PAI-1 at the Heart of Things

By Jason Socrates Bardi

When Cell Biology Professor David Loskutoff came to The Scripps Research Institute in 1975, he came from a laboratory that studied proteases—enzymes that cleave other proteins—and he was interested in continuing this research by looking at one class of proteases in particular, the plasminogen activators. Scientists had observed that tumor cells made plasminogen activators in high levels, suggesting that these enzymes helped tumors grow.

Normally, plasminogen activators are involved in removing blood clots from arteries. They belong to a class of enzymes known as serine proteases, and they cleave a circulating inactive blood protein called plasminogen to make active plasmin—another protease. Plasmin degrades fibrin, the principal protein that forms a blood clot, and this process removes clots from arteries. Plasminogen activators are so effective in removing clots that they have been produced commercially as a new "clot-busting" drug that is routinely administered to patients who have had heart attacks and strokes.

So why would a tumor cell make extra plasminogen activator? Possibly because the plasmin that is generated not only helps to degrade the matrix surrounding the cells but also may cleave and activate other proteins that do the same thing—including matrix metalloproteinases. Thus, tumor cells can use these proteases to help them invade tissues and disseminate.

Plasminogen Activator Inhibitor-1

At The Scripps Research Institute, Loskutoff began his research on the plasminogen activators produced by endothelial cells. In those days, endothelial cells—the cells that line the vasculature—had just been cultured for the first time, and this was a big breakthrough because it enabled scientists to study the growth and behavior of these cells and their potential role in cancer and cardiovascular disease.

Within a few years, Loskutoff and his laboratory discovered the primary inhibitor of plasminogen activator, which they named plasminogen activator inhibitor-1 (PAI-1). PAI-1 is a highly glycosylated protein with a molecular weight of about 50,000 Daltons, which basically controls the levels of plasminogen activator in the body. Throughout the last two decades, Loskutoff and his colleagues have studied the structure and function of PAI-1 in a variety of murine models of cardiovascular disease.

One of the unusual things about the inhibitor is that it is present in the blood only as a trace protein. The blood is rich with other protease inhibitors, and they usually circulate at much higher concentrations. PAI-1, however, is only present in the bloodstream at 5 to 10 nanograms per mL—up to a thousand times less concentrated than other protease inhibitors in the blood.

Another unusual property of the inhibitor involves its stability. Normally, protease inhibitors in the blood are very stable—like little molecular rocks. PAI-1, however, is quite unstable, spontaneously inactivating while in the bloodstream. Finally, although the synthesis of most protease inhibitors in the blood is not regulated, PAI-1 biosynthesis is highly regulated by a diverse number of mechanisms in the body, such as signaling molecules, growth hormones, and changes in physiological state.

In the years since Loskutoff and his colleagues discovered PAI-1, much work has gone into describing its expression under different physiological conditions. Loskutoff has spent a great deal of time studying PAI-1 in models of human disease and in human tissue samples. This work and similar investigations in other laboratories has led to much insight into its possible role in different diseases states.

For instance, PAI-1 levels are high in the early morning, while plasminogen activator protein concentrations are low at these times. People statistically have heart attacks in the early morning, and Loskutoff believes that this may be related to PAI-1. Too much PAI-1 might increase one's risk of a heart attack.

Similarly, since cancers express high levels of the plasminogen activators that PAI-1 inhibits, Loskutoff reasoned, high PAI-1 levels may equate to anticancer activity.

"You might predict that if you have high levels of the inhibitor that blocks those proteases, you might have fewer, less aggressive tumors," says Loskutoff. "In fact, it is the opposite."

PAI-1 and Cancer High PAI-1 levels indicate a poor prognosis for survival in many human cancers. This unexpected observation may be related to the recent observation in the Loskutoff laboratory that PAI-1 is a potent deadhesive molecule and that it can detach cells from their substratum (e.g., the extracellular matrix). One such matrix protein is vitronectin. Cells bind to vitronectin through integrin proteins, which are abundant on the surface of many types of cells.

