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.

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