One of the most noticeable occupants of the bone marrow are the megakaryocytes, hulking cells that contain dozens of times the usual amount of DNA. These giants eventually disintegrate, releasing thousands of small platelets into the bloodstream. The roving platelets must be replenished every seven to 10 days, so their ongoing production is a massive undertaking. The complex process by which platelets are produced is less understood than that of other blood cells, yet problems in platelet production have a role in atherosclerosis, acute coronary events, and bleeding disorders.
A collaboration between Uli von Andrian and Ramesh Shivdasani offers a first view of the process of platelet formation in living animals. With movies capturing the large megakaryocytes releasing platelets into the bloodstream, the study offers a definitive perspective. “The birth of a platelet, seen for the first time,” von Andrian said. (The movies appear online.)
Shivdasani, HMS associate professor of medicine at Dana–Farber Cancer Institute, has been studying platelet formation for more than a decade. In culture dishes, megakaryoctytes will spontaneously release long projections of cell material, as if the contents of the cell were being squeezed out like ribbons of toothpaste. Platelets then pinch off from these structures, called proplatelets. After the platelets are released, all that is left of the megakaryocyte is a membrane and nucleus. Shivdasani said that although the evidence for the proplatelet model was very strong, “there has always been this underlying concern that what we study in culture is an artifact of cultured cells.” Megakaryocytes, in particular, have been observed to contain many internal membranes, and a competing theory is that the platelets form inside the cell until it breaks open, releasing its contents like a burst egg sac.
In order to see the process in vivo, Shivdasani partnered with von Andrian’s lab, which many years ago developed a technique for imaging cells in the bone marrow of a living mouse. Although the lab is focused on studying immune system cells, postdoctoral fellow Tobias Junt led the project of capturing platelet formation. The easiest and least invasive site to view bone marrow is in the skull bone; using multiphoton fluorescence microscopy, the team could image the marrow through the intact overlying bone without disrupting the structures inside. The mice were engineered to express a fluorescent protein unique to megakaryocytes and platelets. Under the microscope, the super cells are bulky and easily detected, but they are rare, so the team treated the mice with thrombopoietin, a drug that causes more cells to form.
By taking three-dimensional time-lapse microscopic images every 15 seconds, the team could link together movies that capture the behavior of megakaryocytes. What they saw was very similar to the model that had been developed based on other evidence. The hulking cells hugged the edge of bone marrow sinuses and closely mingled with these blood vessels, which were filled with a green dye. The team could see the cells send fingers into the microvessels in the marrow. Occasionally, a large fragment of the cell inside the vessels was dislodged and moved away in the direction of blood flow.
Von Andrian, the Edward Mallinckrodt Jr. professor of immunopathology, said that this mechanism of releasing proplatelets directly into the blood helps explain a conundrum of platelet biology: “How do they get into the bloodstream without getting activated?” Though the cells begin to stick together and form clots when they come in contact with the extracellular matrix, they are born in the matrix-rich bone marrow. The delivery system in which megakaryocytes burrow directly into the blood to release their contents circumvents the problem.
Although the study, which appears in the Sept. 21 Science, largely confirms the model that Shivdasani had helped develop in culture, there were some differences. “We thought we’d see thin ribbonlike structures,” he said. “In fact, what is released from megakaryocytes is a larger, more compound structure that gets further processed in the circulation.” In culture, platelets break off neatly from the megakaryocyte fingers in barbell-shaped pairs. The chunks that the movies captured breaking off were much larger. Junt, who now leads a laboratory at the Novartis Institute for Biomedical Research in Vienna, said it makes sense that events in vivo would be more exaggerated. “In vitro, you don’t mimic the blood flow, and you don’t mimic the environment of the bone marrow,” he said. “If you don’t apply flow, nothing gets ripped off. With the flow, you also have a mechanical component to it.”
If platelets are still clustered in large chunks when they enter the blood, something must break them down further on their journey, whether it is simply the mechanical forces of blood flow or a biochemical process. Shivdasani noted that younger platelets have been thought to contribute more to overactive clotting, as if the newer cells are more prone to activate and stick together. He said that the existence of larger proplatelets in the bloodstream could partly explain the differences between newborn and seasoned platelets. “This allows one to think more broadly about how young platelets might behave,” he said.
Kenneth Kaushansky, professor and chair of medicine at the University of California, San Diego, said that the study puts to rest any doubt about the basic mechanism by which platelets form. At the same time, it opens up new questions about how the megakaryocytes release platelets and what interventions might enhance or diminish that step in the process.
There are two major steps in the birth of platelets: first the megakaryocyte, stimulated by the hormone thrombopoietin, duplicates its DNA several times over as if undergoing mitosis—but instead of dividing, it simply swells. Then it releases its contents. Kaushansky said that drugs mimicking thrombopoietin are in development to treat people at a high risk of bleeding. “But we also know from our work and that of others that thrombopoietin only stimulates the early step of growing megakaryocytes, not the later step of making proplatelets.” That second step would also be an attractive target for therapies, either to stimulate platelet production to reduce bleeding or inhibit their production to prevent aberrant clots.
