Partial purification and biochemical characterization of human plasma thrombopoietin.

A factor that stimulates the incorporation of 75Selenomethionine into the newly formed platelets of recipient mice (thrombopoietin, TPO) has been partially purified from the plasma of thrombocytopenic patients. The activity was precipitated at 60-80% ammonium sulfate saturation and further purified with hydrophobic interaction chromatography. Thrombopoietin was retained by concanavalin-A-Sepharose. Using HPLC size-exclusion chromatography, an approximate molecular weight of 40,000 dalton was calculated. The overall purification factor was about 2,100-fold. TPO was stable in a pH range from 5 to 9 and was heat-sensitive, and the biological activity was destroyed by trypsin treatment and by dithiothreitol. The partially purified molecule did not stimulate the proliferation of megakaryocyte progenitors in vitro and had no effect on the growth of erythroid or granulocyte-macrophage colonies; when administered in-vivo, TPO significantly affected the mean platelet volume and increased the number of small acetylcholinesterase cells in the bone marrow. TPO appears to be specific for the megakaryocytic lineage and active on the postmitotic compartment of megakaryocytes.

Recombinant human erythropoietin for treatment of myelodysplastic syndromes.

Nine patients with myelodysplastic syndromes and one patient with agnogenic myeloid metaplasia have been treated with recombinant human erythropoietin (rhEpo), at the dose of 150 U/kg/day. Although serum Epo levels were correlated with hemoglobin concentrations in the whole population of patients, they clearly appeared inadequate in some instances, if compared to those of a group of control subjects with iron deficiency anemia. Moreover, no correlation was found between serum Epo and reticulocytes. Six patients showed a partial or complete response to the treatment and the outcome was not correlated with the pre-therapy serum Epo levels; however, serum Epo was less than 100 mU/ml in three of four patients who achieved a complete response. The mechanism(s) by which Epo stimulated erythrocyte production in myelodysplastic patients is unclear, because the number of both the reticulocytes and erythroid progenitors remained unchanged during and at the conclusion of a three months' therapy. Further studies are needed to better define the optimal dosage required to correct anemia in myelodysplastic syndromes, and to clarify rhEpo mechanism of action in these diseases.

Serotonin incorporation as a marker of murine megakaryocyte proliferation in liquid cultures.

We have developed a new in vitro method for the quantitation of murine megakaryocyte proliferation that is based on the unique property of megakaryocytes to incorporate and store [14C]serotonin in cytoplasmic dense granules. The specificity of the assay was demonstrated by autoradiography of whole bone marrow cell suspensions, which showed evidence of grain accumulation only in megakaryocytes. Bone marrow cells were cultured in liquid cultures in the presence of a stimulator of megakaryocyte growth before the addition of 2.5 microM [14C]serotonin. The amount of serotonin incorporated in cells was evaluated after 3 h. Radioactivity peaked at days 6 and 7 and remained high until day 10; there was a linear relationship between the incorporation of serotonin and the number of cells plated. A dose-response curve between the incorporation of serotonin and the concentration of pokeweed mitogen spleen-conditioned medium (PWM-SCM) was observed, with inhibitory effects becoming predominant at the highest concentrations. The proliferation of megakaryocyte progenitors was also stimulated by partially purified interleukin 3, whereas both human recombinant erythropoietin and human recombinant granulocyte colony-stimulating factor (rG-CSF) failed to modify the incorporation of serotonin in comparison with unstimulated cultures. Finally, in parallel experiments we observed a significant correlation between the number of megakaryocytic colonies grown in agar and the radioactivity in liquid cultures. The method described herein is reproducible, sensitive, and easy to perform; it should be useful for the study and purification of factors affecting megakaryocyte proliferation.

Constitutive expression of a megakaryocytic functional property by murine erythroleukemia (Friend) cells: the incorporation of serotonin.

Murine Friend-derived erythroleukemia cells (MEL) are generally believed to be unipotential progenitors inducible to terminal erythroid differentiation. However, we found that MEL can constitutively incorporate significant amounts of radiolabeled serotonin ([3H]5-HT). Because this process is typical of cells belonging to the megakaryocytic lineage, we investigated the significance and mechanisms of 5-HT incorporation in the MEL system. We observed that: 1) normal murine erythroid cells and erythroid progenitors do not incorporate [3H]5-HT, as well as normal murine myeloid cells and the human myeloid cell line HL-60; on the other hand, the human erythroleukemia cell lines K562 and HEL, which have been shown to constitutively express megakaryocytic features, were able to incorporate [3H]5-HT; 2) MEL incorporated 5-HT by an active and saturable mechanism, dependent on temperature and sodium concentration in the medium; and 3) 5-HT uptake was very rapid. Moreover, because about 65% of cell-associated radioactivity was no longer displaced by the cold substrate, we assumed it to represent "true" cytoplasmic internalization. Finally, 5-HT incorporation by MEL was inhibited by clomipramine, ouabain, and reserpine, which are known inhibitors of 5-HT uptake in platelets. The commitment of MEL to terminal erythroid differentiation by hexamethylene bisacetamide or dimethyl sulfoxide greatly reduced the capacity to incorporate [3H]5-HT. These results seem to suggest that the MEL system, although mainly erythroid as regards its differentiation capability, constitutively expresses features of the megakaryocytic lineage, possibly disclosed by the ability to incorporate 5-HT. This hypothesis was further supported by the findings that 30%-40% of uninduced MEL were labeled by a polyclonal antibody raised against murine platelets that selectively recognized megakaryocytes in murine bone marrow smears.

