This study is directly relevant to hirudotherapy research because it characterizes the molecular interaction of a defined hirudin-derived peptide with thrombin, advancing understanding of how leech secretome components allosterically regulate this key coagulation protease. The caveat is that the work is purely biochemical/in vitro, uses a synthetic hirudin fragment rather than whole hirudin or crude leech secretion, and does not address therapeutic application of hirudotherapy in vivo.
Citation
Allosteric modulation of BPTI interaction with human alpha- and zeta-thrombin.
De Cristofaro et al. · European journal of biochemistry, 1999
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