Previous in vivo studies have established that plasma thrombopoietin (TPO) levels are regulated by binding to c-Mpl on platelets and that, in vitro, platelets bind and degrade TPO. To determine if the in vivo metabolism of TPO was specific and saturable, we injected normal CD-1 mice IV with trace amounts of 125I-rmTPO with or without a saturating concentration of rmTPO. The amount of radioactivity present in the spleen, blood cell fraction, platelet fraction, tibia/fibula, and femur was significantly greater in the mice receiving 125I-rmTPO alone. Conversely, the amount of radioactivity present in the plasma was significantly greater in the mice receiving both 125I-rmTPO and rmTPO, thus suggesting the uptake of rmTPO by the spleen, platelets, and bone marrow in vivo was saturable. Platelet and spleen homogenates from animals receiving 125I-rmTPO alone showed a degradation pattern of 125I-rmTPO similar to that observed in vitro using mouse platelet rich plasma. To determine the in vivo binding dynamics for rmTPO, mice were injected with 125I-rmTPO alone or with increasing concentrations of rmTPO; spleen and blood cell-associated radioactivity was determined at 2 hours postinjection. A 4-parameter curve fit of the data indicated that the "in vivo binding affinity" for rmTPO was approximately 6.4 microg/kg. These data indicate that after a dose of approximately 6.4 microg/kg, 50% of all c-Mpl receptors will be saturated with rmTPO. Electron microscopy indicated that radioactivity was present bound to and within megakaryocytes and platelets in both sternum and spleen and platelets in circulation. Together these data demonstrate that in vivo, 125I-rmTPO is mainly metabolized by platelets and to a small extent by cells of the megakaryocyte lineage, via a specific and saturable mechanism.

Thrombopoietin (TPO) has been established as the major regulator of megakaryocyte and platelet production. In vitro and in vivo studies have demonstrated that TPO affects both megakaryocyte proliferation and maturation. In vitro, TPO has been reported to be essential for full development of megakaryocytes and platelets. These studies are in contrast to results observed in vivo in mice deficient in the TPO or c-mpl gene (TPO-/- and c-mpl-/-). Both TPO-/- and c-mpl-/- mice exhibit a 90% reduction in megakaryocyte and platelet levels. But even with this small number of circulating platelets, these mice do not have any excessive bleeding. Ultrastructural analysis indicates that platelets and megakaryocytes present in the knockout mice are morphologically normal. Characterization of platelet function shows that platelets from knockout mice are functionally identical to the wild-type platelets as measured by upregulation of 125I-fibrinogen binding to platelets in response to adenosine diphosphate (ADP) stimulation and by platelet attachment to the immobilized extracellular matrix proteins, collagen and von Willebrand factor (vWF). These results demonstrate that in vivo, TPO is required for the control of megakaryocyte and platelet number but not for their maturation. Other factors with megakaryocytopoietic activity may be able to compensate for the maturational role of TPO and lead to the formation of normal megakaryocytes and platelets in TPO-/- and c-mpl-/- mice.

Cystic fibrosis (CF) patients frequently experience recurring airway infections characterized by thick, viscous sputum. The consistency and nature of these purulent secretions may produce a significant barrier to the successful delivery of drugs and gene therapy vectors designed to treat CF. We have carried out a series of in vitro studies to determine the distribution of two macromolecular components typically present in purulent sputum, bacterial alginate and neutrophil-derived DNA. Sputum samples were obtained from hospitalized CF patients. DNA and alginate were disrupted, respectively, by the in vitro additions of human recombinant deoxyribonuclease I (rhDNase) or alginate lyase prepared from a mucoid strain of Pseudomonas aeruginosa. N-acetyl-L-cysteine (acetylcysteine) was similarly used to collapse the mucin matrix of these samples for comparison. Using a centrifugation-based rheological method known as the compaction assay, a greater maximal response was observed for rhDNase compared to alginate lyase treatment. A simultaneous addition of these enzymes to purulent sputum produced an additive compaction response. Electron microscopy was used to identify alginate and DNA components within the mucin matrix of sputa and to evaluate changes following treatment with high concentrations of alginate lyase or rhDNase. DNA was more widely distributed throughout purulent samples than alginate. Differences in the distribution of DNA and alginate may explain, at least in part, the larger compaction response to rhDNase versus alginate lyase treatment. An improved understanding of DNA and alginate distribution within purulent CF sputum may lead to improvements in drug and vector delivery to airway epithelial cells.