MOLECULAR BIOLOGY OF BLOOD COAGULATION 
David Ginsburg, M.T)., Assistant Investigator 
The research program of this laboratory focuses 
on the biology of the blood coagulation system and 
the molecular genetics of human hematologic dis- 
eases. 
I. von Willebrand Factor and von Willebrand's 
Disease. 
von Willebrand factor (vWF) is a plasma adhesive 
glycoprotein that plays a central role in hemostasis, 
both as the major mediator of platelet adhesion to 
the blood vessel wall and as the carrier for factor 
Vine (the antihemophilic factor), von Willebrand's 
disease (vWD) is the most common inherited bleed- 
ing disorder in humans, with prevalence estimated 
to be as high as 1-3% of the population. This labo- 
ratory is interested in the molecular basis of vWD 
and structure-function analysis of the vWF protein. 
The study of vWD at the molecular level has been 
difficult because the vWF gene is unusually large 
(>175 kb, >50 introns), a nonprocessed pseudo- 
gene is present, and there is no ready source of 
vWF mRNA from patients. In addition, more than 
20 distinct subtypes of vWD have been reported, all 
with subtle phenotypic differences. This laboratory 
has recently adapted the polymerase chain reaction 
(PGR) to study trace amounts of vWF mRNA present 
in peripheral blood platelets. With this approach, 
two different single-base substitutions were identi- 
fied in two type IIA vWD patients, both resulting in 
nonconservative amino acid substitutions. Both 
substitutions are located immediately adjacent to 
an important new functional domain of vWF in- 
volved in platelet binding, independently identified 
in the laboratory (see below). When introduced 
into full-length vWF cDNA and expressed by trans- 
fection into COS cells, one of these mutations re- 
sulted in loss of the large vWF multimers, similar to 
the pattern observed in type IIA vWD plasma. In 
the other case, a normal multimer pattern was 
seen. The major defect in type IIA vWD may be 
functional, due to the disruption of this novel 
platelet-binding domain, with loss of multimers a 
secondary phenomenon. Other single-base substi- 
tutions, all clustered in the same region, have re- 
cently been identified in three additional type IIA 
patients. In two of these unrelated families the 
identical base substitution was observed. Based on 
sequence differences upstream of the substitution, 
the same mutation seems to occur independently 
in these two families. Five potential mutations have 
now been identified in nine type IIA families. The 
effects of these substitutions on vWF function and 
multimer assembly are currently being investigated. 
The laboratory has continued to study structure- 
function relationships within the vWF molecule. 
vWF is an unusually large and complex adhesive 
glycoprotein that interacts with platelets, the sub- 
endothelium, and several other clotting proteins, 
via a number of distinct functional domains. In col- 
laboration with Dr. Paula Bockenstedt (University 
of Michigan), this laboratory has used a recombi- 
nant approach to localize specific immune epitopes 
precisely within vWF, for a large panel of monoclo- 
nal antibodies (MAbs). To date, 12 MAbs have been 
localized. Several of these identify identical epi- 
topes, suggesting that there may be a limited num- 
ber of strongly immunogenic regions within this 
large molecule. Two of these regions are of particu- 
lar functional interest. In collaboration with Dr. 
David Pass (Mayo Clinic), MAbs observed to block 
the binding of factor VIIIC to vWF were localized to 
a 19-amino acid epitope near the vWF amino termi- 
nus. Further studies are focusing on this region to 
define more precisely the structural requirements 
for the important vWF-factor VIIIC binding inter- 
action. 
Another MAb observed to block vWF binding to 
platelets via the glycoprotein GPIb receptor was lo- 
calized to a region near the midportion of vWF. 
When a vWF segment from this region was ex- 
pressed in Escherichia coli, a marked effect on 
vWF-platelet binding was observed in an in vitro 
assay. This novel region is distinct from any pre- 
viously identified vWF functional domains and may 
be associated with the defect in type IIA vWD (see 
above). These findings could have important impli- 
cations for the therapy of both vWD and hemo- 
philia A and might lead to the development of im- 
portant new anticoagulant drugs. 
II. Plasminogen Activator Inhibitor-1. 
The fibrinolytic system is regulated, in part, by a 
balance between tissue-type plasminogen activator 
(tPA) and its specific, rapidly acting inhibitor, plas- 
minogen activator inhibitor-1 (PAI-1). Abnormal 
plasma levels of PAI-1 may be associated with a vari- 
ety of thromboembolic disorders in humans. Full- 
length cDNA clones for human PAI-1 have pre- 
Continued 
213 
