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 r oles 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 so luble mutants of TM, TMD123 and TMD12, and three deletion mutants lack ing 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 C OS cells and purified. The results of protein C-activating cofactor as say showed that TMD12 Delta e, TMD12 Delta 18, and TMD12 Delta 38, whi ch lack the C-terminal region, had remarkably weak cofactor activities in comparison with TMD123 (9.1, 1.4, and 1.1% of TMD123 activity, res pectively). Similar findings were obtained for anticoagulant activity. These findings indicate that the last loop structure in E6 is require d for full activity of recombinant human TM. We also determined in viv o stabilities of TMD12, TMD123, and TMD12 Delta 6 in a pharmacokinetic study in rats. TMD12 and TMD12 Delta 6, which lack the D3 domain, exh ibited increased clearance (about twice that of D123). This finding su ggested that the D3 domain of TM plays an important role in stabilizin g TM in vivo.