G. Honda et al., THE ROLES PLAYED BY THE D2 AND D3 DOMAINS OF RECOMBINANT HUMAN THROMBOMODULIN IN ITS FUNCTION, Journal of Biochemistry, 118(5), 1995, pp. 1030-1036
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.