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

In this study, thrombin interaction with the basic pancreatic trypsin inhib itor (BPTI) was investigated in the presence of different allosteric modula tors of thrombin, that is the C-terminal hirudin peptide 54-65 (Hh54-65), a recombinant thrombomodulin form (TMEGF4-6) and Na+. BPTI binding to alpha- thrombin is positively linked to Na+ Under low sodium concentration (5 mM N a+) the BPTI affinity for alpha-thrombin was roughly threefold lower than i n the presence of 150 mM sodium (K-i = 320 mu M vs. 100 mu M). 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 mor e 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 wa s also observed for BPTI interaction with zeta-thrombin, in which the amidi c bond between W148 and T149 is cleaved. However, in this case the effect b y Hir54-65 and TMEGF4-6, although qualitatively similar to that observed wi th alpha-thrombin, had a smaller magnitude. Thrombin hydrolysis of Protein C was also differently affected by Hir54-65 and TMEGF4-6 peptides. While th e latter enhanced the Protein C activation, the former caused a reduction o f both alpha- and S-thrombin k(cat)/K-m' for Protein C cleavage. These resu lts showed that (a) Na+ facilitates BPTI interaction with thrombin; (b) Hir 54-65 and TMEGF4-6, though sharing in part the same binding site at the thr ombin FRS, can affect in opposite way thrombin's interaction with BPTI and Protein; (c) such findings along with the results obtained with zeta-thromb in might be explained by admitting that the thermodynamic linkage between F RS and the critical W60-loop is also controlled by ligation and/or conforma tional state of the W148 insertion loop.