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Tracking a Perpetrator Gene
by Paul Muhlrad
A blood clot consists of a plug of platelets enmeshed in a network of insoluble fibrin molecules. In this colorized scanning electron micrograph of a blood clot that formed in vitro, the teal strands are fibrin, the purple clusters are adherent platelets, and the red objects are trapped red blood cells.
On May Day 1999, Jennifer Chamberlin,* a 43-year-old secretary in the Midwest, stayed home to recuperate from back surgery and spend time with her three daughters, on spring break from school. Walking into her kitchen, Jenn fainted and fell to the floor. She soon regained consciousness but never returned to her normal self. "It was like she was in a trance," says Jenn's husband, Tim. "She was always lying down and talked just when talked to." Doctors couldn't detect any physical ailments. "She might need to see a psychologist," a physician told Tim. Over the next 3 weeks, Jenn plunged deeper into mental darkness, crying out in delusional outbursts.
At the end of May, Jenn's platelet count had dropped precipitously to 16,000 per microliter--the minimum for a normal count is 150,000. Follow-up tests revealed that her red blood cells were breaking into shards. With those telltale symptoms, doctors finally diagnosed her with thrombotic thrombocytopenic purpura (TTP), a rare disorder of the blood-clotting system that was almost always fatal until the 1980s and 1990s, when doctors developed a crude but effective blood plasma transfusion treatment.
Scientists understood relatively little about the cause of Jenn's illness until recently. Researchers knew that platelets in TTP patients form spontaneous clots, or thrombi, within the narrowest blood vessels. As a result, circulating platelets become depleted, and red blood cells become shredded as they squeeze through the occluded vessels. The restricted circulation and anemia leave tissues starved for oxygen, leading to strokes, heart attacks, and failure of other critical organs. Jenn's May Day attack, her doctors suggest, was probably the first of several strokes resulting from TTP.
Doctors ordered plasma-exchange therapy, the only known treatment that might save her life. For three and a half hours every day Jenn lay in a hospital bed, hooked up to a machine via a large catheter in her arm vein. The machine filtered Jenn's blood, saving the cells and replacing the plasma portion with about one and a half volumes of donor plasma. The treatment worked. Jenn's platelet count rebounded, and her condition improved over 5 days. But on the sixth day her platelets dropped again, and she endured another plasma exchange. Then another. Ultimately, in just over 2 years, she underwent more than 145 exchanges.
One of Jennifer Chamberlin's hematologists is HHMI investigator J. Evan Sadler, from Washington University School of Medicine in St. Louis. Sadler and HHMI investigator David Ginsburg, at the University of Michigan, were both drawn to investigate TTP by their prior research on a key blood-clotting protein called von Willebrand factor (VWF). In the mid-1980s, the two physician-scientists independently cloned the gene for VWF and since then they have been studying the protein's roles in blood clotting. One of those roles, Sadler explains, is to make platelets adhere to the walls of injured blood vessels.
In 1996, two research groups, one led by Han-Mou Tsai at the Albert Einstein College of Medicine in New York and one by Miha Furlan in Bern, Switzerland, separately discovered that an unidentified enzyme in blood could cleave VWF but only if the protein was slightly unfolded. Such unfolding might occur when VWF, tethering platelets to a blood vessel, gets stretched in the current. Why VWF might get cleaved was anyone's guess, but an important clue soon followed.
The next year, Furlan's group made the crucial finding that children with a rare hereditary form of TTP lacked the VWF-cleaving activity in their blood, suggesting a link between VWF, the enzyme, and the disease. Then, in 1998, Tsai and Furlan, in separate studies, bolstered that notion by showing that most adults with the acquired form of TTP (including, as it turned out, Jennifer Chamberlin) produce antibodies against the still-unidentified enzyme.
The finding shifted researchers into high gear in their efforts to identify the VWF-cleaving activity. "We were then madly working to try to purify, to clone this protein," Sadler recounts. In collaboration with Dominic Chung at the University of Washington, who purified enough of the protein to determine some of its amino acid sequence, Xinglong Zheng, then a postdoctoral fellow in Sadler's lab, used a combination of bioinformatics and DNA sequencing to identify the gene.
*The names of the patient and her husband have been changed.
Image: Yuri Veklich and John W. Weisel, University of Pennsylvania. Cover image, Nature, October 4, 2001.