Tissue Factor in Coagulation and Inflammation
By Jason Socrates
Bardi
"If
Nature, sovereign mistress over wrack, As thou goest onwards,
still will pluck thee back..."
—William Shakespeare, Sonnet 126, circa 1600.
Last week in his center pavilion office, TSRI Associate
Professor Nigel Mackman was explaining how he welcomes new
postdoctoral fellows—by plopping them down in front of
a complicated diagram that shows interactive protease cascades
involved in blood coagulation and inflammation, a pop quiz.
Many of his fellows come to study a protein called tissue
factor, which is the primary molecule that initiates coagulation—the
process of blood clotting. Years of research in many laboratories
across the world have described this process and the role
of tissue factor, the only non-plasma protein in the clotting
cascade, to initiate the formation of blood clots.
The diagram—which has many abbreviated names of blood
factors (proteins) and cells, like TF, PAR, LPS, IL-6, and
Xa, and myriad arrows connecting them all—is not meant
to scare anyone away, but to illustrate how the protein tissue
factor affects many physiological processes in living systems—the
focus of Mackman's research.
"We would like to think that understanding these processes
would be beneficial to the treatment of human diseases—particularly
hemostatic diseases, like hemophilia, and inflammatory diseases
like sepsis," he says.
The Action and Reaction of Coagulation
Coagulation is a complex protease cascade involving about
30 interacting proteins and platelets (those flat, molecule-filled
cytoplasmic disks in the blood). The cascade starts when tissue
factor is exposed to the bloodstream due to a cut or other
injury. Tissue factor activates the coagulation cascade, which
leads to the generation of thrombin, a protein that circulates
in the bloodstream as an inactive "zymogen" protein called
prothrombin.
Thrombin is a very efficient proteolytic enzyme—it
activates various proteins by proteolytic cleavage at specific
points in their amino acid sequences. One of the proteins
it cleaves is fibrinogen, which generates fibrin, the sticky,
clot-forming protein that, together with platelets, forms
a stable clot.
Interestingly, this coagulation cascade is counterbalanced
with an anti-coagulation cascade, which is necessary for maintaining
homeostasis in the bloodstream. "Basically," says Mackman,
"so that we don't clot to death."
Thrombin is one of the most interesting molecules involved
because it can switch from a coagulation-promoting molecule
to an anti-coagulant. Thrombin can bind a cell surface protein
called thrombomodulin. When it does, it's game over for making
fibrin.
Thrombin bound to thrombomodulin undergoes a specificity
change and activates a plasma protein called protein C. Activated
protein C begins to shut down the clotting cascade by deactivating
the cofactors required to make thrombin, which in turn reduces
the amount of activated protein C. Thus, the two pathways
work together in a feedback loop to balance each other. "We
are interested in this balance between how [the body] clots,
but also how this is counterbalanced by the anti-coagulant
system," says Mackman.
The blood clotting cascade is relevant to diseases such
as hemophilia, where patients are deficient in one of the
blood proteins necessary for clotting. It is also linked to
vascular diseases like heart attack and stroke, where blood
clotting can lead to the occlusion of blood vessels. Clotting
is also involved in inflammation and septic shock.
Mackman came to TSRI in 1987. At the University of Leicester
in the U.K., he had been studying a toxin secreted by the
bacterium E.coli that lyses red blood cells.
He came here because he wanted to work with eukaryotic cells—like
human cells—rather than the prokaryotic E.coli.
"I saw myself getting more into the medical side," says
Mackman, and he came to TSRI to study monocytes and the molecules
they express.
When he arrived, TSRI Professor Thomas Edgington and his
associates had just become the first group to clone tissue
factor. This was no small feat, and took two years of dedicated
effort.
In the following years, Edgington, Mackman, and Associate
Professor Wolfram Ruf directed much of their efforts towards
characterizing tissue factor, its gene and its regulation,
the protein's structure and mechanisms of action, and the
complicated cascade of physiological reactions that tissue
factor directs in hemostasis, thrombosis, inflammation, certain
immune reactions, and even in tumor biology.
"Tissue factor is potentially playing a pivotal role in
many physiological processes," says Mackman.
Tissue-Specific Clotting
About seven years ago, as many of these pathways were being
worked out in vitro, Mackman decided that he wanted to study
them in vivo—in models that are created in his
laboratory. And in the years since, he has spent a great deal
of time creating models to try to see what happens when they
take out the different parts of the pathways. However, a complete
deficiency in tissue factor resulted in embryonic lethality.
The challenge was to rescue this embryonic lethality and generate
models that could be used to study the role of tissue factor
in various physiological processes. For instance, Mackman
and his colleagues made a model that rescued the embryonic
lethality by expressing human tissue factor from a transgene.
The first attempts produced a model with low levels of tissue
factor expression (about one percent of normal levels). A
second strategy produced a model expressing normal levels
of human tissue factor. Currently, Mackman is generating models
in which tissue factor can be selectively deleted in different
tissues.
Analysis of these different models led them to the discovery
of tissue-specific differences in the control of the clotting
cascade, which went against the predominant dogma that the
clotting cascade would be the same regardless of the tissue
involved.
"All tissues are not equal," says Mackman, "and the clotting
cascade cannot just be viewed as a global machine."
In their models, Mackman and his colleagues identified specific
areas where having low levels of tissue factor created bleeding
problems. For instance, tissue factor is expressed in cardiac
muscle but not in skeletal muscle. Mackman reasons that this
is so that the TF expressed by cardiac muscles can offer extra
protection against a bleed into the heart, which would be
more devastating than a bleed almost anywhere else in the
body.
Open Heart
A few years ago, Mackman gained some important insights
into the clotting problems faced by clinicians when he traveled
to Seattle, Washington, to meet with a few of his collaborators
at the University of Washington Cardiovascular Center. The
cardiothoracic surgeons provided Mackman with tissue samples,
and he provided basic science input on their research program
that addressed the problem of cardiac ischemia-reperfusion
injury (how to salvage cardiac tissue after a heart attack).
No amount of expertise could have prepared him for what
he saw, though.
Soon after walking off the plane, Mackman was asked to put
on surgical scrubs and join the doctors in the operating room
for a first-hand demonstration of what they do. They were
performing a triple bypass operation that day, and Mackman
saw a patient on the operating table with his chest open and
a heart–lung machine hooked up to his aorta. "Seeing
a patient's blood flowing through a heart-lung machine...
that really brought it home for me," says Mackman. In this
surgery, the heart is completely isolated so that no blood
is flowing through it at all while the surgical team deals
with coronary artery blockages by grafting bypass vessels
in place on the heart to bring blood to cardiac muscles. Because
the heart is no longer pumping blood, the blood from the patient's
body is circulated through a machine, oxygenated, and then
returned into the patient's body.
The consequences of putting the blood through this heart–lung
machine are more then mere oxygenation. There is activation
of inflammation and coagulation as well because cells like
platelets and monocytes are very sensitive to being outside
the body, and they become activated as these cells come into
physical contact with the foreign surfaces inside the heart–lung
machine. Because of this, anti-coagulant drugs are given to
patients undergoing these surgeries. Mackman hopes that the
new anti-coagulants currently under development may also reduce
the inflammatory complications associated with the heart-lung
machine.
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