The roles played by the D2 and D3 domains of recombinant human thrombomodulin in its function.
Thrombomodulin (TM) is composed of five domains. We investigated the roles of the sixth epidermal growth factor (EGF)-like structure (E6) in the second domain (D2) and of an O-glycosylation site rich domain (D3) in the function of TM in more detail using deletion analysis. Two soluble mutants of TM, TMD123 and TMD12, and three deletion mutants lacking respectively 6, 16, and 38 C-terminal residues of the E6 portion, TMD12 delta 6, TMD12 delta 16, and TMD12 delta 38, were expressed in COS cells and purified. The results of protein C-activating cofactor assay showed that TMD12 delta 6, TMD12 delta 16, and TMD12 delta 38, which lack the C-terminal region, had remarkably weak cofactor activities in comparison with TMD123 (9.1, 1.4, and 1.1% of TMD123 activity, respectively). Similar findings were obtained for anticoagulant activity. These findings indicate that the last loop structure in E6 is required for full activity of recombinant human TM. We also determined in vivo stabilities of TMD12, TMD123, and TMD12 delta 6 in a pharmacokinetic study in rats. TMD12 and TMD12 delta 6, which lack the D3 domain, exhibited increased clearance (about twice that of D123). This finding suggested that the D3 domain of TM plays an important role in stabilizing TM in vivo.
Honda
G
G
Laboratory for Biological Research, Asahi Chemical Industry Co. Ltd., Fuji, Shizuoka.
Masaki
C
C
Zushi
M
M
Tsuruta
K
K
Sata
M
M
D000925:Anticoagulants / Q000378:metabolism; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D009154:Mutation; D011486:Protein C / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology; D012995:Solubility; D013329:Structure-Activity Relationship; D018180:Thrombomodulin / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
D000925:Anticoagulants; D011486:Protein C; D011994:Recombinant Proteins; D018180:Thrombomodulin; D004815:Epidermal Growth Factor