Allosteric modulation of BPTI interaction with human α‐ and ζ‐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 (TM EGF4–6 ) and Na + . BPTI binding to α‐thrombin is positively linked to Na + . Under low sodium concentration (5 m m Na + ) the BPTI affinity for α‐thrombin was roughly threefold lower than in the presence of 150 m m sodium ( K i  = 320 µ m vs. 100 µ m ). The hirudin fragment, which binds to the fibrinogen recognition site (FRS) of thrombin, induced a progressive and saturable decrease (3.6‐fold) of α‐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 TM EGF4–6 was also observed for BPTI interaction with ζ‐thrombin, in which the amidic bond between W148 and T149 is cleaved. However, in this case the effect by Hir54‐‐65 and TM EGF4–6 , although qualitatively similar to that observed with α‐thrombin, had a smaller magnitude. Thrombin hydrolysis of Protein C was also differently affected by Hir54‐‐65 and TM EGF4–6 peptides. While the latter enhanced the Protein C activation, the former caused a reduction of both α‐ and ζ‐thrombin k cat / K m ′ for Protein C cleavage. These results showed that (a) Na + facilitates BPTI interaction with thrombin; (b) Hir54‐‐65 and TM EGF4–6 , 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 ζ‐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.