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Enhancement of inflammation by the clotting cascade is of
great interest to Mackman because of his interest in diseases
related to clotting. In addition to cleaving fibrinogen, thrombin
also cleaves receptor proteins displayed on vascular cells.
The first such receptor, identified in 1991, was the thrombin
receptor or protease activated receptor 1 (PAR1), a receptor
of the broad class of G protein-coupled receptors. Since 1991,
three other PARs have been discovered. During coagulation,
thrombin activation of platelets is mediated by PARs. In addition,
thrombin activation of PAR1 on other vascular cells, such
as monocytes and endothelial cells, initiates a multitude
of pro-inflammatory signals that contribute to an inflammatory
response. Similarly, PAR2 is activated by the blood coagulating
proteins factor VIIa and factor Xa.
"That means in the local environment of a clot, you are
going to get activation of these PARs simultaneous to the
activation of coagulation," says Mackman.
Sepsis is a fast-moving, dramatic, and often fatal disease
and is a major problem in the United States, where it is one
of the ten leading causes of both infant and adult mortality
and directly caused over 120,000 deaths in 2000 alone, according
to the Centers for Disease Control and Prevention (CDC). And
the prognosis is especially bad for children.
In a widespread infection, the response of the immune system
is triggered by components of microorganisms, such as endotoxin
or lipopolysaccharide (LPS). LPS activates innate immune cells
known as monocytes that induce inflammation at the site of
infection.
Monocytes release pro-inflammatory cytokines like TNF-alpha
and Interleukin-6 (IL-6), which makes a person feverish. This
inflammation is a first line of defense. Without it, the body
cannot fight off the bacterial infection.
During a bacterial infection, monocytes also upregulate
tissue factor, which increases thrombin levels and drives
blood clotting.
"Why do you need that coagulation?" asks Mackman. "Presumably
to wall off an infection so that it won't spread into the
systemic circulation."
However, sepsis is caused by these processes spinning out
of control. In patients with sepsis, the levels of inflammatory
cytokines like IL-6 stay high. The release of these inflammatory
molecules that fight infection can become too widespread and
lead to complications, such as multi-organ failure.
Another problem with sepsis is the activation of coagulation
within the vasculature. Widespread coagulation in the blood
vessels of vital organs leads to blockade of the microcirculation
and organ shut down. Frequently, the vital function of kidneys
and lungs are affected.
Treatment to reduce inflammation proved to make patients
worse off because the therapies compromised their immune response
to the bacteria. For many years, the best treatment has been
to administer broad antibiotics to try to quell the infection.
The rise of antibiotic-resistant bacteria in the last few
decades may exacerbate the problem.
A new form of treatment for sepsis arrived in 2001 when
the United States Food and Drug Administration (FDA) approved
the recombinant form of the anti-coagulant activated protein
C for use in severe sepsis. Today, the drug is manufactured
by Eli Lilly and sold under the brand name Xigris.
Now Mackman is looking at the effect of other anti-coagulants,
such as antibodies against tissue factor. He is interested
in the mechanism by which these anti-coagulants reduce inflammation
as well as coagulation, and whether they might also be used
to protect against sepsis in humans. Recent studies in the
Mackman laboratory have shown that PARs mediate cross talk
between coagulation and inflammation during endotoxemia. Thus,
PARs represent a new therapeutic target for the treatment
of sepsis.
More generally, he is also asking by which pathways different
anti-coagulation molecules influence inflammation. Are they
the same pathways? Do they overlap or are they distinct? In
his laboratory, he is interested in the cross talk between
coagulation and inflammation.
"If we can understand these mechanisms, that would be very
beneficial," says Mackman.
A Hot Topic
Mackman is also studying the intriguing possibility of a
second source of tissue factor in the body. This second source,
says Mackman, is blood-borne.
The idea of blood-borne tissue factor also goes against
the traditional dogma, which holds that tissue factor is solely
linked to tissue at the point it is exposed. In the traditional
view of tissue factor, the protein is not expressed on the
surfaces of endothelial cells that line blood vessels, but
on the layer of cells underneath. There it initiates clotting
when it is exposed to clotting factors by an injury to the
vessel wall.
Significantly, blood-borne tissue factor may play a different
role in the formation of blood clots—propagating them
rather than initiating them.
In 1993, Yale Nemerson first proposed a blood-borne source
of tissue factor, when he flowed human blood across a porcine
aortic media and observed that human tissue factor functionally
contributed to thrombus formation.
"Clearly, this experiment demonstrated that blood-borne
human tissue factor played a major role in propagation of
the thrombus," says Mackman. This immediately interested him
because he knew blood-borne tissue factor would be tightly
regulated or else we would form clots all over the vasculature.
This summer, the International Society on Thrombosis and
Haemostasis held their semi-annual congress in Birmingham,
England. The Birmingham meeting was one of the largest in
the field of vascular biology this year, attended by some
20,000 doctors and scientists. These sorts of meetings are
important to keep up on the rapid changes in the field, says
Mackman, but also to communicate with medical doctors.
"You can talk to people who work in the clinic and get a
better understanding of how what we do in the laboratory applies,"
he says. "At the conference," says Mackman, "[blood-borne
tissue factor] was a hot topic."
A blood-borne source of tissue factor suggests that the
protein plays multiple roles in the formation of a blood clot.
It is involved in the initiation of clotting, and the cell-anchored
tissue factor leads to the production of fibrin and the cross-linking
of platelets. And it is involved in the propagation of this
clotting, as blood-borne tissue factor is incorporated into
the growing clot.
Still being debated was where the blood-borne tissue factor
comes from. Is it associated with platelets? Is there some
alternatively spliced form of tissue factor that is secreted
in the blood? Or does it, as Mackman and his colleagues believe,
come from circulating microparticles—very small membrane
and protein blobs that pinch off the surface of a cell.
Mackman says that Yale Nemerson was asked directly about
the source of blood-borne tissue factor, and that the next
day, he was able to present an answer in his own talk.
"We had done experiments to address that," he says.
In collaboration with Dr. Bruce Furie's laboratory at Harvard,
Mackman used his models and bone marrow transplantation to
demonstrate that the blood-borne tissue factor microparticles
are coming from monocytes. It is still not known how the blood-borne
tissue factor is activated or what regulates it.
"Still," he says, "this is important because any antithrombotics
have to consider this blood-borne tissue factor pool."
Importantly, upregulating pro-coagulant microparticles could
promote clotting and be beneficial to hemophiliacs. On the
other hand, inhibiting these pro-coagulant microparticles
could inhibit clotting and inflammation and might be beneficial
during sepsis.
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