October 04, 2001
Researchers Pinpoint Cause of Deadly Blood-Clotting Disorder
Researchers have determined the cause of a potentially deadly
inherited blood-clotting disorder that can lead to kidney failure or
stroke.
The researchers found that the disorder, thrombotic thrombocytopenic
purpura (TTP), is caused by mutations in a gene that render the
ADAMTS13 enzyme ineffective. The research suggests that it might be
possible to treat TTP by administering an active form of the enzyme, in
much the same way that people with hemophilia receive clotting
factor.
“[TTP] was known as a dangerous disorder, in which patients suddenly became very ill and could be treated only by replacing their blood plasma. No one was really sure, in fact, whether such treatment was removing something bad or adding something good that was missing.”
David Ginsburg
In an article published in the October 4, 2001, issue of
Nature, a research team that included Howard Hughes Medical
Institute investigator David
Ginsburg at the University of Michigan reported that mutations in
the ADAMTS13 gene were responsible for causing TTP in nearly all
of the affected families studied.
"Until about four or five years ago, the cause of the disease was a
mystery," Ginsburg said. "It was known as a dangerous disorder, in
which patients suddenly became very ill and could be treated only by
replacing their blood plasma. No one was really sure, in fact, whether
such treatment was removing something bad or adding something good that
was missing."
Several earlier studies had implicated a clotting-related protein
known as von Willebrand factor (VWF) in the disorder. These studies
found that the blood of patients with TTP showed an abnormally large
form of the VWF protein that had not been cleaved into two smaller
sizes, as is normally the case. Thus, said Ginsburg, many scientists
believed that a defect in a protein-clipping enzyme known as a protease
might be responsible for the disorder.
One of the keys to identifying the gene mutations that underlie TTP
was the development of a precise assay for detecting VWF protease
activity. Han-Mou Tsai, a senior author of the Nature paper, and
colleagues at Montefiore Medical Center and Albert Einstein College of
Medicine developed the assay and applied it to blood samples that were
provided by members of four families that had an inherited form of TTP.
The assays clearly revealed that within these families, those who had
TTP showed low VWF protease activity, while carriers of the disease
showed medium levels of protease activity, and unaffected individuals
showed normal levels.
"The findings seemed almost too good to be true," said Ginsburg.
"They clearly showed the presence of a recessive gene in which all the
carriers, who had one good copy and one bad copy of the gene, had about
half the level of protease activity."
Using results from the assay as a guide, Gallia G. Levy, lead author
of the Nature article, performed linkage analyses of the family
members and determined which of known genomic markers were inherited
with the disease gene. These studies enabled her to narrow down the
region containing the disease gene to a specific region of chromosome
9.
A search of the human genome database found several fragments of
genes resembling proteases that were attractive candidates, said
Ginsburg, but they could not be sure because the database was
incomplete for that region of chromosome 9. When Levy studied the TTP
patients for mutations in the target region, she found mutations in a
gene that coded for a protease that showed DNA sequence similarity to
members of a family of zinc metalloproteinases, called ADAMTS.
Levy then obtained the full gene sequence and proceeded to test the
other patients for mutations in the gene, which they named
ADAMTS13. Levy subsequently identified a dozen mutations in the
gene among the patients, accounting for nearly all the cases of TTP.
According to Ginsburg, Levy’s findings open the way to
understanding how and why the ADAMTS13 protease cleaves VWF and
how the failure to cleave the protein causes disease.
"The current hypothesis in the field is that the large form of VWF
that is initially made is too sticky. Unless it is cleaved, it
spontaneously sticks to blood platelets and clogs vessels," said
Ginsburg.
The discovery of the role of the ADAMTS13 enzyme also suggests a
relatively straightforward therapy for TTP, said Ginsburg. "It
doesn’t appear to take much of this protease to treat this
disease, and it lasts for a while in the blood" he said. "So, it might
be possible to give people with TTP a periodic injection of the enzyme
to maintain their protease activity. Such treatment would work better
and be safer than plasma exchange because of the risk of complications
from transfusions."
Ginsburg also noted that a form of acquired TTP clotting disorder
can be a complication in patients receiving bone marrow transplants and
those with lupus or AIDS. "While the carriers seem normal, it could be
that they are more susceptible to acquired TTP in such cases," he
said.
In other independent studies, the structure of the ADAMTS13 protease
and identification of its gene have also been deduced by a
collaboration of HHMI investigator Evan Sadler and researchers Dominic
Chung and Kazuo Fujikawa.
In that work, Fujikawa and Chung, who are at the University of
Washington in Seattle, purified the ADAMTS13 protein, obtained a
partial amino acid sequence, and identified the gene based on data in
the Human Genome Project database. Starting with that partial sequence,
Sadler and his colleagues at Washington University School of Medicine
in St. Louis collaborated with the Seattle group to determine the
complete cDNA and protein sequence. Their results have been published
online by the Journal of Biological Chemistry.
"Our data are entirely in agreement with David Ginsburg’s,"
said Sadler of the findings by Ginsburg and his colleagues. "But Gallia
Levy and David have made a further, major advance in the field by
identifying mutations in this gene that actually cause a serious and
sometimes fatal human disease."
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