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HEMATOLOGY New Device Documents Clot Formation in Living Mice Using a specialized instrument they designed, researchers have observed new details in the real-life drama of one of the most deadly events in life: a thrombus forming in a vessel. Perhaps not surprisingly, some of the key players in the early stages of coagulation behave differently in a living animal than they do in a test tube. Barbara and Bruce Furie (above) and their colleagues report how three components of a thrombus (below) assemble in real time in a living animal. Click on the image to see a 60-second movie which shows a thrombus forming in a tiny vessel. Blood flow is from right to left. In response to a laser-induced injury of a normal vessel wall, red-labeled platelets and green-labeled tissue factor rush to the scene. About 20 seconds into the thrombosis, blue-labeled fibrin form and fill in the bulk of the thrombus. In the composite movie (and image below), colors overlap where the components mix: Tissue factor shows up in the turquoise, white and yellow; fibrin shows up in the turquoise, white and magenta; platelets show up in the yellow, white and magenta. (Photo by Pam Murray; image and movie courtesy of Shahrokh Falati) In the October 2002 Nature Medicine, Bruce and Barbara Furie, both HMS professors of medicine at Beth Israel Deaconess Medical Center, report on the timing and assembly of three components in arterial thrombosis in mice. Their findings suggest a revised model of the earliest steps of blood coagulation. The Furie lab is focusing on normal coagulation and thrombus formation, but the same mechanisms can plug up the plumbing pathologically, causing heart attacks, strokes, deep vein thrombosis, and pulmonary embolisms, said Barbara Furie. In the new study, postdoctoral fellows Shahrokh Falati and Peter Gross labeled tissue factor, platelets, and fibrin with colored fluorescent antibody tags in a normal mouse. Tissue factor and platelets work independently toward the same end--forming a thrombus dominated by platelets and fibrin. Video and Data Capture To observe the live action, the researchers peeked through the paper-thin membrane of the mouse scrotum using a new system that combines high-speed digital video microscopy with spectroscopy. The system snaps more than 1,000 confocal and widefield images a minute. In this study, the instrument recorded a dynamic 60-second show. More important for the researchers, it also captured the spectroscopic data for later analysis. In response to a laser-induced injury of a normal vessel wall, red-labeled platelets and green-labeled tissue factor accumulated within seconds, as expected. In about 20 seconds, blue-labeled fibrin formed and filled in the bulk of the thrombus. In the final analysis, some tissue factor was integrated throughout the clot but, unexpectedly, most of the tissue factor lined the interface between the thrombus and the vessel wall. Tissue factor is known to be one of the first proteins on the scene in the quick scramble of cells and proteins in coagulation, but its source has been mysterious. A teetering classic model of coagulation says tissue factor comes from nonvascular cells and leaks into the vascular system only in cases of vascular injury. The new Furie study supports an alternative--that tissue factor may arrive instead from the endothelial cell lining and smaller particles careening through the vessels. Dozens of other proteins and cells are also known to interact in a precise cascade of events leading to blood clots. For their next study, the Furies and their associates reached back in their past to examine how another protein, P-selectin, figures into the kinetics of thrombosis. Discovered in the Furie lab 20 years ago, the cell-adhesion molecule lives in the membrane storage granules of platelets and endothelial cells. When activated, it flips to the outside of the cell and acts as molecular Velcro. In intervening years, P-selectin has become best known for helping to catch rolling leukocytes and adhere them to the vessel wall, whence they migrate into the tissue during the inflammatory response. But P-selectin has also been implicated in thrombosis, including a study in baboons in the Furie lab 10 years ago, in which antibodies to P-selectin blocked fibrin generation during clot formation. This time, the researchers used the new instrument to compare thrombus formation in mice missing P-selectin or its receptor, PSGL-1, and wild type mice. Detailed findings will be presented at the December meeting of the American Society of Hematology. Preliminary results have led the Furies to flesh out a proposed in vivo model for blood coagulation based on new data. The Clot Thickens According to their working hypothesis, the tissue factor in the earliest phase of blood clotting might come from microparticles, small pieces of cell membrane that circulate in normal blood. Some particles contain the receptor PSGL-1 and tissue factor, and they stick to P-selectin in platelets and endothelial cells. The particles may concentrate sufficient tissue factor within a growing platelet thrombus to catalyze the enzymatic reactions of blood coagulation, setting in motion the chain of events leading to a fibrin clot, the Furies said. In another application of the new technique to be presented at the December hematology meeting, postdoctoral fellow Derek Sim tested a newly identified potent inhibitor of thrombosis. Sim works jointly in the BID labs of the Furies and of former Furie postdoc Robert Flaumenhaft, now an HMS assistant professor of medicine. The small molecule was identified by a screen of more than 16,000 molecules at the Institute of Chemistry and Cell Biology. The inhibitor appeared to work in a mouse within the first 30 seconds following laser injury by interfering with the cAMP level in platelets. Going Digital The idea for the new instrument was born several years ago when the Furies' eldest son recommended his parents "go digital" with their video intravital microscopy. The computer engineer, who worked on visual effects for filmmaker George Lucas, is now in graduate school at the University of Southern California working on his own computer-animated film. The Furies finally found the centerpiece of their custom system thanks to a tip from Shinya Inoué, a renowned microscopist and a colleague of the Furies at Woods Hole, Mass., where they all have labs at the Marine Biological Laboratory. Inoué told them about a high-speed confocal system he helped to design. It took more than a year and dozens of configurations with different components to refine the system to allow multicolor visualization of components of the microcirculation of a living mouse. The Furies developed their instrument to move biochemical experiments of platelet biology and blood coagulation from test tubes into living animals where they can observe behavior in normal physiological conditions. Their work is part of a general trend in biology to figure out how individually well-studied proteins, cells, and genes all work together in living animals. --Carol Cruzan Morton