Elements of the primary structure of thrombomodulin required for efficient thrombin-activable fibrinolysis inhibitor activation.
Deletion and point mutants of soluble thrombomodulin were used to compare and contrast elements of primary structure required for the activation of thrombin-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 loss of function in protein C activation for mutations within the c-loop of EGF3. 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 reductions 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 rate 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 activation but only marginally influenced that of TAFI. A Q387P mutation, however, severely reduced both activities. TAFI activation was shown to be Ca(2+)-dependent. The response, unlike that of protein C, was monotonic and was half-maximal at 0.25 mm Ca(2+). Like protein C activation, TAFI activation was eliminated by a monoclonal antibody directed at the thrombin-binding domain (EGF5) but was not affected by one directed at EGF2. Thus, elements of structure in the thrombin-binding domain are needed for the activation of both 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.
Wang
W
W
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
Nagashima
M
M
Schneider
M
M
Morser
J
J
Nesheim
M
M
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D025901:Carboxypeptidase B2; D002268:Carboxypeptidases / Q000494:pharmacology*; D005342:Fibrinolysis / Q000187:drug effects*; D017354:Point Mutation; D011486:Protein C / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D011486:Protein C; D018180:Thrombomodulin; D002268:Carboxypeptidases; D025901:Carboxypeptidase B2; D013917:Thrombin; D002118:Calcium