Thrombocytopenic c-mpl−/− mice can produce a normal level of platelets after administration of 5-fluorouracil: the effect of age on the response

Abstract

Administration of 5-fluorouracil (5-FU) to mice results in a marked increase in the level of circulating platelets in 10 days. Mice lacking Mpl, the receptor for thrombopoietin (TPO), are thrombocytopenic. To gain insight into the mechanism by which 5-FU produces such a substantial stimulation of platelet production, this study investigated whether 5-FU (150 mg/ kg) produced thrombocytosis in c-mpl ؊/؊ mice, thus establishing whether TPO was required for this response. A 5-to 6-fold increase in platelet levels in c-mpl ؊/؊ mice (to approximately 1000 ؋ 10 9 /L) was observed on days 20 and 25 after 5-FU injection. Thus, at the peak of the response, c-mpl ؊/؊ mice had platelet levels comparable to those in normal mice. Administration of 5-FU also produced thrombocytosis in previously splenectomized c-mpl ؊/؊ mice. Comparison of the platelet response to 5-FU in young (6-12 weeks) and old (33-46 weeks) c-mpl ؊/؊ mice found that older mice produced a much more marked response than younger mice, with a mean maximum platelet level of approximately 1700 ؋ 10 9 /L. To determine whether this increase in circulating platelets was preceded by an increase in hematopoietic progenitors, serial cultures of bone marrow and spleen were evaluated. A considerable increase in all colony types studied was observed on days 15 and 20 in spleens of c-mpl ؊/؊ mice, but no similar elevations were detected in bone marrow. These results indicate that c-mpl ؊/؊ mice can achieve a normal level of platelets after 5-FU injection, by means of a TPO-independent mechanism, and that they respond to 5-FU myelosuppression by producing large numbers of megakaryocytic, myeloid, and erythroid progenitors. IntroductionThe myelosuppressive agent fluorouracil (5-FU) produces a unique effect on megakaryocytopoiesis. Administration to mice of a single dose of 150 mg/kg results in gradual development of moderate thrombocytopenia, with the nadir in platelet count occurring approximately 4 to 8 days after administration. [1][2][3] Subsequently, the level of circulating platelets increases rapidly and thrombocytosis develops by day 10. 1-3 Platelet levels as high as 3000 ϫ 10 9 /L to 3500 ϫ 10 9 /L are reached (normal platelet count in mice is 1000 ϫ 10 9 /L to 1200 ϫ 10 9 /L), and thrombocytosis persists from approximately day 10 to day 19.Changes in platelet levels are associated with a marked increase in the number of megakaryocytes (MKs) in both bone marrow and spleen, with the increase occurring in the bone marrow after 7 or 8 days 3,4 but in the spleen only after 12 days. 4 A marked increase in the frequency and total numbers of MK colony-forming cells (MK-CFCs) occurs in the spleen concurrent with the period of thrombocytosis. 2 Despite the increase in spleen size and numbers of splenic MK-CFCs, it was shown that the spleen is not required for production of the marked thrombocytosis after administration of 5-FU. 3,5 The mechanism by which 5-FU produces marked thrombocytosis is unknown. The minimal brief reduction in the concentration of recognizable MK...

BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling

“…The pivotal role of TPO is supported by the abrogation of either TPO or its receptor (c-mpl) in mice displaying almost 85% reduction in marrow MK numbers and circulating platelets. 38 However, the presence in these mice of spare normal MK and platelets 39 and the correction of thrombocytosis by 5-fluorouracil treatment 40 suggest that TPO is important but not essential for megakaryopoiesis. Together with other observations, 41 these findings indicate that cytokines or microenvironment signals can elicit a TPO-independent megakaryopoiesis.…”

Synergistic effects on erythropoiesis, thrombopoiesis, and stem cell competitiveness in mice deficient in thrombopoietin and steel factor receptors

“…36 Yet double mutants involving c-Mpl and a host of other genes have failed to identify the in vivo thrombopoietic regulator. G-CSF, GM-CSF, IL-3, IL-5, IL-6, leukemia inhibitory factor (LIF), and IL-11, many of which stimulate megakaryocyte growth in vitro, all failed to exacerbate the thrombopoietic defect of c-Mpl Ϫ/Ϫ mice.…”

VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis

“…30 We injected mice with a single dose of 5-FU and then treated the animals with VEGF-C-Cys. We found that VEGFR-3 activation transiently, but significantly (P Ͻ .002), limited 5-FU-induced thrombocytopenia and thrombocytosis (P ϭ .005) compared with the control group ( Figure 5B).…”

“…In the blocking Ab experiments, mice were injected with 600 g/animal/injection of mF4-31C1 VEGFR-3-blocking Ab, isotype control Ig, or PBS on a MondayWednesday-Friday schedule for 6 weeks. Blood was taken on days 0, 8,13,16,20,23,27,30,34,37,41, and 44 and analyzed. In each experiment, all animals were treated at the same time and on the same day and all animals were bled at each time point.…”