Claudia C. Folman

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.

The role of thrombopoietin in post‐operative thrombocytosis

Thrombopoietin (Tpo), the main regulator of thrombocytopoiesis, is a probable candidate to play a role in the increase in platelet counts that is frequently seen after surgery. In the current study, serial blood samples of patients that underwent major surgery were analysed with respect to Tpo kinetics, platelet turnover and inflammatory cytokines. Platelet Tpo content and plasma Tpo levels rose before platelet counts increased, suggesting that Tpo was indeed responsible for the elevation in platelet counts. In addition, an increase in interleukin 6 (IL‐6) levels, but not in IL‐11 and tumour necrosis factor alpha levels, was seen before the rise in Tpo concentration. In vitro, IL‐6 was shown to enhance Tpo production by the HepG2 liver cell line. Thus, increased Tpo levels after surgery, possibly resulting from enhanced Tpo production under the influence of IL‐6 or other inflammatory cytokines, are involved in an enhanced thrombocytopoiesis.

The Arg633His substitution responsible for the private platelet antigen Groa unravelled by SSCP analysis and direct sequencing

We have previously described the private or family platelet antigen, Groa, which was identified in a case of neonatal alloimmune thrombocytopenia. The Groa antigen was found to be located on the GP IIIa (β3) subunit of the GP IIb/IIIa complex, the most prominent fibrinogen receptor of platelets. Initial experiments to characterize the Groa antigen at the molecular genetic level were unsuccessful. We therefore decided to use a different strategy to unravel the molecular basis of this antigen. Platelet GP IIIa mRNA of a Gro(a+) and a Gro(a−) donor was amplified with suitable primers in a reverse transcriptase–polymerase chain reaction (RT‐PCR) and subjected to single‐strand conformational polymorphism (SSCP) analysis. Three regions of the amplified GP IIIa cDNA derived from the Gro(a+) donor showed a different SSCP pattern when compared to that of the Gro(a−) donor. Direct nucleotide sequence analysis of these three segments revealed that two of them contained silent substitutions, A1163C, A1553G and G1565A. The first and the latter changes were described previously. In the third segment a G1996A mutation was found, predicting an arginine  →  histidine substitution at position 633 of the mature glycoprotein. PCR‐ASRA (allele‐specific restriction enzyme analysis) performed on cDNA as well as on genomic DNA with the restriction enzyme MaeIII showed that the His633 form of GPIIIa is restricted to the Gro(a+) phenotype. The observed mutation is three amino acids upstream of the mutation underlying the HPA‐8/Sr system (Arg636Cys), suggesting this region of GP IIIa to be susceptible for mutations. Moreover, the presence of a silent mutation and two low‐frequency forms of the silent polymorphisms strongly suggests that the G1996A mutation did not occur in a direct ancestral allele.

Donors with a rare pheno (geno) type

H. W. Reesink, C. P. Engelfriet, H. Schennach, C. Gassner, S. Wendel, R. Fontão-Wendel, M. A. de Brito, P. Sistonen, J. Matilainen, T. Peyrard, B. N. Pham, P. Rouger, P. Y. Le Pennec, W. A. Flegel, I. von Zabern, C. K. Lin, W. C. Tsoi, I. Hoffer, K. Barotine-Toth, S. R. Joshi, K. Vasantha, V. Yahalom, O. Asher, C. Levene, M. A. Villa, N. Revelli, N. Greppi, M. Marconi, Y. Tani, C. C. Folman, M. de Haas, M. M. W. Koopman, E. Beckers, D. S. Gounder, P. Flanagan, L. Wall, E. Aranburu Urtasun, H. Hustinx, C. Niederhauser, E. Massey, A. Gray, M. Needs, G. Daniels, T. Callaghan, C. Flickinger, S. J. Nance & G. M. Meny

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.

