Thrombopoiesis (""""""""platelet formation"""""""") is a subject of increasing interest. Despite numerous studies of megakaryocyte progenitors and maturation, actual and verifiable production of platelets has not been directly observed in vivo or in vitro. The mechanism of platelet shedding is unknown; the site may be intravascular and thus difficult to study. The objective of this proposal is to reproduce the phenomenon of platelet production in vitro. In the short term, it is proposed to study different conditions which might induce and support platelet shedding. Isolated megakaryocytes mature in culture, but do not make platelets. When anchored to a substrate, or when exposed to flow in suspension, megakaryocytes also show no evidence of platelet formation. However, when circulating megakaryocytes are allowed to attach to a subendothelial extracellular matrix and continue to be exposed to shear stress, they develop a long pseudopod similar to that seen in vivo, extending into marrow sinusoids. These processes have long been interpreted as the penultimate step in platelet shedding, although the separation of platelets from such a process has never been confirmed. Additional factors seem necessary to complete the process. Investigation of several conditions, individually as well as in combination, should be of intrinsic value in pursuit of this goal. Appropriate substrates (subendothelial extracellular matrix, endothelial cells and marrow derived extracellular matrix) within a well characterized flow chamber will be observed continuously by time-lapse video microscopy. Attached or circulating cells will also be examined by transmission or scanning electron microscopy. Isolated guinea pig megakaryocytes will be used fresh, or after pre-incubation (to yield more mature cells). The effect of thrombopoietin on mature megakaryocytes will be tested under static and flow conditions. Non specific cell growth factors, such as oxygen tension and insulin, will also be assayed. By defining and manipulating these conditions, we will gain insight into how the supply of platelets is controlled, and eventually some clinical tools to influence platelet counts.
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