PAI-2 was cloned in Dr. Sadler's laboratory and 
shown to be a new member of the serine protease 
inhibitor gene family that is particularly similar to 
chicken ovalbumin. The human PAI-2 gene has now 
been cloned and its structure determined. Six of 
the seven intron positions in the PAI-2 gene are 
identical to those in the ovalbumin gene. Thus PAI- 
2 is the closest known mammalian homologue of 
avian ovalbumin. 
Ovalbumin is unusual among eukaryotic proteins 
because it is secreted efficiently without cleavage of 
an amino-terminal signal peptide. The similarity of 
PAI-2 to ovalbumin suggested that PAI-2 might 
share this property. This was confirmed by study of 
PAI-2 synthesis in a human monocytic cell line. It is 
not known whether this has significance for the 
physiological function of PAI-2 or is only a fortu- 
itous consequence of the homology between PAI-2 
and ovalbumin. 
Endothelial cells were known to make PAI-1 but 
were generally thought not to express PAI-2. Dr. 
Sadler has shown that the levels of PAI-2 mRNA and 
protein are increased in endothelial cells by either 
phorbol esters or TNF-a. This occurs without a de- 
tectable change in PAI-2 transcription. The mRNA 
for PAJ-2 is unstable in endothelial cells but is stabi- 
lized by protein synthesis inhibitors and appears to 
be stabilized transiently by treatment of cells with 
phorbol esters or TNF-a. Thus PAI-2 may be in- 
duced in part by inhibition of mRNA degradation. 
B. Tissue factor. Tissue factor is a membrane recep- 
tor that binds coagulation factor VII, and the tissue 
factor-factor VII complex is the most important 
physiological activator of blood coagulation. Tissue 
factor is found in many tissues but is not normally 
expressed by cells that are in contact with blood. 
However, exposure of endothelium or monocytes 
to inflammatory mediators such as TNF-a rapidly 
induces tissue factor, and this apparently contrib- 
utes to the pathophysiology of disseminated intra- 
vascular coagulation. 
Human tissue factor was cloned in Dr. Sadler's 
laboratory, the gene was localized to human chro- 
mosome 1, and RFLPs with Mspl and Taq\ were 
identified. These cDNA probes were used to study 
the regulation of tissue factor in cultured endothe- 
lial cells. As was found for PAI-2, tissue factor mRNA 
is unstable but can be stabilized by inhibitors of 
protein synthesis. Unlike PAI-2, tissue factor gene 
transcription is induced by phorbol esters or TNF- 
a. Thus tissue factor is regulated at the level of 
transcription and also potentially at the level of 
mRNA stability. The structural elements required 
for this regulation are under study. 
C. Thrombomodulin. Thrombomodulin is an endo- 
thelial cell membrane receptor that binds thrombin 
and alters its substrate specificity. This activity 
serves an essential anticoagulant function. 
ThrombomoduHn is expressed in endothelial cells 
of most blood and lymphatic vessels. Thrombo- 
modulin expression is decreased by inflammatory 
mediators such as TNF-a and interleukin-1, and this 
may contribute to intravascular thrombosis at sites 
of inflammation. 
Human thrombomodulin cDNA clones were iso- 
lated in Dr. Sadler's laboratory, and the gene was 
localized to chromosome 20cen-pl2. These clones 
have been employed to study the regulation of 
thrombomodulin in cultured endothelial cells. The 
thrombomodulin gene appears to be transcribed 
constitutively and to be unaffected by phorbol es- 
ters or TNF-a. Treatment of cells with phorbol 
esters does not alter thrombomodulin activity or 
mRNA levels. However, treatment with TNF-a for 
24 hours causes an ~90% reduction in total and 
cell surface thrombomodulin, with no apparent de- 
crease in mRNA. These data suggest that 
thrombomodulin is not regulated significantly by 
controlling mRNA concentration but must be regu- 
lated during or subsequent to translation. 
Thrombomodulin presumably interacts with 
membrane or cytoskeletal components to undergo 
endocytosis and degradation in response to TNF-a. 
Recombinant thrombomodulin was expressed in 
COS-7, CV-1, and K562 cells and appeared to be 
identical structurally and functionally to nonrecom- 
binant thrombomodulin. Whether endocytosis re- 
quires specific sequences in the cytoplasmic tail or 
in other domains of thrombomodulin is under in- 
vestigation by the expression of thrombomodulin 
mutants in such heterologous cells. 
Further study of tissue factor, PAI-2, and 
thrombomodulin will clarify the endothelial cell's 
role in inflammation and hemostasis and may sug- 
gest a strategy for altering the expression of individ- 
ual proteins to achieve a therapeutic effect in pa- 
tients with bleeding or thrombosis. 
Dr. Sadler is also Associate Professor of Medicine 
and Assistant Professor of Biochemistry and Molec- 
ular Biophysics at the Washington University 
School of Medicine. 
Continued 
102 