Serum levels of thrombopoietin, IL-11, and IL-6 in pediatric thrombocytopenias

Abstract We measured serum levels of thrombopoietin (TPO), interleukin (IL)-11, and IL-6 in 90 different samples from 67 pediatric patients with thrombocytopenia (TP). The cytokine levels were determined by enzyme-linked immunosorbent assays (ELISA), and the biological activity of TPO was measured using a cell line transfected with human c-mpl. In patients with impaired megakaryocytopoiesis, as found in diseases such as aplastic anemia, amegakaryocytic TP, or TP with absent radii, we found TPO levels which were highly elevated compared with normal values (mean=261 AU/ml, n=52, vs. 22 AU/ml in healthy controls). In contrast, patients suffering from idiopathic thrombocytopenic purpura (mean=16 AU/ml, n=31) or platelet function defects (mean=23 AU/ml, n=7) demonstrated normal TPO levels. The biological activity tested in the bioassay correlated well with the ELISA data. However, sera of some patients with amegakaryocytic TP demonstrated a remarkably higher biological activity of TPO than expected from the ELISA data. Within the different groups there was no correlation between platelet counts and TPO levels. Only 27% of all samples had elevated levels of IL-11 (mean=450 pg/ml, n=20). Elevated IL-6 serum levels were detected in only 13% of all samples analyzed (mean=42 pg/ml, n=12). We conclude that megakaryocytopoiesis is regulated mainly by TPO, that it is dependent on the platelet and the megakaryocytic mass, and that IL-11 plays an additional role in supporting the platelet production. IL-6 does not appear to be up-regulated in children with thrombocytopenia.

High levels of reticulated platelets and thrombopoietin characterize fetal thrombopoiesis

To characterize fetal thrombopoiesis, we determined plasma thrombopoietin (TPO) and glycocalicin levels, platelet counts and reticulated platelets (RP) of fetuses and compared them with the respective values of their mothers. Percutaneous umbilical vein sampling in abnormal pregnancies revealed twofold higher thrombopoietin levels and 20‐fold higher reticulated platelet counts, but lower levels of glycocalicin in fetuses compared with their mothers (P < 0·05). Neither the expression of platelet glycoprotein Ib and IIb on platelets nor the platelet counts were different between mothers and their fetuses. These data indicate enhanced thrombopoiesis and/or increased platelet turnover in fetuses.

Analysis of the kinetics of TPOuptake during platelet transfusion

BACKGROUND: It has been shown in several studies that platelets play a role in the removal of TPO from the circulation. For instance, in vitro studies have shown that platelets can bind and internalize TPO, and transfusion studies have shown that the concentration of circulating TPO decreased after platelet transfusion. In the current study, the in vivo kinetics of plasma TPO levels and TPO uptake by transfused platelets is analyzed in more detail.

Fetal and neonatal thrombopoietin levels in alloimmune thrombocytopenia

Thrombopoietin (Tpo) is the main hematopoietic growth factor for platelet production. Plasma Tpo levels in autoimmune thrombocytopenic patients are normal or slightly elevated. Although thrombocytopenia exists, Tpo levels are not increased because the produced megakaryocytes and platelets can bind circulating Tpo, thereby normalizing Tpo levels. In this study, plasma samples from fetuses and neonates with neonatal alloimmune thrombocytopenia (NAIT), a different form of immune thrombocytopenia, were measured. Umbilical cord samples from 50 fetuses before treatment because of severe thrombocytopenia and 51 fetuses after treatment, and peripheral blood samples of 21 untreated newborns with NAIT were analyzed. As controls, plasma Tpo levels were determined in 21 umbilical cord samples of 14 nonthrombocytopenic fetuses with hemolytic disease resulting from red blood cell alloimmunization and in umbilical cord samples of 51 healthy newborns. The values were also compared with the plasma Tpo levels in 193 healthy adults. Mean Tpo levels from the groups of fetuses and neonates, including both NAIT and control plasma, were slightly but significantly elevated compared with levels in healthy adults. Tpo levels in NAIT samples were not significantly different from the levels in hemolytic disease samples or in samples from healthy newborns. Thus, as in autoimmune thrombocytopenic patients, normal Tpo levels are present in NAIT patients.

Molecular analysis of the York antigen of the Knops blood group system

BACKGROUND: Antigens of the Knops blood group system are present on complement component (3b/4b) receptor 1 (CR1/CD35), which is a transmembrane glycoprotein encoded by the CR1 gene. Eight of the nine known antigens of this system are linked to polymorphisms in Exon 29. The molecular background of one antigen, York (Yka), has not yet been described.