THROMBIN-THROMBOMODULIN INTERACTION - ENERGETICS AND POTENTIAL ROLE OF WATER AS AN ALLOSTERIC EFFECTOR

The interaction of rabbit lung thrombomodulin (TM) and C-terminal hiru din 54-65 fragment (Hir(54-65)) with human alpha-thrombin were investi gated by exploiting their competitive inhibition of thrombin-fibrinoge n interaction. Measurements of K-i values for TM and H-54-65 interacti ons with human alpha-thrombin performed over a temperature range spann ing from 10 to 40 degrees C showed a constant enthalpy for both ligand s. The enthalpic and entropic contributions to the free energy of bind ing, however, are different for TM and the hirudin peptide. The calcul ated values of Delta H and Delta S, in fact, were -47.3 +/- 2.51 kJ (- 11.3 +/- 0.6 kcal)/mol and -42.7 +/- 7.9 J (-10.2 +/- 1.9 cal)/mol . K for the hirudin peptide, while being -22.9 +/- 2.09 kJ (-5.47 +/- 0.5 kcal)/mol and 102.50 +/- 6.69 J (24.5 +/- 1.6 cal)/mol . K respective ly for TM binding. These findings indicate that the interaction betwee n thrombin and Hir(54-65) is largely driven by the enthalpic contribut ion, whereas the positive entropy change is the driving force for the formation of the thrombin-TM complex. In other experiments performed i n the presence of various concentrations of either sorbitol or sucrose it could be demonstrated that the value of the equilibrium associatio n constant for thrombin-TM interaction increases as a function of the osmotic pressure, while the thrombin-Hir(54-65) interaction was not af fected by the same conditions. Moreover, control experiments showed th at no major conformational changes are produced on TM by osmotic press ures used in the present study. From these experiments it was calculat ed that roughly 35 water molecules are released into the bulk water up on TM binding. Such a phenomenon, which is likely to be responsible fo r the entropic change described above, indicates the relevance of hydr ation processes for the formation of the thrombin-TM adduct.