platelets. On binding to thrombomodulin, however, 
the procoagulant activities of thrombin are reduced, 
and its ability to activate protein C, a serine protease 
zymogen, is greatly increased. Activated protein C 
degrades clotting factors Va and Villa. Thus the for- 
mation of the thrombin-thrombomodulin complex 
converts thrombin from a procoagulant into an anti- 
coagulant enzyme, and the normal balance between 
these opposing activities is critical to the regulation 
of hemostasis. 
Inhibition of thrombomodulin expression by 
the thrombogenic agent homocysteine. Homocys- 
teinemia caused by cystathionine |8-synthase defi- 
ciency is associated with thrombosis and atheroscle- 
rosis, but the mechanisms responsible for this 
association are unknown. Studies in cultured human 
endothelial cells, or CV-1 cells expressing recombi- 
nant human thrombomodulin, suggest that homo- 
cysteine can reduce thrombomodulin cell surface 
expression. Homocysteine produced slight in- 
creases in thrombomodulin mRNA and thrombomo- 
dulin synthesis without affecting cell viability. The 
newly synthesized thrombomodulin was not trans- 
ported to the cell surface but was retained in the 
endoplasmic reticulum. By altering the expression 
of hemostatic proteins in endothelial cells, homo- 
cysteine may contribute to the thrombosis in pa- 
tients with cystathionine /3-synthase deficiency. 
Thrombomodulin structure-function relation- 
ships. Thrombomodulin contains an amino-terminal 
extracellular domain, one transmembrane domain, 
and a short cytoplasmic tail. The extracellular do- 
main is composed of a lectin-like domain, six tan- 
demly repeated epidermal growth factor (EGF)- 
like domains, and an 0-glycosylated Ser/Thr-rich 
domain. EGF-like domains 5 and 6 contain a 
major thrombin-binding site. Thrombin-binding se- 
quences were further localized by employing syn- 
thetic peptides derived from thrombomodulin to in- 
hibit thrombin binding. Each EGF-like domain 
contains ~40 amino acids that form three disulfide 
loops. The most active peptide inhibitors corre- 
sponded to the third loop of the fifth EGF-like do- 
main and to parts of the second and third loops of 
the sixth EGF-like domain. Replacement of the 
Ser/Thr-rich domain with a segment devoid of gly- 
cosylation sites did not affect the cofactor function 
of thrombomodulin, whereas substitution of this 
domain with segments of decreasing length progres- 
sively decreased both cofactor activity and throm- 
bin-binding affinity. Thus the Ser/Thr-rich domain 
is required to position the thrombin-binding site 
optimally above the membrane surface, and O- 
linked glycosylation is not required for cofactor 
activity. 
Ligand specificity of human thrombomodulin. 
Thrombomodulin has been proposed to bind pro- 
teases other than thrombin and thereby to regulate 
blood coagulation by several independent mecha- 
nisms. Both bovine factor Xa and bovine mei- 
zothrombin, an intermediate product of prothrom- 
bin activation, have been reported to catalyze 
thrombomodulin-dependent activation of protein 
C. To examine the ligand specificity of human 
thrombomodulin, equilibrium binding assays were 
performed with human thrombin, thrombin S205A 
(wherein the active-site serine is replaced by ala- 
nine), meizothrombin S205A, and human factor Xa. 
Human meizothrombin did not compete with 
thrombin for binding to recombinant human throm- 
bomodulin, nor did meizothrombin bind directly to 
thrombomodulin. Similarly, human factor Xa did 
not bind to thrombomodulin, nor did it catalyze the 
thrombomodulin-dependent activation of protein 
C. These results suggest that, in contrast to results 
reported for the bovine proteins, human mei- 
zothrombin and factor Xa are unlikely to be impor- 
tant thrombomodulin-dependent activators of pro- 
tein C and that thrombin is the physiological 
protease ligand for human thrombomodulin. 
These studies will improve understanding of how 
endothelial ceil proteins may participate in inflam- 
mation and hemostasis. Such knowledge may sug- 
gest new strategies for the treatment of patients with 
bleeding or thrombosis. (The projects described in 
this section are supported by a grant from the Na- 
tional Institutes of Health.) 
Dr. Sadler is also Associate Professor of Medi- 
cine and Assistant Professor of Biochemistry and 
Molecular Biophysics at the Washington Univer- 
sity School of Medicine, St. Louis. 
Articles 
Inbal, A., Seligsohn, U., Kornbrot, N., Brenner, B., 
Harrison, P., Randi, A., Rabinowitz, I., and 
Sadler, J. E. 1992. Characterization of three mu- 
tations causing von Willebrand disease type HA in 
five unrelated families. Thromb Haemost 
67:618-622. 
Jorieux, S., Tuley, E.A., Gaucher, C., Mazurier, 
C., and Sadler, J.E. 1992. The mutation 
Arg(53)^Trp causes von Willebrand disease 
Normandy by abolishing binding to factor VHI. 
Studies with recombinant von Willebrand factor. 
Blood 79:563-567. 
Lentz, S.R., and Sadler, J.E. 1991. Inhibition of 
thrombomodulin surface expression and protein 
CELL BIOLOGY AND REGULATION 
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