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Tissue Factor in Coagulation and Inflammation
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 CoagulationCoagulation 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 ClottingAbout 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 HeartA 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. 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 TopicMackman 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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