ROLE OF THE THROMBIN INSERTION LOOP-144-155 - STUDY OF THROMBIN MUTATIONS W148G, K154E AND A THROMBIN-BASED SYNTHETIC PEPTIDE

Thrombin is a multifunctional serine protease that plays a critical ro le in hemostasis. Crystallographic studies revealed that the insertion loop, residues 144-155 (human thrombin B chain numbering) located on the surface of thrombin, might be involved in the access of substrates to the active-site of the enzyme. This loop has also been proposed as a potential candidate for a binding site for thrombomodulin and selec ted thrombin substrates. In order to examine this hypothesis, we have introduced single amino acid substitutions into the loop 144-155 (W148 G, K154E). These point mutations did not result in major changes in th rombin specificity. However, the mutant thrombins presented slight mod ifications in their catalytic activity on the tripeptidic substrate on -benzyloxycarbonyl)-Pro-Arg-NH-{[K154E]thrombin} or to syl-Gly-Pro-Arg -NH-nitro anilide {[W148G]thrombin}, and in the second-order rate cons tants of inhibition by antithrombin III {[K154E]thrombin} and {[W148G] thrombin} compared to recombinant wild-type thrombin. Kinetics of fibr inogen hydrolysis were minimally affected by the K154E mutation and we re not affected by the W148G mutation. Neither of the mutations affect ed thrombin interaction with hirudin or its C-terminal tail, protein C activation by thrombin or thrombin-thrombomodulin, or platelet activa tion. We also examined the properties of a synthetic peptide correspon ding to the sequence T147-S158. The synthetic peptide T147-S158 did no t inhibit thrombin interaction with fibrin, thrombomodulin or protein C. Together, our results indicate that the thrombin loop 144-155 is in directly involved in the catalytic function of the enzyme, most probab ly by limiting the access of the substrates to the catalytic site, and argue against the presence of a recognition exosite for fibrin(ogen), thrombomodulin or platelets within the loop.