Sequences required for thrombomodulin cofactor activity within the fourth epidermal growth factor-like domain of human thrombomodulin.
Activation of protein C by thrombin is stimulated by the endothelial cell cofactor thrombomodulin. The structural regions of thrombomodulin necessary for cofactor activity have been localized to the fourth through sixth epidermal growth factor (EGF)-like domains. The fourth EGF-like domain is unnecessary for high affinity thrombin binding, but is required for cofactor activity. To identify essential sequences within the fourth EGF-like domain, a series of recombinant human thrombomodulins consisting of EGF-like domains four through six were expressed in human kidney cells. These mutants contain replacements of disulfide loops within the fourth EGF-like domain, thereby conserving overall disulfide bond structure. All of the mutants bound to thrombin with high affinity, and inhibited the fibrinogen-clotting activity of thrombin to a similar extent. Two regions of the fourth EGF-like domain were identified to be essential for cofactor activity: 1) the sequence consisting of amino acids Glu-357, Tyr-358, and Gln-359 shared by the overlapping 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 thrombomodulin cofactor activity are located near the junction between the two subdomains of the fourth EGF-like domain.
Department of Internal Medicine, University of Iowa, Iowa City 52242.
S R
SR
Lentz
Y
Y
Chen
J E
JE
Sadler
D000595:Amino Acid Sequence; D000925:Anticoagulants; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004220:Disulfides / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000737:chemistry*; D005340:Fibrinogen / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011486:Protein C / Q000378:metabolism; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D018179:Receptors, Thrombin; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000378:metabolism
D000925:Anticoagulants; D004220:Disulfides; D011486:Protein C; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D005340:Fibrinogen; D013917:Thrombin