PAI-1 at the Heart of Things
By Jason Socrates
Bardi
When Cell Biology Professor David Loskutoff came to The Scripps
Research Institute in 1975, he came from a laboratory that
studied proteases—enzymes that cleave other proteins—and
he was interested in continuing this research by looking at
one class of proteases in particular, the plasminogen activators.
Scientists had observed that tumor cells made plasminogen
activators in high levels, suggesting that these enzymes helped
tumors grow.
Normally, plasminogen activators are involved in removing
blood clots from arteries. They belong to a class of enzymes
known as serine proteases, and they cleave a circulating inactive
blood protein called plasminogen to make active plasmin—another
protease. Plasmin degrades fibrin, the principal protein that
forms a blood clot, and this process removes clots from arteries.
Plasminogen activators are so effective in removing clots
that they have been produced commercially as a new "clot-busting"
drug that is routinely administered to patients who have had
heart attacks and strokes.
So why would a tumor cell make extra plasminogen activator?
Possibly because the plasmin that is generated not only helps
to degrade the matrix surrounding the cells but also may cleave
and activate other proteins that do the same thing—including
matrix metalloproteinases. Thus, tumor cells can use these
proteases to help them invade tissues and disseminate.
Plasminogen Activator Inhibitor-1
At The Scripps Research Institute, Loskutoff began his research
on the plasminogen activators produced by endothelial cells.
In those days, endothelial cells—the cells that line
the vasculature—had just been cultured for the first
time, and this was a big breakthrough because it enabled scientists
to study the growth and behavior of these cells and their
potential role in cancer and cardiovascular disease.
Within a few years, Loskutoff and his laboratory discovered
the primary inhibitor of plasminogen activator, which they
named plasminogen activator inhibitor-1 (PAI-1). PAI-1 is
a highly glycosylated protein with a molecular weight of about
50,000 Daltons, which basically controls the levels of plasminogen
activator in the body. Throughout the last two decades, Loskutoff
and his colleagues have studied the structure and function
of PAI-1 in a variety of murine models of cardiovascular disease.
One of the unusual things about the inhibitor is that it
is present in the blood only as a trace protein. The blood
is rich with other protease inhibitors, and they usually circulate
at much higher concentrations. PAI-1, however, is only present
in the bloodstream at 5 to 10 nanograms per mL—up to
a thousand times less concentrated than other protease inhibitors
in the blood.
Another unusual property of the inhibitor involves its stability.
Normally, protease inhibitors in the blood are very stable—like
little molecular rocks. PAI-1, however, is quite unstable,
spontaneously inactivating while in the bloodstream. Finally,
although the synthesis of most protease inhibitors in the
blood is not regulated, PAI-1 biosynthesis is highly regulated
by a diverse number of mechanisms in the body, such as signaling
molecules, growth hormones, and changes in physiological state.
In the years since Loskutoff and his colleagues discovered
PAI-1, much work has gone into describing its expression under
different physiological conditions. Loskutoff has spent a
great deal of time studying PAI-1 in models of human disease
and in human tissue samples. This work and similar investigations
in other laboratories has led to much insight into its possible
role in different diseases states.
For instance, PAI-1 levels are high in the early morning,
while plasminogen activator protein concentrations are low
at these times. People statistically have heart attacks in
the early morning, and Loskutoff believes that this may be
related to PAI-1. Too much PAI-1 might increase one's risk
of a heart attack.
Similarly, since cancers express high levels of the plasminogen
activators that PAI-1 inhibits, Loskutoff reasoned, high PAI-1
levels may equate to anticancer activity.
"You might predict that if you have high levels of the inhibitor
that blocks those proteases, you might have fewer, less aggressive
tumors," says Loskutoff. "In fact, it is the opposite."
PAI-1 and Cancer High PAI-1 levels indicate a poor prognosis
for survival in many human cancers. This unexpected observation
may be related to the recent observation in the Loskutoff
laboratory that PAI-1 is a potent deadhesive molecule and
that it can detach cells from their substratum (e.g., the
extracellular matrix). One such matrix protein is vitronectin.
Cells bind to vitronectin through integrin proteins, which
are abundant on the surface of many types of cells.
Integrins bind to what is called the somatomedin B domain,
which is on the end of vitronectin, next to a portion of vitronectin
known as the RGD site (named for its characteristic triad
of Arginine–Glycine–Aspartic Acid residues). "PAI-1
has the ability to block those sites," says Loskutoff.
PAI-1 also has the ability to detach cells from vitronectin,
and Loskutoff is collaborating with Molecular Biology Professor
Jane Dyson to solve the structure of the somatomedin B domain
of vitronectin, which is composed of about 20 percent cysteine
residues.
Like any protein with a large number of cysteines, there
are many disulfides that form within the protein—covalent
crossbridges where two cysteines attach to one another through
their side chains. The crossbridge structure of the somatomedin
B domain turned out to be linear, which is highly unusual.
"That was totally unexpected," says Loskutoff, adding that
NMR studies with Dyson showed that all the cysteines were
tightly packed within the core of the domain, with the PAI-1
binding site on the surface.
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