An experimental strategy based on solution viscosity perturbation allowed us to study the energetics of amide substrates, p-aminobenzamidine ( p-ABZ) and proflavin binding to the catalytic site of two proteolyzed forms of α-thrombin, i.e. ζ- and γ T-thrombin. These thrombin derivatives are cleaved at the Leu144-Gly150 loop and at the fibrinogen recognition exosite (FRS), respectively. A phenomenological analysis of thermodynamic data showed that the amide substrates and p-ABZ interactions with ζ-thrombin were respectively, associated with a chemical compensation (i.e. the linear relationship between entropy and enthalpy of binding) and a hydrophobic phenomenon (i.e. a change in the standard heat capacity). The latter was slightly lower than that previously observed for a α-thrombin (0.78±0.25 versus1.01±0.17 kcal/mol K). Both phenomenon were absent in γ T-thrombin. The interaction of a α-, ζ- and γ T-thrombin with macromolecular substrates that “bridge-bind” to both the catalytic site (CS) and fibrinogen recognition exosite (FRS), such as fibrinogen and the cleavable platelet receptor (CPR), was also evaluated. These interactions ere studied by following fibrinopeptide A (FpA) release and by measuring intraplatelet Ca 2+changes induced by thrombin-CPR interaction. It was found that the free energy of activation ( RTln k cat/ K m) for both fibrinogen and CPR hydrolysis followed the same hierarchy, i.e. α>ζ>γ. Moreover, the values of ΔC pfot α-, δ- and γ T- thrombin interaction with p-ABZ were found to be linearly correlated to the free energy of activation for both fibrinogen and CPR cleavage. In conclusion, these data demonstrate that: (1) the Leu144-Gly150 loop and the FRS are both involved in the conformational transition linked to the binding of p-aminobenzamidine to the thrombin active site; (2) the extent of thrombin's capacity to undergo conformational transitions in α-, ζ and γ Tforms is positively correlated to the free energy of activation for hydrolysis of macromolecular substrates interacting with both the catalytic domain and the FRS. f2 f2 |em|Corresponding author at: Centro Ricerche Fisiopatologia dell'Emostasi, Universitaè Cattolica S. Cuore, Largo F. Vito 1, 00168 Roma, Italy. f3 f3 |em|Abbreviations used: CS, catalytic site; p-ABZ, p-aminobenzamidine; CPR, cleavable platelet receptor; FRS, fibrinogen recognition site; PEG, polyethylene glycol; FpA, fibrinopeptide A; fluo-3-Am, fluo-3-acetoxymethyl ester; PRP, platelet-rich plasma. Numbering of thrombin residues was done according to the chymotrypsin nomenclature.