ENERGETICS OF THROMBIN-THROMBOMODULIN INTERACTION

Temperature and salt dependence studies of thrombin interaction with t hrombomodulin, with and without chondroitin sulfate, and two fragments containing the EGF-like domains 4-5 and 4-5-6 reveal the energetic si gnatures and the mechanism of recognition of this physiologically impo rtant cofactor, Binding of thrombomodulin is affected drastically by t he particular salt present in solution and is positively linked to Na binding to thrombin and the conversion of the enzyme from the slow to the fast form, but is opposed by Cl- binding to the fibrinogen recogn ition site and especially to the heparin binding site. Binding of thro mbomodulin has an unusually large salt dependence (Gamma(salt) = -4.8) contributed mostly by the polyelectrolyte-like nature of the chondroi tin sulfate moiety that binds to the heparin binding site and increase s the affinity of the cofactor by almost 10-fold. On the other hand, t he chondroitin sulfate has no effect on the Delta C-p of binding, whic h is determined predominantly by contacts made by the EGF-like domains 5 and 6 with the fibrinogen recognition site. The modest heat capacit y change (-0.2 kcal mol(-1) K-1) observed when thrombomodulin binds to the fast form suggests a rigid-body association of the cofactor with the enzyme. In the slow form, however, the heat capacity change is sig nificantly more pronounced (-0.5 kcal mol(-1) K-1) and signals the pre sence of a conformational transition of the enzyme linked to binding o f the cofactor that mimics the slow-->fast conversion. These results d emonstrate that recognition of thrombomodulin by thrombin is steered e lectrostatically by the highly charged regions of the fibrinogen recog nition site and the heparin binding site, to which the chondroitin sul fate moiety binds and enhances the affinity of the interaction. The re cognition event also involves conformational changes of the enzyme in the slow form mediated by binding of the EGF-like domains 5-6 to the f ibrinogen recognition site. Consistent with this model, binding of thr ombomodulin to the fast form has only a small effect on the hydrolysis of nine chromogenic substrates carrying substitutions at P1, P2, and P3 aimed at probing the environment of the specificity sites S1, S2, a nd S3 of the enzyme. Binding to the slow form, on the other hand, enha nces the specificity toward all substrates up to 15-fold. For substrat es carrying a Gly at P2, binding of thrombomodulin changes the relativ e specificity of the slow and fast forms and makes the slow form more specific, Interestingly, these effects are not specific of thrombomodu lin and depend solely on binding to the fibrinogen recognition site of the enzyme. In fact, they are also observed with the hirudin C-termin al fragment 55-65. The characterization of the mechanism of thrombin-t hrombomodulin interaction and the effects of the cofactor on the hydro lysis of chromogenic substrates probing the interior of the catalytic pocket bear on the thrombomodulin-induced enhancement of protein C cle avage by thrombin. We propose that this enhancement is due predominant ly to an effect of thrombomodulin on the bound protein C in the ternar y complex. Therefore, thrombomodulin would carry out its physiological function by making protein C a better substrate for thrombin, rather than making thrombin a better enzyme for protein C.