Compound Inhibits Clotting

What do Wisconsin dairy cattle, rat poison, and former President Dwight Eisenhower have in common?

The answer is warfarin, the most prescribed anticoagulant in North America that unites these seemingly disparate phenomenon with two vastly different medical issues: anticoagulation and tuberculosis.

In the laboratory of Jonathan Beckwith, a professor of microbiology and molecular genetics at Harvard Medical School, a team of researchers have discovered that warfarin—a compound found in blood thinners—interferes with not only the process of blood coagulation, but also the growth of the bacteria molecule that causes tuberculosis.

The discovery has profound consequences for the future of new anticoagulants and tuberculosis antibiotics, the researchers said.

Warfarin was first discovered during the Great Depression when dairy cattle on a Wisconsin farm suffered excessive bleeding from consuming spoiled sweet red clover hay, which was later found to contain a warfarin-like compound that inhibits blood clotting.

Recognized for its anticoagulant abilities, warfarin was later used in 1955 to treat Eisenhower after a heart attack. Now, warfarin is used in rat and mice pesticides.

Warfarin works as a blood thinner by interfering with VKOR, a bacterial version of the human enzyme that recycles vitamin K in the liver. Through the recycling process, clotting factors are produced and secreted into the blood, piling up to act as coagulant agents.

Coagulation, which stops the loss of blood at vascular injury sites, is essential for the process of stopping bleeding, or hemostasis, but it can be dangerous in the case of blood clotting complications like deep vein thrombosis. Warfarin, an anticoagulant, inhibits the function of the VKOR enzyme.

In addition to its role as an anticoagulant, warfarin inhibits the VKOR homologue of mycobacterium tuberculosis—a productive agent of tuberculosis.

The VKOR homologue forms disulfide bonds in proteins secreted by the bacteria. The bonds lock the bacteria into peak fighting form by stabilizing the proteins, helping them to survive in harsh conditions outside the cell, according to HMS Professor of Cell Biology Tom Rapoport.

But the “caveat” of using warfarin to inhibit tuberculosis growth is the need to apply very high concentration of the drug to produce desired results—and high dosages of the drug may cause a patient to bleed to death, according to graduate student and lead researcher Rachel Dutton.

Rapoport said that his hope is to find reagents that “can kill TB cells but not cause the person to bleed too much.”

Beckwith’s team worked off of discoveries made in Rapoport’s lab, including the structure and function of the VKOR protein, its formation of disulfide bridges, and its method of inhibiting the VKOR enzyme.

The next step of the HMS researchers’ work will require collaboration between the discoveries made in Beckwith’s and Rapoport’s labs. The identification and understanding of the VKOR structure will help the researchers in their search for new tuberculosis antibiotics, Rapoport said.

Beckwith’s team is currently developing screening assays for various anticoagulants and antibiotics to test whether they inhibit VKOR’s enzymatic activity. Dutton said they hope to “find other small molecules that would be stronger inhibitors that could potentially serve as starting points for new TB antibiotics.”