Sonja van den Oudenrijn

Mutations in the thrombopoietin receptor, Mpl, in children with congenital amegakaryocytic thrombocytopenia

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder of undefined aetiology. The disease presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. Furthermore, CAMT patients may develop bone marrow aplasia. To obtain more insight into the mechanism underlying CAMT, five children were analysed. All patients had increased plasma thrombopoietin (Tpo) levels, indicating a platelet production defect. Bone marrow‐derived CD34+ stem cells from three patients were cultured in an in vitro liquid culture system to study megakaryocytopoiesis. CD34+ cells from two of the three patients failed to differentiate into megakaryocytes. The lack of megakaryocyte formation could imply that a defect in the c‐mpl gene, encoding the Tpo receptor, exists. Sequencing of c‐mpl revealed mutations in four of five patients. Three patients had point mutations and/or a deletion in the coding regions of c‐mpl. All point mutations led to an amino acid substitution or to a premature stop codon. In one patient, a homozygous mutation in the last base of intron 10 was found that resulted in loss of a splice site. This study showed that mutations in c‐mpl could be the cause of thrombocytopenia in CAMT in the majority of patients. Furthermore, Tpo has been shown to have an anti‐apoptotic effect on stem cells. Therefore, mutations in c‐mpl might not only affect megakaryocyte formation but may also impair stem cell survival, which could explain the occurrence of bone marrow failure as final outcome in patients with CAMT.

Differences in megakaryocyte expansion potential between CD34+ stem cells derived from cord blood, peripheral blood, and bone marrow from adults and children

Abstract Objective Reinfusion of ex vivo expanded autologous megakaryocytes together with stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. We compared the megakaryocyte expansion potential of CD34 + stem cells derived from different sources: cord blood (CB), peripheral blood (PB), bone marrow from adults (ABM), and bone marrow from children (ChBM). Three different growth factor combinations were tested to identify the best combination for each of the sources. Materials and Methods CD34 + cells were isolated from CB, PB, ABM, or ChBM and cultured in an in vitro liquid culture system in the presence of thrombopoietin (Tpo), Tpo + interleukin (IL-1), or Tpo + IL-3. After 8 days, proliferation was determined and the cultured cells were identified with lineage-specific surface markers by flow cytometry. Results Cultures with ChBM-derived CD34 + cells showed the lowest level of expansion of megakaryocytes and gave rise to more profound formation of myeloid and monocytic cells. In cultures with BM- or PB-derived cells, presence of IL-3 reduced the number of immature megakaryocytes (CD34 + CD41 + cells). However, in CB cultures, the number of CD34 + CD41 + cells was highest in cultures with Tpo + IL-3. Overall, cultures with CB CD34 + cells yielded the highest number of megakaryocytes, but these cells showed reduced ploidization and lower level of CD41 expression, suggesting less maturation. Conclusions Each of the different CD34 + cell sources responded differently to cytokine stimulation. For PB and ABM, the cytokine combination Tpo + IL-1 is most suitable to obtain high numbers of both immature and mature megakaryocytes for transfusion purposes. For CB, Tpo + IL-3 is better.

A combination of megakaryocyte growth and development factor and interleukin‐1 is sufficient to culture large numbers of megakaryocytic progenitors and megakaryocytes for transfusion purposes

Chemotherapy‐induced thrombocytopenia is a major risk factor in cancer treatment. The transfusion of autologous ex vivo expanded megakaryocytes could be a new therapy to shorten the period of thrombocytopenia. Therefore we investigated, in a liquid culture system, the effect of various cytokine combinations composed of pegylated megakaryocyte growth and development factor (PEG‐rHuMGDF), interleukin‐1 (IL‐1), IL‐3, IL‐6, IL‐11 and stem cell factor (SCF) on the proliferation and differentiation of CD34+ cells, in order to define the most optimal and minimum levels of cytokine combinations for megakaryocyte expansion.

