MOLECULAR BIOLOGY OF BLOOD COAGULATION 
David Gbnsburg, M.D., Associate Investigator 
The research program of Dr. Ginsburg's labora- 
tory focuses on the biology of the blood coagulation 
system and the molecular genetics of associated hu- 
man diseases. 
von Willebrand Factor 
and von Willebrand Disease 
von Willebrand factor (vWF) is an adhesive 
plasma glycoprotein that plays a central role in he- 
mostasis, both as the major mediator of platelet ad- 
hesion to the blood vessel wall and as the carrier for 
factor VIII (the antihemophilic factor), von Wille- 
brand disease (vWD) is the most common inherited 
bleeding disorder in humans with prevalence esti- 
mated to be as high as 1-3% of the population. Over 
20 distinct subtypes of vWD have been reponed, all 
with subtle phenotypic differences. 
Quantitative defects in plasma vWF, type I and 
type III, are the most common forms of vWD. Al- 
though gene deletions have been identified in rare 
cases, more detailed analysis has been difficult, 
given the large size of the gene. Dr. Ginsburg's labo- 
ratory has developed a novel polymerase chain reac- 
tion (PGR) approach using a panel of exonic DNA 
sequence polymorphisms that can be scored both at 
the DNA and mRNA level. Once a heterozygous 
polymorphism is identified in patient genomic 
DNA, PGR analysis of platelet vWF mRNA can detect 
decreased or absent expression from one or the 
other vWF allele. To date, such "null alleles" have 
been identified in 3 of 3 type III vWD families (re- 
cessive inheritance) but only 1 of 9 type I patients 
(dominant inheritance), suggesting a difference in 
the molecular mechanisms responsible for these 
two disorders. 
The laboratory has recently begun to study an ani- 
mal model for type I vWD in the RIIIS/J mouse, in 
collaboration with Dr. Richard Swank (Roswell 
Park). Surprisingly, early linkage data suggest that 
the murine disorder is not linked to the vWF gene. 
Defects outside of the vWF locus, potentially inter- 
fering with vWF biosynthesis or secretion, may also 
account for some or all of human type I vWD. 
Type IIA vWD is associated with a selective loss of 
the large, most functionally active vWF multimers 
from plasma. Dr. Ginsburg and his colleagues have 
now identified seven distinct mutations in 9 of 11 
unrelated type IIA vWD patients. All seven are clus- 
tered within a 1 23-amino acid segment of the vWF 
A2 domain, and one has been identified on three 
distinct genetic backgrounds. Analysis of these type 
IIA mutations by expression in heterologous cells 
has identified two distinct mechanisms responsible 
for the type IIA phenotype. In one group the muta- 
tion results in a block to intracellular transport at 
the level of the endoplasmic reticulum, more se- 
verely affecting larger vWF multimers. In the second 
group, normal intracellular processing is observed 
with the loss of large multimers occurring after se- 
cretion, presumably due to proteolysis in plasma. 
Interestingly, two type IIA vWD mutations have 
been identified in the same codon, one resulting in a 
secretory and the other a nonsecretory defect. 
Type IIB vWD is characterized by markedly in- 
creased vWF binding to platelets, with the subse- 
quent clearance of large multimers from plasma. A 
panel of four missense mutations, all clustered 
within a 35-amino acid segment of the vWF Al do- 
main, account for all 1 4 of the type IIB vWD patients 
studied to date. Insertion of the most common of 
these mutations into recombinant vWF confers mark- 
edly increased binding to platelets, accounting for 
the phenotype of this disorder. Again, several of 
these mutations have been shown to occur on dis- 
tinct restriction fragment length polymorphism 
(RFLP) backgrounds, consistent with independent 
genetic origins and suggesting that only a limited 
number of mutations can produce this unique 
"gain-of-function" defect. 
The information derived from these studies 
should provide the tools for precise genetic diagno- 
sis and classification of vWD. Along with ongoing 
analysis of vWF structure and function, these studies 
may also lead to novel therapeutic strategies for the 
treatment of thromboembolic disease. This work 
has been supported in pan by a grant from the Na- 
tional Institutes of Health. 
Plasminogen Activator Inhibitor- 1 
Plasminogen activator inhibitor- 1 (PAI-1), a 
member of the serine protease inhibitor (SERPIN) 
supergene family, serves as the major regulator for 
the plasminogen activators uPA (urokinase plasmin- 
ogen activator) and tPA (tissue plasminogen activa- 
tor) . Abnormal plasma levels of PAI- 1 may be asso- 
ciated with a variety of thromboembolic disorders 
in humans. In addition, PAI-1 has been hypothesized 
to play a critical role in diverse biologic processes, 
including ovulation, embryogenesis, tissue remodel- 
ing, and tumor metastasis. Current studies of PAI-1 
GENETICS 197 
