CONFORMATIONAL TRANSITIONS LINKED TO ACTIVE-SITE LIGATION IN HUMAN THROMBIN - EFFECT ON THE INTERACTION WITH FIBRINOGEN AND THE CLEAVABLE PLATELET RECEPTOR

An experimental strategy based on solution viscosity perturbation allo wed us to study the energetics of amide substrates, p-aminobenzamidine (p-ABZ) and proflavin binding to the catalytic site of two proteolyze d forms of alpha-thrombin, i.e. zeta- and gamma(T)-thrombin. These thr ombin derivatives are cleaved at the Leu144-Gly150 loop and at the fib rinogen recognition exosite (FRS), respectively A phenomenological ana lysis of thermodynamic data showed that the amide substrates and p-ABZ interactions with zeta-thrombin were respectively, associated with a chemical compensation (i.e. the linear relationship between entropy an d enthalpy of binding) and a hydrophobic phenomenon (i.e. a change in the standard heat capacity). The latter Mras slightly lower than that previously observed for a alpha-thrombin (0.78 +/- 0,25 versus 1.01+/- 0.17 kcal/mol K). Both phenomenon were absent in gamma(T)-thrombin. Th e interaction of a alpha-, zeta- and gamma(T)-thrombin with macromolec ular substrates that ''bridge-bind'' to both the catalytic site (CS) a nd fibrinogen recognition exosite (FRS), such as fibrinogen and the cl eavable platelet receptor (CPR), was also evaluated. These interaction s were studied by following fibrinopeptide A (FpA) release and by meas uring intraplatelet Ca2+ changes induced by thrombin-CPR interaction. It was found that the free energy of activation (RT In K-cat/K-m) for both fibrinogen and CPR hydrolysis followed the same hierarchy, i.e. a lpha > zeta > gamma. Moreover, the values of Delta C-p for alpha-, zet a- and gamma(T)-thrombin interaction with p-ABZ were found to be linea rly correlated to the free energy of activation for both fibrinogen an d CPR cleavage. In conclusion, these data demonstrate that: (1) the Le u144-Gly150 loop and the FRS are both involved in the conformational t ransition linked to the binding of p-aminobenzamidine to the thrombin active site; (2) the extent of thrombin's capacity to undergo conforma tional transitions in alpha-, zeta- and gamma(T) forms is positively c orrelated to the free energy of activation for hydrolysis of macromole cular substrates interacting with both the catalytic domain and the FR S.