Three parameters, plasma thrombopoietin levels, plasma glycocalicin levels and megakaryocyte culture, distinguish between different causes of congenital thrombocytopenia

Summary.  Fourteen children with congenital thrombocytopenia were analysed in order to unravel the mechanisms underlying their thrombocytopenia and to evaluate the value of new laboratory tests, namely measurement of plasma thrombopoietin (Tpo) and glycocalicin (GC) levels and analysis of megakaryocytopoiesis in vitro. Three groups of patients were included. The first group (n = 6) was diagnosed with congenital amegakaryocytic thrombocytopenia. They had no megakaryocytes in the bone marrow, three out of four patients showed no megakaryocyte formation in vitro, and all had high Tpo and low GC levels. Mutations in the thrombopoietin receptor gene, c‐mpl, were the cause. The second group of patients (n = 3) had normal Tpo and severely decreased GC levels. In bone marrow, normal to increased numbers of atypical, dysmature megakaryocytes were present. In vitro megakaryocyte formation was quantitatively normal. A defect in final megakaryocyte maturation and subsequent (pro‐)platelets may be the cause of the thrombocytopenia. The patients in the third group (n = 5) had Wiskott–Aldrich syndrome (WAS). They had normal Tpo and GC levels and normal megakaryocyte formation both in vivo and in vitro. This corresponded with the generally accepted hypothesis that thrombocytopenia in WAS is due to increased platelet turnover. In conclusion, different causes of congenital thrombocytopenia can be distinguished using three parameters: Tpo and GC plasma levels and in vitro analysis of megakaryocytopoiesis. Therefore, these parameters may be helpful in early diagnosis of different forms of congenital thrombocytopenia.

Functional analysis of single amino-acid mutations in the thrombopoietin-receptor Mpl underlying congenital amegakaryocytic thrombocytopenia.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder that presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. The disease may develop into bone marrow aplasia. Genetic defects in the gene encoding the thrombopoietin (Tpo) receptor, MPL, are the cause of this disease. In a previous study, we identified four missense mutations in CAMT patients, predicting Arg102Pro, Pro136His, Arg257Cys and Pro635Leu. To investigate whether these mutations result in defective Tpo‐binding and/or signalling, full‐length wildtype and mutant MPL were transduced into K562 cells. Expression levels and the ability to activate the mitogen‐activated protein kinase, Janus kinase‐signal transducer and activator of transcription and phosphoinositide‐3 kinase pathways upon Tpo‐binding were studied. The results predicted that MPL carrying the P136H or P635L mutation was not properly expressed, whereas the R102P and R257C mutations resulted in impaired signal transduction. Our results indicate that a severe clinical course may be expected when these mutations lead to absent Mpl expression or signalling in CAMT patients with missense mutations.

Influence of medium components on ex vivo megakaryocyte expansion.

Reinfusion of ex vivo-expanded autologous megakaryocytes together with a stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. In this study, we analyzed several serum-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover, two thrombopoietin (Tpo)-mimetic peptides were tested to evaluate whether they could replace Tpo in an expansion protocol. To analyze the effects of different media on megakaryocyte expansion, we used an in vitro liquid culture system. For this purpose, CD34+ cells were isolated from peripheral blood and cultured for 8 days in the presence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was analyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure, megakaryocyte medium (MK medium) supplemented with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded higher numbers of megakaryocytes, implying the pr...

Thrombopoietin and its receptor: structure, function and role in the regulation of platelet production

Although thrombopoietin (Tpo) was already described in the early sixties, it took more than 30 years before it was cloned. It became possible after the recognition and cloning of its receptor, Mpl. In the past few years new information about Tpo and Mpl has accumulated rapidly. Structure, biosynthesis and tissue expression have been elucidated. The central role of Tpo in the regulation of megakaryocytopoiesis and platelet production has become clear, as has the way in which this is accomplished. Tpo appears to be an important growth factor for haematopoietic stem cells as well. Thus, it may become one of the most important factors in stem cell transplantation. Finally, the signal transduction mechanisms involved and the way in which Tpo affects platelets and their precursors functionally have been studied in detail. The cloning and characterization of thrombopoietin and its receptor Mpl is one of the most important advances in the haematology of the nineties.

11 Thrombopoietin: its role in platelet disorders and as a new drug in clinical medicine

The cloning and characterization of thrombopoietin and its receptor Mpl in the past few years has been a major advance in haematology. It opens new ways of studying congenital and acquired platelet disorders, leading to new insights in the pathogenesis and treatment of this group of diseases. The Tpo level of the blood appears to be a useful marker for the differentiation between thrombocytopenia due to peripheral destruction and that due to thrombocytopoietic failure. No simple clinical parameter exist for this important differential diagnostic problem. When recombinant thrombopoietin becomes available for therapy it will rapidly become the drug of choice for many of our patients. However, because of all the legal and commercial issues at stake it is expected that it will still take a few more years before general availability is realized. Short peptides with Tpo activity ( Cwirla et al, 1997 ; Kimura et al, 1997 ), which can be synthesized chemically, may form a more easily obtainable alternative.