SEATTLE -- For the first time, researchers have described the structure of a key blood-clotting protein implicated in hemophilia A, the most prevalent and serious type of hemophilia, an inherited disorder that causes recurrent, uncontrolled bleeding, most often into the joints.
Knowing the structure of this protein, called factor VIII, gives scientists a first-hand glimpse at the subtle changes -- often involving just a single errant atom -- that cause this devastating bleeding disorder, which affects about one in 10,000 American males. This discovery also provides a key to the development of new drugs -- better clotting agents for hemophiliacs and improved anticoagulants for those at risk of stroke and heart attack.
The discovery, a collaborative effort between researchers at the Fred Hutchinson Cancer Research Center and the University of Washington, will be reported tomorrow (Thursday, Nov. 25) in the British journal Nature.
The Hutchinson Center team was led by senior author Barry L. Stoddard, Ph.D., and included Kate Pratt, Ph.D., and Betty Shen, Ph.D., all of the Center's Basic Sciences Division. The UW group was led by Earl W. Davie, Ph.D., and Kazuo Fujikawa, Ph.D., both of the Biochemistry Department within the School of Medicine.
"Solving the structure of this protein tells us a great deal about how it works. It also explains very specifically, at the atomic level, why factor VIII doesn't work in many people with hemophilia A," says Stoddard, a member of the Hutchinson Center's Basic Sciences Division and an affiliate associate professor of biochemistry at UW.
Using a technique called X-ray crystallography -- an ultra-high-powered form of microscopy that reveals the structure of matter at the atomic level -- the researchers uncovered the architecture of a region within factor VIII called the C2 domain. This particular region of the protein harbors many functional genetic mutations -- and corresponding structural defects -- responsible for hemophilia A.
"A huge percentage of the total function of the factor VIII protein is tied up in this little domain at the end of the protein chain," Stoddard says, referring to the string of some 2,300 amino acids that comprises the huge factor VIII protein. Along this chain are sections of amino acids folded into six independent regions, or domains, each with a highly specific function. The job of the C2 domain is to seek out any injury to the circulatory system and immediately help trigger a clotting response in that precise location.
The researchers are particularly excited about the potential medical applications of their discovery.
"Now that we understand the structure of this critical regulatory protein, which is absolutely specific to blood clotting, we can hopefully exploit it as a target for developing anti-clotting agents," Stoddard says. Unique compounds could be identified as candidates for drug development against stroke, heart attack and thrombosis within the next three years, he predicts.
Currently, the only available blood thinners are "non-specific" -- they can produce a wide range of side effects because they act on a vast number of proteins and enzymes throughout the body, not just those that regulate blood clotting. Knowing the structure of the factor VIII protein could allow researchers to develop drugs that act much more selectively and thus produce few or no side effects.
Unlocking the structure of factor VIII also could lead to the development of improved therapies to treat hemophilia A. The current standard of treatment for hemophilia A involves injecting recombinant human factor VIII at the time of injury, or when symptoms of hemorrhage surface. While this form of protein-replacement therapy works in the majority of cases, about 30 percent of patients develop an allergic reaction to the protein. As a result, they develop antibodies that inhibit the drug so it no longer works. The only recourse at this point is to give such patients factor VIII from pigs; subtle differences on the surface of the porcine protein can prevent an immune response in a small number of patients.
A better alternative to the pig protein, Stoddard and colleagues suggest, would be to give patients access to a well-stocked "medicine chest" containing multiple versions of genetically tweaked human factor VIII, each designed to evade the immune response while still allowing the protein to do its clotting job.
"Now that we have the structure, we can identify every amino acid on the surface of the protein that potentially could be mutated in the laboratory. In theory, we could make a whole battery of variants of recombinant human factor VIII that could be administered one after another," Stoddard says. "When a patient develops an immune response to one, they could switch to another version of the protein. The variations could be practically endless."
The researchers have filed a patent on methods of drug design, based on the structure of the factor VIII protein, to develop anticoagulants and mutated versions of factor VIII designed to skirt the immune response.
"This has been an exciting project," says Davie, UW professor of biochemistry. "It has combined the efforts of scientists in X-ray crystallography and molecular biology to provide insight into a medical problem with a long and interesting history," he says, referring to the impact of the disease throughout the royal families of Europe -- the genetic legacy of Queen Victoria.
"Indeed, Queen Victoria of England was a carrier of hemophilia and this was passed on to her granddaughter, Princess Alexandria. The princess was married to Czar Nicholas of Russia, and their son, the Czarevitch, was also affected with the disease. The princess and the monk Rasputin, who treated the Czarevitch, had considerable political influence on Czar Nicholas, and this influence may have been responsible for precipitating the Russian Revolution."
The National Heart, Lung, and Blood Institute, a branch of the National Institutes of Health, supported this research.
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