NEWS

By DAVID WILLIAMSON
UNC News Services

CHAPEL HILL – For the second time in the past 13 years, University of North Carolina at Chapel Hill scientists have identified and cloned one of the two known genes required for vitamin K metabolism and blood coagulation.

In 1991, they purified and copied the first of those -- gamma-glutamyl carboxylase. That work has led to better understanding of how vitamin K works in coagulation and promises to lead to cheaper production of clotting factors to treat hemophiliacs.

"Gamma-glutamyl carboxylase is required for the biological functioning of a number of important proteins that cause coagulation, including human clotting factors IX, X and VII," said Dr. Darrel W. Stafford, professor of biology and a member of the UNC School of Medicine's Center for Thrombosis and Hemostasis. "Absence of any of these factors results in hemophilia."

Now, the research team Stafford heads has found and cloned the gene for another closely related enzyme -- vitamin K epoxide reductase (or VKOR) -- which converts vitamin K to a usable form, he said. This newest enzyme has great medical significance since it is the enzyme targeted by warfarin, a frequently prescribed anticoagulant for preventing or reducing clotting problems.

Warfarin, also known as coumadin, works by blocking the conversion of vitamin K to its active form, thereby reducing clotting and cutting the risk of heart attacks and stroke. Millions of patients in the United States alone are on long-term warfarin therapy, the UNC scientists say.

A report on the UNC team's latest success appears in the Feb. 5 issue of the journal Nature.

Besides Stafford, authors are Drs. Tao Li and Pen-Jen Lin, who recently completed their Ph.D. degrees in biology at UNC, technicians Chung-Yun Chang and Da-Yan Jin and Dr. Anastasia Khvorova of Dharmacon Inc. of Lafayette, Colo.

Khvorova and Dharmacon provided genetic particles called "short interferring RNAs" tailored to each gene tested and greatly boosted the team’s chance of success, Stafford said.

"Fourth among heart drugs and the 11th most often prescribed drug in the United States, warfarin is commonly used to prevent blood clots following certain types of irregular heartbeats, heart attacks and heart valve replacement surgery," said Li, the first author of the report. "Earlier studies have shown that it prevents 20 strokes for every bleeding episode it causes, but we have never known enough about how it works."

Because individuals respond differently to warfarin, treatment is tricky, and careful patient monitoring is required, he said. Besides excessive internal bleeding, risks include bruising, unexplained fever and stomach pain.

"Identification and characterization of the VKOR gene will shed much light on how warfarin works and will benefit future research on thrombosis and heart disease," Li said. "It may tell us why different people respond differently to the drug and allow physicians to learn about the best doses for individual patients by analyzing their genes. Before long, we also should be able to create enough enzyme to analyze its structure and later design more effective anticoagulants with fewer side effects."

In their latest research, the UNC team first relied on previously published studies on rodents and humans to help narrow their search for the VKOR gene to one region of one chromosome, he said. Then, rather than using older, more time-consuming methods, they employed a new technique to pinpoint it.

After excluding genes similar to known genes with other functions and those not believed to be associated with internal cell membranes, they were left with 13 genes. In turn, they inhibited each of those genes in human lung cancer cells using short pieces of the "interfering" RNA to see which test led to the greatest loss of enzyme activity. The gene they found is called gi:13124769.

"To our knowledge, this is the first time short interfering RNAs have been used in mammalian cells to identify an unknown gene," Li said.

In the same issue of Nature, German scientists led by Dr. Johannes Oldenburg of Wurzburg University announced that they too have identified the VKOR gene.

"Dr. Stafford has turned out to be the world's leading expert in vitamin K metabolism," said Dr. Harold R. Roberts, Kenan professor of medicine at UNC. "He not only was the first to isolate and clone the vitamin K carboxlase, but he and his group were the first to isolate and clone the elusive protein Vitamin K epoxide reductase with a unique technique involving a modified form of ribonucleic acid."

UNC is renown for its pioneering research in blood-related disorders. The late Dr. Kenneth Brinkhous, for example, developed the first effective treatment for hemophilia, identified previously unknown clotting factors and developed the partial thromboplastin test, an assay revealing clotting problems that now is used millions of times a year in hospitals worldwide.

Hemophilia is an inherited bleeding disorder resulting from the body's inability to produce proteins that cause blood to clot. About one in 10,000 baby boys are born with the condition, best known for afflicting European royalty. Until the development of genetically engineered clotting factors, first tested at UNC, hemophiliacs had to rely on pooled clotting factors derived from 2,000 to 5,000 blood donors. During the early years of the AIDS epidemic, many hemophiliacs became infected with HIV, the AIDS virus, from those blood concentrates.

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Note: Contact Stafford or Li at (919) 962-0597, Roberts at (919) 966-4305.

Contact: David Williamson, (919) 962-8596