Abstract
Thrombomodulin (TM) is a cofactor for activation of protein C by thrombin. We showed that 80-90% of this cofactor activity is lost by oxidation of Met388, located within the short interdomain loop between epidermal growth factor-like domains 4 and 5 (Glaser, C. B., Morser, J., Clarke, J. H., Blasko, E., McLean, K., Kuhn, I., Chang, R.-J., Lin, J.-H., Vilander, L., Andrews, W. H., and Light, D. R. (1992) J. Clin. Invest. 90, 2565-2573). For each of the 3 amino acids of the loop, site-specific mutants are described in which, 1) all possible single amino acid substitutions are made, 2) deletions are made, or 3) alanine is inserted adjacent to each residue of the loop. Most substitutions within the loop (38/57) result in a > 50% decrease in cofactor activity, while changes in the length of this region result in > 90% loss of activity. Only the Met388-->Leu mutant has higher cofactor activity (2-fold) than wild-type TM. A number of soluble and full-length TM analogs with the Met388-->Leu substitution are improved thrombin cofactors, whether produced in bacteria, insect, or mammalian cells. Detailed kinetic analysis of a soluble TM analog consisting of the six EGF-like domains secreted from insect cells shows that the enhanced activity of the Met388-->Leu mutant results from an increased catalytic efficiency (kcat/Km). This enhancement is maximal at physiological concentrations of calcium. The loss of activity following Met388 oxidation in the wild-type protein is the result of both decreased binding to thrombin (Kd effect) and a decreased interaction of the TM.thrombin complex with protein C (Km effect). We demonstrate the critical role of this interdomain loop in the biological anticoagulant properties of TM.
Highlights
Protein C by thrombin.We showed that SO-90% of this andVIIIa(Fulcher et al, 1984) andtherebyinhibitsthe cofactor activity is lost by oxidationof Metsss, located generation of thrombin
Metm is the central amino acid in the interdomain loop connecting two essential EGF-like domains in thrombomodulinandthismethionineis very susceptibletooxidation (Glaser eat l., 1992).EGF-like domains arehighly constrained by the threedisulfide bonds by which they aredefined
T M structure the interdomainloop joining EGF4 and EGF5 is sandwichedbetweenthe 2 cysteines which arepart of disulfide bonds forming the C-loop of EGF4 and the A-loop
Summary
Protein C by thrombin.We showed that SO-90% of this andVIIIa(Fulcher et al, 1984) andtherebyinhibitsthe cofactor activity is lost by oxidationof Metsss, located generation of thrombin.
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