SEQUENCES REQUIRED FOR THROMBOMODULIN COFACTOR ACTIVITY WITHIN THE 4TH EPIDERMAL GROWTH FACTOR-LIKE DOMAIN OF HUMAN THROMBOMODULIN

Activation of protein C by thrombin is stimulated by the endothelial c ell cofactor thrombomodulin. The structural regions of thrombomodulin necessary for cofactor activity have been localized to the fourth thro ugh sixth epidermal growth factor (EGF)-like domains. The fourth EGF-l ike domain is unnecessary for high affinity thrombin binding, but is r equired for cofactor activity. To identify essential sequences within the fourth EGF-like domain, a series of recombinant human thrombomodul ins consisting of EGF-like domains four through six were expressed in human kidney cells. These mutants contain replacements of disulfide lo ops within the fourth EGF-like domain, thereby conserving overall disu lfide bond structure. All of the mutants bound to thrombin with high a ffinity, and inhibited the fibrinogen-clotting activity of thrombin to a similar extent. Two regions of the fourth EGF-like domain were iden tified to be essential for cofactor activity: 1) the sequence consisti ng of amino acids Glu-357, Tyr-358, and Gln-359 shared by the overlapp ing first and second disulfide loops, and 2) the amino-terminal region of the third disulfide loop containing amino acids Glu-374, Gly-375, and Phe-376. These results suggest that amino acids critical for throm bomodulin cofactor activity are located near the junction between the two subdomains of the fourth EGF-like domain.