Elements of the primary structure of thrombomodulin required for efficientthrombin-activable fibrinolysis inhibitor activation

Deletion and point mutants of soluble thrombomodulin were used to compare a nd contrast elements of primary structure required for the activation of th rombin-activable fibrinolysis inhibitor (TAFI) and protein C. The smallest mutant capable of efficiently promoting TAFI activation contained residues including the c-loop of epidermal growth factor-3 (EGF3) through EGF6. This mutant is 13 residues longer than the smallest mutant that functioned well with protein C; the latter consisted of residues from the interdomain loop connecting EGF3 and EGF4 through EGF6. Alanine point mutants showed no los s of function in protein C activation for mutations within the c-loop of EG F3. In TAFI activation, however, alanine mutations cause a 50% reduction at Tyr-337, 67% reductions at Asp-338 and Leu-339, and 90% or greater reducti ons at Val-340, Asp-341, and Glu-343. A mutation at Asp-349 in the peptide connecting EGF3 to EGF4 eliminated activity against both TAFI and protein C . Oxidation of Met-388 in the peptide connecting EGF5 to EGF6 reduced the r ate of protein C activation by 80% but marginally, if at all, affected the rate of TAFI activation. Mutation at Phe-376 severely reduced protein C act ivation but only marginally influenced that of TAFI. A Q387P mutation, howe ver, severely reduced both activities. TAFI activation was shown to be Ca2-dependent. The response, unlike that of protein C, was monotonic and was h alf-maximal at 0.25 mM Ca2+. Like protein C activation, TAFI activation was eliminated by a monoclonal antibody directed at the thrombin-binding domai n (EGF5) but was not affected by one directed at EGF2. Thus, elements of st ructure in the thrombin-binding domain are needed for the activation of bot h protein C and TAFI, but more of the primary structure is needed for TAFI activation. In addition, some residues are needed for one of the reactions but not the other.