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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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Role of Tissue Factor and Protease Activated Receptors in a Mouse Model of Endotoxemia. Click to enlarge.