Protein Research Provides Clues to How Blood Clots, Wounds Heal

They are proteins that cut other proteins, enabling a wide range of essential functions such as wound healing, blood clotting and formation of muscle and nerve cells.
But serine proteases also can cut a path of destruction, contributing to the plaques involved in heart disease and Alzheimer’s and to extensive birth defects as well when something goes awry. Understanding this sort of physiological crescendo called a protease cascade is a goal of Dr. Ellen K. LeMosy, developmental biologist at the Medical College of Georgia. From the Medical College of Georgia:
Protein Research Provides Clues to How Blood Clots, Wounds Heal

They are proteins that cut other proteins, enabling a wide range of essential functions such as wound healing, blood clotting and formation of muscle and nerve cells.

But serine proteases also can cut a path of destruction, contributing to the plaques involved in heart disease and Alzheimer’s and to extensive birth defects as well when something goes awry.

Understanding this sort of physiological crescendo called a protease cascade is a goal of Dr. Ellen K. LeMosy, developmental biologist at the Medical College of Georgia.

This M.D.-Ph.D. saw poignant examples of cascades gone bad while still a medical student at Duke University taking a clinical coagulation course; post-surgery patients would unexpectedly end up in intensive care because their clotting systems went berserk, causing clots in the tiny vessels inside organs and uncontrolled bleeding elsewhere. Physicians gave the patients more blood clotting factor because it was the bleeding that was killing them. “They might lose their kidneys, but they also might survive,” Dr. LeMosy said. “It just seemed like we should know more.”

Dr. LeMosy, who just received a Basil O’Connor Starter Scholar Research Award from the March of Dimes for her work, is using the comparatively simple fruit fly — which remains transparent as it speeds through embryogenesis in about 24 hours — as a model for learning how protease cascades work and how they might be better regulated.

Her work with researchers at Washington University in St. Louis is geared at engineering more active proteases, specifically a more active version of the protease clot buster TPA, used to reduce the damage of heart attacks and stroke. Their work was published in the online Journal of Biological Chemistry in December and is scheduled for the print version early this year.

But much of her research focuses on defining the inner workings of these proteins that cut and the carbohydrates that signal them to action.

Her work already has shown that the first protease is activated very early; in fact, it’s tied to ovulation. “As soon as the egg is pushed out of the ovary, this pathway is activated,” Dr. LeMosy said.

She’s following that pathway across the body, using the proteases involved in nerve and muscle cell formation as a model for how proteases are turned on and off throughout the body. It’s a scientific road not yet well-traveled: about 125 proteases have been identified in humans and only a handful of cascades, such as blood-clotting and clot-busting, have been well studied, she said.

Proteases activate or deactivate other proteins by cutting with a process that resembles digestion; some of the digestive enzymes in the stomach actually are proteases. “The blood-clotting proteases are in your body all the time, but they are activated in response to certain cues,” Dr. LeMosy said. “So what makes that stopwatch go off after surgery?” she said of the clotting/bleeding chaos that can follow surgery. “And, what controls the amount of activity through the cascade? Even if you have a stopwatch go off, could you limit the amount of activation somewhat further down the line to only get a little bit of reactivity, instead of a massive, uncontrolled response?”

When all goes right in the developing embryo, proteases help with cell-fate determination, giving cells an identity and, ultimately, direction on where they should go. Early in development, the formation of two axes ? head to toe and front to back ? is critical. “Proteases are involved in directing that,” Dr. LeMosy said of front-to-back development. “If you don’t get these signals, all the cells develop as if they were part of the back and basically development just stops. You just get a hollow tube of dorsal cells.”

She knows that four proteases form a signal transduction pathway that tells cells on the belly side to become primarily muscle and nerve cells. “If you don’t form these initial belly cells all you end up with essentially is skin,” Dr. LeMosy said.

The researcher, who joined the MCG faculty in January 2002 after completing her postdoctoral research work at Yale University, also has characterized the function and activation of the first two.

Now she is focusing on the final two, looking for the “matchmaker,” the final signal that comes only from the belly or ventral side that puts the final two proteases of the pathway together and ensures that the cells get to the proper place for development.

Genetic evidence indicates the matchmaker here is a big carbohydrate molecule called heparan sulfate proteoglycan which also has many important roles in development and is involved in uniting many molecules. One of Dr. LeMosy’s many goals is to interfere with the action of this matchmaker in her animal model and see if the cells still form properly. Of particular interest are resulting defects such as the failure of kidneys to form and heart defects.

“We think that this is the signal that tells cells what to be ? and if you don’t get the signal, you don’t get muscle or nerves. (So) we need to know what gene encodes the heparan sulfate proteoglycan involved in this process, what gene makes it,” she said.

Bigger questions are how these carbohydrates, or sugars, that are so pervasive in the body get made, why there are so many of them and what they do. The human genome has about 30,000 genes, most of which make proteins, little engines involved in countless body functions. Carbohydrates, in turn, are involved in how proteins interact and what function they ultimately perform. “It’s like you have little blocks on a string, a sulfate group here and another here,” Dr. LeMosy said. “For whatever reason, this makes a binding site for a protein that makes the protein do something. We know almost nothing about how that works,” she said, noting that carbohydrates are more complex in their structure than the proteins they bind and that there probably are more of them.

In fact, now that the human genome is mapped and proteomics, the study of how and what these genes ultimately do, is under way, scientists such as Dr. LeMosy are more fervently exploring the structural complexity of carbohydrates, the glue that seems to organize and hold many critical pieces of the human puzzle together. The field is so new that some tools aren’t yet available to learn what’s needed, so Dr. LeMosy also is working on developing methods to identify sections of carbohydrates important for different developmental processes.

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