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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"Tissue factor is potentially playing a pivotal role in many physiological processes," says Associate Professor Nigel Mackman. Photo by Jason S. Bardi.