W. Wang et al., Elements of the primary structure of thrombomodulin required for efficientthrombin-activable fibrinolysis inhibitor activation, J BIOL CHEM, 275(30), 2000, pp. 22942-22947
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.