My father-in-law woke up one morning with his right calf muscle twice as big as the left. He called his doctor, who said, “Go to the emergency room right now.”
It was a deep-vein thrombosis, a blood clot that could have broken into chunks, traveled to his lungs, and killed him on the spot. Doctors immediately put him on warfarin, a common blood thinner. For three days, they slowly increased the dosage, trying to find the correct level to make his blood the proper viscosity and slowly dissolve the clot. Too much of the drug could’ve caused him to bleed internally. Too little wouldn’t have helped at all.
After five stressful days the clot dissipated and he was allowed to go home. When I asked him why it took so long, he said that doctors don’t really know how much warfarin to give patients at first because everyone reacts to it differently.
Carolina biologist Darrel Stafford found out why, and he has developed a way to test patients before they receive the drug.
His team isolated the gene that encodes vitamin K epoxide reductase (VKOR), the enzyme that warfarin targets to inhibit normal blood coagulation. Stafford then devised a method to check patients for certain types of mutations on the VKOR gene that make people less or more sensitive to warfarin.
“Being able to predict whether someone is likely to be a bleeder might help get someone started on a warfarin regimen,” Stafford says.
Isolating the VKOR gene could also help researchers make better blood thinners with fewer side effects.
Warfarin has been in use for a long time, but until now scientists knew very little about the genetics behind coagulation.
In the 1920s, farmers in Canada and the northern United States reported that a mysterious disease was killing their cattle. Some cows bled to death from only minor injuries. Some showed no signs of external injury but still died of internal hemorrhaging.
“Turns out the cows were eating fermented sweet clover,” Stafford says, “which inhibited coagulation.”
In the 1940s, scientists at the University of Wisconsin discovered the anticlotting culprit in the moldy clover and named it dicoumerol. The lead investigator, Karl Paul Link, synthesized a stronger version of the drug and named it warfarin after his funding agency — the Wisconsin Alumni Research Foundation. Link’s main goal was to use warfarin as rat poison. Today a version of dicoumerol is still used for just that, but researchers also turned the compound into a blood thinner that was approved for use in humans in 1954. A year later doctors prescribed warfarin to President Dwight D. Eisenhower after he had a heart attack, and it became a commonly prescribed drug.
Warfarin — or Coumadin, the most popular brand of the drug — is now the fourth-most-prescribed heart drug and eleventh-most-prescribed drug overall in the United States.
In 1978 researchers discovered that warfarin inhibited the VKOR enzyme and foiled proper vitamin K metabolism. But no one had been able to isolate the VKOR gene, which researchers suspected caused some patients to be more sensitive to warfarin.
Stafford’s team gathered all the previous studies on VKOR and managed to narrow down its location to one region of a chromosome. Still, that region contained 194 genes. Instead of using older, more time-consuming methods to find the right gene, they decided to use a new technique.
First, the team took sixteen different cell lines, ruptured the cells, and used high-pressure liquid chromatography to check them for measurable amounts of epoxide reductase. Second, graduate student Tao Li used a computer program to translate the 194 genes into protein sequences. This helped Stafford narrow his search to thirteen likely candidates for the production of epoxide reductase. Third, Stafford’s team made small interfering RNA (siRNA) for each gene. SiRNA is found in many organisms, and in 2001 scientists figured out how to make synthetic siRNA for specific genes to turn off or reduce gene expression.
Stafford made siRNA to turn down the expression of epoxide reductase. When he added the siRNA to the genes, he saw that one had a significant decrease in reductase activity.
“So then we put that gene into cells and it had a gigantic increase in epoxide reductase activity,” he says.
This was the first time anyone had used siRNA as a screening technique to isolate a gene. Stafford’s discovery made the cover of Nature, which published his paper alongside an article by Johannes Oldenburg, a German scientist who had used an older, more laborious method to isolate the same gene.
After this, Stafford wanted to isolate the enzyme that completes the vitamin K metabolism cycle. But his wife, a physician, pushed him to figure out why certain people are extremely sensitive to even low doses of warfarin.
“She just said, ‘Look, this is a big deal. Millions of people are on warfarin. You’ve got to go ahead and figure this out.’”
Thanks to Stafford’s long-standing friendships with several blood researchers at Carolina, including Harold Roberts and Roger Lundblad, he knew that UNC had a lot of patients’ records that he could check for warfarin resistance.
“Some of these patients required thirty-five milligrams of warfarin and some needed two milligrams,” he says. “We took the data and looked for genetic polymorphisms in these patients — places on the gene where there were differences.”
He found that patients were less or more sensitive to warfarin depending on their sequence of genetic mutations. Researchers can use a polymerase chain reaction, a common genetic-testing method, to determine if a patient has one kind of nucleotide sequence or a different kind at a particular site in the genome. This, essentially, is how Stafford’s patent-pending diagnostic test works. It has been licensed to four different companies who are developing tests for clinical use.
Stafford is now setting his sights back on vitamin K enzymatic activity. He’s trying to figure out which enzyme is responsible for actually reducing the VKOR at the end of each cycle — a reduction that is crucial for proper blood coagulation.
“We’ve used a lot of techniques to try to figure this out,” he says. “But so far we still don’t know.”