Allosteric modulation of BPTI interaction with human alpha- and zeta-thrombin.

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.