Hepatitis-free interval after clotting factor therapy in first infused haemophiliacs.

Post-infusion hepatitis is known to occur very frequently in haemophiliacs after treatment with unheated commercial clotting factor concentrates, obtained from large plasma donation pool. On the contrary, single-donor cryoprecipitate is likely to carry a lower risk of transmitting hepatitis. To evaluate this hypothesis, we retrospectively reviewed the medical records of 25 first infused haemophiliacs (from 1981 to 1984) treated with unheated commercial clotting factor concentrates (n = 19) or cryoprecipitate (n = 6). The hepatitis-free interval after the beginning of therapy was expressed as exposure days. The end point of each patient, i.e. the hepatitis occurrence, was defined as an increase of amino-transferases (ALT and AST) and/or the seroconversion of HBV-markers, which were checked every three months. The life-table method and log-rank test showed that cryoprecipitates had a significantly longer hepatitis-free interval (p = 0.0131, log-rank test) and a lower risk of transmitting hepatitis (p = 0.01-0.05, life-table method) than the commercial concentrates. However, the safety of cryoprecipitate therapy was shown to cover only a few exposure days, and so the real advantage of this product depends on the bleeding frequency of the patient concerned. We believe that these methods and our findings may be useful to assess and compare the safety of the new "heat-treated" clotting factor concentrates.

Serum erythropoietin levels in patients undergoing autologous bone marrow transplantation.

Serum erythropoietin (sEpo) levels were serially measured with a radioimmunoassay in 14 patients undergoing autologous bone marrow transplantation (BMT), starting before the institution of the conditioning regimen up to day +45. An increase in sEpo levels was observed soon after starting the chemotherapy regimen, and before an evident fall in hemoglobin (Hb) levels took place. The peak in sEPo levels (221 +/- 181 mU/ml) was reached at day 0 in 9/14 patients, and was delayed up to day + 10 in the remaining five. There was a negative correlation between loge sEpo and Hb values (r = -0.730; p less than 0.01); the regression line of this correlation was comparable to the one obtained in a group of 15 iron-deficiency anemic subjects. Therefore, patients undergoing autologous BMT appear to be able to develop adequately increased sEpo levels in response to the severity of anemia. No correlation was found between sEpo and white blood cell or platelet count. On the other hand, sEpo value at day 0 was significantly related to the day of neutrophil recovery (r = -0.806; p less than 0.001): patients with the highest sEpo levels at day 0 showed significantly faster (p less than 0.001) neutrophil recovery.

Protein content and factor VIII complex in untreated, treated and monoclonal factor VIII concentrates.

Replacement therapy with clotting factor concentrates may expose the recipients not only to virus contamination but also to continuous stimulation of the immune system by repeated infusions of allogenic proteins. Concentrate purity is now a very important prerequisite to be taken into account in choosing what product can better meet the patient's needs. We compared protein content (albumin, fibrinogen, fibronectin, immunoglobulins) and factor VIII:C/vWF:Ag complex in untreated, treated and monoclonal factor VIII concentrates. Protein content is dramatically decreased in new treated ultrapure concentrates. Improved traditional fractionation methods allowed to obtain very high Factor VIII specific activity. New fractionation methods with immunoaffinity chromatography by means of monoclonal antibodies can give highly pure concentrates even if deliberately added albumin decreases factor VIII specific activity in final formulation. Otherwise monoclonal concentrates show a very high specific activity in terms of fibrinogen and immunoglobulin content, which, unlike albumin, are affecting the immune system in hemophiliacs.

Effects of miocamycin and erythromycin on polymorphonuclear cell function.

Previous studies have shown that erythromycin can enter phagocytic cells, stimulating their functional activity. In this work we compared the effects of erythromycin and another newer macrolide antibiotic, miocamycin, on a series of in vitro tests aimed at evaluating their influence on polymorphonuclear cell (PMN) functions. Results indicate that erythromycin induces an increase in leukotriene B4 production in PMNs, while chemotaxis, killing of Candida albicans and respiratory burst are not influenced, at least at the doses used in this study. On the contrary, all these activities are significantly enhanced following incubation with miocamycin, and the response varies according to the antibiotic concentration.

