Thrombopoietin in thrombocytopenic mice: evidence against regulation at the mRNA level and for a direct regulatory role of platelets

Abstract

Thrombopoietin (TPO), originally described as an activity in the serum of thrombocytopenic animals that leads to increased production of platelets, has recently been isolated and cloned. Its closest relative in the cytokine superfamily, erythropoietin (EPO), is transcriptionally regulated during anemia, and it was expected that TPO would similarly be regulated during thrombocytopenia. We induced thrombocytopenia in mice and confirmed that TPO activity was upregulated, as determined by a bioassay. Liver and kidney were found to be the major sources of TPO mRNA. Surprisingly, TPO mRNA in these tissues was not upregulated in thrombocytopenic mice. Using a sensitive RNase protection assay that can distinguish between TPO isoforms, we found no change in the profile of mRNA for these isoforms. A semiquantitative reverse transcription- polymerase chain reaction assay also did not demonstrate upregulation of TPO mRNA in the spleen. Thus, the increase of TPO activity during thrombocytopenia is not caused by regulation at the level of TPO mRNA. Furthermore, isolated mouse platelets absorbed high amounts of bioactive TPO out of TPO-conditioned medium in a dose-dependent fashion. Our results are consistent with TPO protein being regulated at a posttranscriptional level and/or directly through absorption and metabolism by platelets.

Circulating thrombopoietin concentrations in thrombocytopenic patients, including cancer patients following chemotherapy, with or without peripheral blood progenitor cell transplantation

“…0001 by Fisher's Exact Test). This is consistent with the model in which the TPO gene is constitutively expressed and the circulating level of TPO is regulated by platelet mass (Kuter & Rosenberg, 1995;Stoffel et al, 1996;de Sauvage et al, 1996). However, the correlation between TPO concentrations and platelet counts in the thrombocytopenic patients was weak, suggesting that factors other than platelet mass also contribute to the regulation of circulating TPO levels (Fig 1).…”

“…This led the authors to speculate that TPO is not regulated at the gene level. Indeed, in thrombocytopenic c-mpl-deficient mice in which circulating TPO is elevated, no increase in TPO mRNA was found in either the liver or kidney (Stoffel et al, 1996), the main sources of TPO mRNA Lok et al, 1994;Bartley et al, 1994). In the studies cited, relative TPO activity was assayed by a semi-quantitative bioassay using factor-dependent murine cells expressing human c-mpl.…”

Interaction of thrombopoietin with the platelet c‐mpl receptor in plasma: binding, internalization, stability and pharmacokinetics

“…It is this mechanism which has been the subject of our studies. As initially proposed , circulating TPO levels appear to be regulated primarily by the clearance of TPO after binding to c-mpl receptors on platelets and possibly megakaryocytes (Chang et al, 1996;Shivdasani et al, 1997), and not by changes in TPO production (Stoffel et al, 1996). Our studies have sought to de®ne the critical interaction of TPO with its platelet receptor using both radiochemical and standard pharmacokinetic methods.…”

The role of thrombopoietin in post‐operative thrombocytosis

“…A third mechanism that might be involved is enhanced production of Tpo. To date, no regulation at the mRNA level has been reported for the liver and kidney in response to thrombocytopenia or thrombocytosis (Stoffel et al, 1996). In contrast, Tpo production by BM stromal cells can be induced.…”