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Hematology

Adding Sugar to Blood Platelets Enables Longer, Safer Storage

Chilled Platelet Study Sweetens Expectations in Transfusion Medicine

hen Thomas Stossel, codirector of the hematology division at Brigham and Women’s Hospital, gives grand rounds, he likes to ask how many doctors think blood platelets are refrigerated between donation and transfusion. Many hands go up. They are wrong.

But their answer eventually may be correct if a new method to treat and chill platelets in mice also works in human trials. In the Sept. 12 Science, Stossel and his colleagues report how chilled platelets are recognized and removed from circulation by hungry immune cells in the livers of mice. They also demonstrate how this clearance mechanism can be blocked by adding a dash of the proper sugar to the chilled platelets.

The added sugar acts like an invisibility cloak, allowing the platelets to evade detection by immune cells and circulate for the normal seven-day life span of platelets, said cardiologist Karin Hoffmeister, first author of the paper and HMS instructor in medicine at the Brigham. In mice, the sugar treatment doubled the useful storage period of platelets to 12 days. Refrigeration seemed to improve the function as well, showing higher platelet counts days after transfusion.

“Assuming it works in people, what we think we can deliver, with refrigeration, is going to be a better quality platelet,” said Stossel, senior author and professor of medicine at HMS and BWH.

When platelets are chilled, the von Willebrand receptors on their surface break formation to huddle closer, also clustering the top sugar residues of the receptor N-acetylglucosamine. The lectin domain of a mouse liver macrophage receptor recognizes the concentrated sugars as a tasty treat. Researchers (from left) Thomas Stossel, Karin Hoffmeister, and John Hartwig found that adding another sugar to platelets (not illustrated) makes them unpalatable to macrophages and allows them to be refrigerated for safer, more effective transfusion into mice.

The Big Chill

Unlike whole red blood, which can be stored for a month in the fridge, cooling is bad for platelets. After refrigeration, platelets transfused into people—even the donor—do not last long enough in circulation to prevent bleeding. Doctors prescribe platelets most often for people whose own platelets have been wiped out by cancer chemotherapy, bone marrow transplants, or severe trauma or surgery.

So platelets reluctantly are stored at room temperature, where they are more vulnerable to bacterial growth and cell deterioration. “You wouldn’t leave a steak out on the counter for a week and then eat it,” said cell biologist John Hartwig, co-author of the paper and an HMS professor of medicine at BWH.

Platelets can be stored for only five days, by order of the U.S. Food and Drug Administration. Still, the risk of bacterial infection from these cells may be 50 times higher than from refrigerated red blood cell products. The constant need to restock the supply creates a chronic shortage. To preserve inventory, blood services use the oldest platelets first, Hartwig said, and “it’s well known that by day five platelets have only 40 percent of the function of fresh platelets.”

When platelets are chilled, they change shape from flying saucers to sloppily fried eggs. The cells also change shape when they activate to clot blood. In fact, chilled platelets still function well, but it was a moot point because they are cleared so quickly from the body.

Shape was the starting premise for Stossel and Hartwig, who collaborate on cell morphology and motility studies. They discovered how cold works on the actin cytoskeleton to change the shape and how two common lab chemicals can prevent the transformation for up to a month in refrigeration. Unfortunately, the preserved disk-shaped platelets did not hang around any longer in baboon transfusions.

Hoffmeister arrived as they were trying to make sense of their surprisingly disappointing results. She repeated the experiments in mice with a similar outcome. Eventually, the team had to accept their experimental results and reject 40 years of prevailing dogma.

So Hoffmeister began to ask different questions. Strangely, chilled platelets were cleared in the liver, she found. A review of the scientific literature told her yeast and bacteria also exit through the liver, courtesy of macrophages. And there was evidence that a platelet receptor can stick to a macrophage integrin receptor.

Transfusion studies of mice missing various receptors on their macrophages confirmed the connection. In knockout mice missing the alpha-M-beta-2 integrin receptor, chilled platelets circulated as long as those at room temperature, compared to a 70 percent drop in minutes in control animals. Likewise, on the platelet side, Hoffmeister identified the von Willebrand receptor complex as the key player. Normally, the platelet receptors line up in short rows that decorate the surface in all directions. In the cold, they break formation to huddle together. Something about the receptor clustering makes macrophages gobble them up like candy, the researchers reported in the January 2003 Cell.

The Carbohydrate Corps

Meanwhile, Hoffmeister was convinced that the surface of platelets held something in common with sugar-coated yeasts. Sugars were the key. When the receptors clustered in the cold, the top sugar, beta-N-acetylglucosamine, also concentrated enough to tickle the sweet tooth of the macrophage receptor’s lectin domain.

As Hoffmeister learned from Danish co-author Henrik Clausen, a carbohydrate scientist from the University of Copenhagen School of Dentistry, sugars attach to each other in a strict order, like a string of pearls, each attached by different catalytic enzymes. In this case, topping the exposed sugar on the clustered receptors with the next one in the hierarchy, galactose, took away the macrophages’ appetite. Both galactose and its attachment enzyme are naturally found in the body, but apparently cannot act quickly enough on their own after transfusion.

Preliminary test tube studies show the mechanism may extend to human macrophages and platelets. Hoffmeister and her colleagues are collaborating with the BWH blood bank. “More preclinical testing is needed to ensure safety, but now we’re working to see if we can take this method and scale it up to the real-world manufacturing requirements of blood banks,” said Richard Kaufman, HMS instructor in pathology and medical director of the BWH adult transfusion service. Until then, Kaufman said, most of BWH’s precious supply comes from a dedicated group of 800 people at the Kraft Family Donor Center who donate platelets as often as every two weeks. New volunteers are welcome; call 617-632-3206 to schedule an appointment.

—Carol Cruzan Morton