MOLECULAR AND CELL BIOLOGY OF BLOOD COAGULATION 
J. Evan Sadler, M.D., Ph.D., Associate Investigator 
Dr. Sadler is interested in the structure, function, 
and regulation of proteins that are required for he- 
mostasis. In particular, he studies how cells that 
contact the blood maintain a balance between stim- 
ulating and inhibiting thrombosis and how this bal- 
ance is disrupted in many human diseases. Dr. 
Sadler has concentrated on two areas: 1) von 
Willebrand factor and von Willebrand disease and 
2) the regulation of blood clotting reactions by en- 
dothelial cells and monocytes. 
L Molecular Biology of Human von Willebrand Fac- 
tor and von Willebrand Disease. 
Von Willebrand factor is a multimeric plasma gly- 
coprotein that is required for platelet adhesion to 
sites of injury and for normal survival of factor VIII 
in the circulation. Deficiency of von Willebrand fac- 
tor (von Willebrand disease) may cause bleeding 
that resembles platelet dysfunction or hemophilia. 
Von Willebrand disease is the most common inher- 
ited bleeding disorder of humans and is phenotypi- 
cally heterogeneous. Knowledge of the molecular 
defect in variants of von Willebrand disease will il- 
luminate structure-function relationships of von 
Willebrand factor and will assist the development 
of improved therapy. 
A. Structure of the von Willebrand factor gene. The 
von Willebrand factor gene was cloned, and its 
structure was determined by restriction mapping 
and DNA sequencing. The gene spans —178 kb and 
contains 52 exons. The von Willebrand factor sub- 
unit has a highly repeated structure, and the gene 
segments that encode homologous domains are 
similar in structure. This is consistent with the evo- 
lution of the protein by gene duplication or exon 
shuffling. Some features of the gene structure have 
found immediate clinical use. A polymorphic BKm 
(banded krait minor) repeat or VNTR (variable 
number of tandem repeats) region was identified in 
intron 40 and was used as a marker to diagnose se- 
vere von Willebrand disease in a fetus for whom the 
known RFLP (restriction fragment length polymor- 
phism) markers were not informative. 
Knowledge of the gene structure permitted Dr. 
Sadler to determine the molecular defect in several 
patients with severe von Willebrand disease type 
III. Deletions of the entire gene were found in two 
unrelated patients. Three other patients were 
shown to have partial gene deletions. One of these 
patients had a small homozygous deletion that in- 
cluded only a single exon. These patients are un- 
usual because they have produced alloantibody in- 
hibitors of transfused von Willebrand factor. In 27 
families with von Willebrand disease type III that do 
not have alloantibody inhibitors, no gene deletions 
have been found. In contrast, in 10 families with al- 
loantibody inhibitors, six deletions have been iden- 
tified. Thus gene deletions may predispose patients 
to this rare complication of therapy. 
B. Structure of the von Willebrand factor pseudo- 
gene. In addition to the von Willebrand factor gene 
on chromosome 12, a partial unprocessed pseu- 
dogene on chromosome 22 was cloned and se- 
quenced. The pseudogene corresponds to exons 
23-34 of the authentic gene. The von Willebrand 
factor gene and pseudogene differ in sequence by 
only ~3%, which suggests a recent origin for the 
pseudogene. With this structural information, gene 
sequences can now be amplified selectively and 
characterized without interference from the 
pseudogene. 
II. Regulation of Blood Coagulation by Endothelial 
Cells and Monocytes. 
The endothelium and monocytes normally do 
not promote blood clotting reactions. However, in- 
flammatory mediators such as interleukin-1 and 
tumor necrosis factor-a (TNF-a) can induce these 
cells to stimulate blood clotting, thereby linking the 
initiation of blood coagulation to activation of the 
immune system. Dr. Sadler has studied three hemo- 
static proteins that are regulated in endothelial 
cells by inflammatory mediators: plasminogen ac- 
tivator inhibitor-2, tissue factor, and thrombo- 
modulin. 
A. Plasminogen activator inhibitor-2 (PAI-2). 
Blood clot destruction may be initiated by uroki- 
nase or tissue-plasminogen activator. In turn these 
proteases are regulated by several plasminogen ac- 
tivator inhibitors. PAI-1 is characteristic of endothe- 
lial cells; PAI-2 is found mainly in placenta and 
monocytes. PAI-2 is induced in monocytes by in- 
flammatory mediators and may regulate fibrin de- 
position that accompanies cell-mediated immune 
responses. 
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