Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity.
Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich domain as an important spacer, in addition to EGF-like domains 4-6, for efficient protein C activation.
Tsiang
M
M
Howard Hughes Medical Institute, Jewish Hospital of Saint Louis, Washington University School of Medicine, Missouri 63110.
Lentz
S R
SR
Sadler
J E
JE
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002872:Chromosome Deletion; D004815:Epidermal Growth Factor / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism*; D018179:Receptors, Thrombin; D015183:Restriction Mapping; D012694:Serine; D013912:Threonine; D013917:Thrombin / Q000378:metabolism*; D014162:Transfection
D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D013912:Threonine; D012694:Serine; D004815:Epidermal Growth Factor; D013917:Thrombin