Thrombopoietin level in young patients is related to megakaryocyte frequency and platelet count.

PURPOSE: To examine the relationships among platelet counts, bone marrow megakaryocyte frequency, and circulating thrombopoietin (TPO) levels. PATIENTS AND METHODS: TPO levels in 17 children and one young adult with chronic or recurrent thrombocytopenia were measured by ELISA and megakaryocyte frequency was analyzed by light microscopy. Three groups of patients were studied: Group I patients had aplastic anemia and absent or decreased megakaryocytes; Group II patients had intermittent periods of chemotherapy-induced thrombocytopenia; and Group III patients had normal or increased megakaryocytes. Controls consisted of 77 healthy adults. RESULTS: Patients in Group I had markedly increased TPO levels compared to normal controls. Their levels were significantly different (p = 0.03) from those of patients in Group III. The latter had normal or only mildly increased TPO levels except for one patient with myelodysplastic syndrome. Patients in Group II had markedly elevated TPO levels. After their bone marrow and platelet counts recovered from chemotherapy, their TPO levels decreased. In all three groups, a transient increase in platelet count (e.g., after platelet transfusion or anti-D immune globulin therapy) was associated with a moderate decrease in TPO. CONCLUSIONS: From this study, three conclusions can be made: 1) TPO levels are inversely related to megakaryocyte frequency; 2) platelet counts have a modest influence on TPO level; and 3) TPO levels may have clinical utility in diagnosis and management and further our understanding of the pathobiology of the disorders that cause thrombocytopenia.

Serum thrombopoietin levels in patients with aplastic anaemia.

Endogenous serum thrombopoietin (TPO) levels were measured in 31 patients with aplastic anaemia (AA) using an enzyme immunoassay with a sensitivity of 20 pg/ ml. The median platelet count for all AA patients was 30 +/- 29 x 10(9)/l (range 5-102) compared with a median of 284 +/- 59 x 10(9)/l (range 148-538) for normal controls. Serum TPO levels were significantly elevated in all patients compared with normals (1706 +/- 1114.2, range 375-5000 v 78 +/- 54, range 16.5-312.9, P < 0.0001). There was no correlation between serum TPO levels and the degree of thrombocytopenia in AA patients, but TPO levels were significantly higher in patients who were platelet transfusion dependent than in patients who were transfusion independent (P < 0.01). There was a trend for higher TPO levels in patients with severe AA compared with non-severe AA patients. Clinical trials of TPO and a related truncated, pegylated molecule, megakaryocyte growth and development factor (PEG-rHuMGDF), are awaited to determine whether treatment with these drugs will result in increased platelet counts in patients with AA.

Purification and biologic characterization of plasma-derived megakaryocyte growth and development factor.

The isolation and cloning of the ligand for the cytokine receptor, Mpl, have been recently described. In this report we present details of the purification of this novel cytokine (megakaryocyte growth and development factor [MGDF]) from aplastic canine plasma. Two forms of canine MGDF, with apparent molecular weights of 25 kD and 31 kD and sharing a common N-terminal amino acid sequence, were isolated. The sole contaminant detected in purified 25-kD or 31-kD MGDF was canine Ig. Canine MGDF is characterized as a human megakaryocyte colony-stimulating factor that acts synergistically with human recombinant stem cell factor but not interleukin-3. MGDF also appears to be physiologically regulated in response to platelet demand. In canine and murine models, serum levels of MGDF activity peak during the thrombocytopenic periods after irradiation, 5-fluorouracil, or antiplatelet antisera injections. These data indicate that the megakaryocyte-stimulating activity that accumulates in plasma in response to platelet losses is a novel cytokine that functions through an interaction with the Mpl cytokine receptor.

Platelets generated in vitro from proplatelet-displaying human megakaryocytes are functional.

An in vitro culture system demonstrating the transitions from megakaryocyte progenitors to functional platelets is described. CD34-selected cells from normal human peripheral blood are cultured under conditions that promote megakaryocyte formation. After 8 to 11 days, enriched populations of mature megakaryocytes are replated under conditions that favor the development of proplatelets. Proplatelets express the platelet-specific proteins, glycoproteins Ib and IIb (GPIb and GPIIb), and fibrinogen and also contain microtubule coils equal in size to those found in plasma-derived platelets. In addition, proplatelets have ultrastructural features in common with plasma-derived platelets. Platelet-sized particles from the proplatelet culture supernatants are examined. Ultrastructurally, these particles are identical to plasma-derived platelets. Functionally, these culture-derived platelets aggregate in response to both thrombin and adenosine diphosphate (ADP) plus fibrinogen. This aggregation is specifically inhibited by the addition of a function-blocking anti-GPIIbIIIa antibody. Culture-derived platelets stimulated with agonists also express the activation-dependent antigens P-selectin and functional fibrinogen receptor. This is the first description of an in vitro culture system that sequentially demonstrates megakaryocyte growth, development, and platelet production.

Enrichment and characterization of peripheral blood-derived megakaryocyte progenitors that mature in short-term liquid culture.

A population of peripheral blood-derived cells that mature into megakaryocytes within four to eight days of liquid culture is described. This population was enriched from normal leukapheresis units by counterflow centrifugal elutriation and CD34 selection. The cells were incubated in suspension with known megakaryocyte growth or maturation factors. Megakaryocytes were identified within the cultures with antibodies to platelet-glycoproteins (Ib and IIb) and cytologically classified as stage I-IV cells. Plasma from aplastic dogs (APK9) or human recombinant interleukin 3 (IL-3) were the only culture additives which reproducibly resulted in megakaryocyte development. The activity present in APK9 was relatively megakaryocyte-specific while IL-3 was not. The phenotype of the short-term megakaryocyte progenitor cell population was determined by FACS and found to be CD34bright but not CD34dull or CD34-. The population was further characterized as CD34+/CD38+ and CD34+/HLA-DR+. Both CD34+/CD41- and CD34+/CD41+ populations contained megakaryocyte progenitor cells, although megakaryocytes that developed from the latter population were fewer in number. In summary, we have developed an enrichment protocol that, when coupled with a liquid culture assay system, enabled us to characterize short-term megakaryocyte progenitors from peripheral blood. These cells may be important for early platelet recovery in transplantation settings.