Integrins bind to what is called the somatomedin B domain, which is on the end of vitronectin, next to a portion of vitronectin known as the RGD site (named for its characteristic triad of Arginine–Glycine–Aspartic Acid residues). "PAI-1 has the ability to block those sites," says Loskutoff.

PAI-1 also has the ability to detach cells from vitronectin, and Loskutoff is collaborating with Molecular Biology Professor Jane Dyson to solve the structure of the somatomedin B domain of vitronectin, which is composed of about 20 percent cysteine residues.

Like any protein with a large number of cysteines, there are many disulfides that form within the protein—covalent crossbridges where two cysteines attach to one another through their side chains. The crossbridge structure of the somatomedin B domain turned out to be linear, which is highly unusual.

"That was totally unexpected," says Loskutoff, adding that NMR studies with Dyson showed that all the cysteines were tightly packed within the core of the domain, with the PAI-1 binding site on the surface.

As the roles of PAI-1 in different physiological states were being discovered in the 80s and 90s, Loskutoff became interested in what seemed to be a connection between PAI-1 and obesity. High PAI-1 levels were observed in obesity.

"Nobody knew why it was there or where it came from," says Loskutoff.

It turns out that the PAI-1 was being produced in adipose tissue—fat itself—in response to specific changes that occur in obesity. This is interesting, says Loskutoff, because it has caused him and many of his colleagues to reassess how they think of the adipose tissue.

"It was originally thought of as just a place to store fat," he says. "[Similarly], the endothelium, blood vessels, have been thought of historically as pipes for carrying blood—that's all."

Scientists have since discovered that endothelial cells are not just static structures that carry blood, but are very dynamic. They secrete proteins into blood, change the composition of blood, and exert a profound influence on tissues, organs, and disease states. Similarly, the cells of the adipose tissue (the adipocytes) are extremely active biologically. They make a lot of proteins and change the composition of blood, and in obesity this can have profound consequences.

In various murine models of obesity, Loskutoff has observed a consistent and very dramatic increase in PAI-1 levels, and he thinks it may be related to the increased risk for cardiovascular disease associated with this condition. As adiopose tissue expands, the adipocytes produce increasing amounts of this prothrombotic molecule.

Leptin and Atherosclerosis

Fat, says Loskutoff, is really another organ in the body—perhaps the biggest organ in a person's body by far—and this may be where some of the problems associated with obesity arise. If fat cells make a protein like PAI-1, and the fat cells enlarge and increase in number as a person becomes obese, then the levels of that protein can become abnormally high.

In the last two decades, there has been a dramatic increase in obesity, now a major public health concern in the United States. According to the U.S. Centers for Disease Control and Prevention, there are currently more than 44 million Americans who are obese, which carries with it an increased risk of type II diabetes, stroke, and other thromboembolic diseases.

Recently, Loskutoff has been looking at another molecule related to obesity, leptin, which was heralded with much fanfare when it was discovered about 10 years ago. Leptin is a protein produced by fat cells that tells the brain when a person has had enough to eat, and it is thus involved in a negative feedback loop that controls food intake. When a person eats, leptin levels increase, the protein binds to its receptor in the hypothalamus, and the body gets the signal to stop eating.

In recent studies, Loskutoff found a link between leptin and atherosclerosis, the coronary artery disease marked by the accumulation of deposits of fat inside the walls of arteries. Long-term obesity often leads to accelerated atherosclerosis and work in the Loskutoff laboratory showed that leptin promoted this process. This may be related to his observation that platelets and endothelial cells contain leptin receptors on their surfaces.

"It turns out that leptin promotes platelet aggregation, and this may also contribute to the risk for atherothrombotic disease in obesity," says Loskutoff.

Though he is still working out the details of this story, Loskutoff says that his findings demonstrate a direct link between leptin and vascular disease.

"We're really excited about that," he says.

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Assistant professor Fahumiya Samad (left) and professor David Loskutoff.