Platelet function and coagulation studies in patients with mitral valve prolapse.

Twenty-eight consecutive patients with mitral valve prolapse (MVP), seven of whom had previous cerebrovascular disorders (CVD), were studied for platelet function and coagulation tests. While platelet function tests were found to be normal with the exception of platelet aggregation rate (PAR), there was a significant rise of factors VIII vWF:Ag (Von Willebrand) and (FPA) fibrinopeptide A. Six cases had high levels of both these factors, suggesting the existence of a particular subset of patients with MVP, with a higher risk of thromboembolic episodes, although only three out of seven patients with previous CVD had either FPA or VIII vWF:Ag levels. The broad spectrum of subjects with MVP probably explains the different results obtained when studying platelet function and coagulation factors. Therefore, larger population studies and prolonged follow-up of cases with either coagulation abnormalities similar to the ones found in the present report and/or altered platelet function tests are suggested to discover if it is possible to detect patients with a potential for thromboembolism.

Does ergometric stress test induce a procoagulative condition in patients with previous myocardial infarction?

A regularly scheduled physical training program seems to have antithrombotic effects. Moreover, the hemostatic changes occurring in patients with coronary artery disease during acute exercise have not been clearly elucidated. Since stress testing is routinely performed in clinical cardiology, it would be helpful to assess whether patients with coronary artery disease are exposed to acute coronary thrombosis during or soon after sustained physical exercise. This study was designed to evaluate the effect of acute physical exercise (stress test by bicycle ergometer) on blood coagulation in a group of patients with previous myocardial infarction, and to determine whether the antithrombotic therapy commonly administered favorably influences hemostatic equilibrium. Our results suggest that exercise testing is not harmful to patients with previous myocardial infarction in regard to hemostasis and fibrinolysis and that antithrombotic therapy reduces postexercise increase in platelets.

In vivo stimulation of megakaryocytopoiesis by recombinant murine granulocyte-macrophage colony-stimulating factor.

Murine recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) was injected in mice, and the effects on bone marrow, splenic megakaryocytes, megakaryocyte precursors (megakaryocyte colony-forming units [CFU-Meg]) were evaluated. In mice injected three times a day for 6 days with 12,000 to 120,000 U rGM-CSF, no significant modification of both platelet levels and mean platelet volume was observed, while there was a twofold increase in blood neutrophils. However, the rate of platelet production, as assessed by the measurement of 75selenomethionine incorporation into blood platelets, was On the contrary, administration of up to 384,000 U rGM-CSF two times a day for 2 days, as for a typical "thrombopoietin assay," failed to modify platelet production. A significant dose-related increase in the number of splenic megakaryocytes occurred in mice receiving 60,000 to 120,000 U rGM-CSF, while a slight increase in the number of bone marrow megakaryocytes was observed in mice injected with 120,000 U rGM-CSF. The proportion of bone marrow megakaryocytes with a size less than 18 microns and greater than 35 microns resulted significantly higher in mice receiving rGM-CSF in comparison with controls; an increase in the percentage of splenic megakaryocytes greater than 35 microns was also observed. A statistically significant increase in the total spleen content of CFU-Meg was observed after administration of 90,000 and 120,000 U rGM-CSF three times a day for 6 days, while no effect on bone marrow CFU-Meg was recorded, irrespective of the dose delivered. Finally, 24 hours after a single intravenous injection of rGM-CSF, there was a significant increase in the proportion of CFU-Meg in S-phase, with the splenic progenitors being more sensitive than bone marrow-derived CFU-Meg. These data indicate that rGM-CSF has in vivo megakaryocyte stimulatory activity, and are consistent with previous in vitro observations. However, an effective stimulation of megakaryocytopoiesis in vivo, bringing about an increase in the levels of blood platelets, may require interaction of rGM-CSF with other cytokines.

Binding of recombinant human 125I-erythropoietin to CFU-E from the spleen of anemic mice.

Erythroid precursors were obtained from the spleen of mice made anemic with phenylhydrazine and used to study the binding of bioactive 125I-rEp to membrane receptors. Kinetic analysis, using splenic cells obtained at different times following the induction of anemia, showed that the maximum binding was reached at day 3, and decreased thereafter; minimal amounts of 125I-rEp were bound to the splenic cells of normal mice. Splenic cells of day 3 anemic mice were fractionated using continuous Percoll density gradients, resulting in a fraction enriched in CFU-E (delta = 1.065-1072 g/ml) which showed the highest 125I-Ep binding on a per cell basis. The amount of 125I-rEp bound was greatly reduced in the densest fractions, which were comprised of maturing erythroblasts. The binding was time- and temperature-dependent, and a significant correlation was found with cell concentration up to 12 X 10(6). The amount of radioactivity specifically bound rose with increasing concentrations of 125I-rEp until a plateau was reached (2.5 nM), whereas non specific binding increased slightly and linearly. The binding of 125I-rEp was susceptible to competitive inhibition by unlabeled rEp, while other hematopoietic growth factors were ineffective. The calculated receptor density on these purified immature erythroid progenitors was 570 molecules with a Kd = 0.5 nM. Overall, these results suggest that the expression of the Ep receptor is reduced with increasing maturation.

Occurrence of haemorrhagic and thrombotic events in myeloproliferative disorders: a retrospective study of 108 patients.

Raised platelet count, impaired platelet function, and high PCV values are all considered risk factors for the haemorrhagic and/or thrombotic events frequently complicating the course of myeloproliferative disorders. Nevertheless, the outcome in young patients is reported as being frequently uncomplicated and, in these cases, treatment could be deferred. We examined retrospectively the occurrence of haemorrhagic and/or thrombotic events at the onset of myeloproliferative disorders in 108 patients and found that 67.4% of patients, with either raised platelet count or isolated high PCV, were symptomatic at the onset and the rate was similar regardless of disease, sex, age, platelet number and PCV values. We were unable to locate a patient group with a lower rate of complications and, in light of the frequency of symptoms, it seems reasonable to attempt a normalization of blood values in every case.

Recombinant human erythropoietin has little influence on megakaryocytopoiesis in mice.

The availability of a preparation of recombinant human erythropoietin (rEp) prompted us to investigate the role of Ep in the regulation of megakaryocytopoiesis in mice, using experimental procedures by which the effects on the mitotic and post-mitotic compartment of megakaryocytes could be evaluated separately. In agar cultures of murine bone marrow cells, either serum-depleted or serum-supplemented, rEp (0.1-2 U/ml) did not stimulate megakaryocyte colony formation and when it was added to suboptimal amount of spleen cell-conditioned medium (SCM), it failed to show a significant synergistic activity. On the contrary, rEp increased the number of megakaryocytic colonies developed from splenic precursors in the presence of suboptimal amounts of SCM, although it was unable per se to stimulate colony formation. The effects of rEps on megakaryocyte maturation and platelet production were studied in vivo evaluating the incorporation of 75Se-selenomethionine into platelets, the platelet count and platelet size, and the number of megakaryocyte precursors (small acetylcholinesterase positive cells, sAchE) in the bone marrow of mice injected with 1-8 U of rEp. No modification of these parameters was found in comparison with control mice. On the other hand, rEp increased the number of recognizable splenic megakaryocytes in a dose-dependent fashion. These data suggest that rEp has little influence on megakaryocytopoiesis, at least at the doses we used and which are known to elicit a maximal response of erythropoiesis. However, a subset of megakaryocytes with particular kinetic properties, such as those in the spleen of mice, may be responsive to relatively high doses of rEp. The significance of this observation in the overall regulation of megakaryocytopoiesis remains to be determined.

Inhibition of erythropoietin production in vitro by human interferon gamma.

The effects of interferon-gamma (IFN-gamma), alone and in combination with IL-1, IL-6 and tumour necrosis factor-alpha (TNF-alpha), on in vitro erythropoietin (Epo) production by the human hepatoma Hep3B cell line were evaluated. The addition of IFN-gamma to either unstimulated or cobalt chloride (CoCl2)-treated Hep3B cells resulted in a dose-dependent inhibition of Epo release in the medium by as much as 70% at 1000 U/ml. Half-maximal inhibition was observed at around 50 U/ml. According to previous observations, IL-6 had a stimulatory effect on Epo production by CoCl2-treated Hep3B cells; however, the simultaneous addition of IFN-gamma and IL-6 resulted in a reversal of the stimulatory effects due to IL-6. IFN-gamma and IL-1 had an additive inhibitory effect, whereas IFN-gamma and TNF-alpha acted in a synergistic fashion in inhibiting Epo production by Hep3B cells. The inhibitory effect of IFN-gamma appeared to be due to a down-modulation of Epo mRNA levels in CoCl2-treated Hep3B cells, as shown by Northern blot analysis. These data indicate that Epo production by hepatoma cells in vitro is inhibited by IFN-gamma, and that a complex network of interacting cytokines may regulate Epo production in response to an hypoxic stimulus. Overall, these results also suggest that IFN-gamma might have a role in the defective Epo production observed in several inflammatory and immunemediated disorders characterized by relatively high IFN-gamma plasma levels.