T. P. McDonald

Bioassay for Thrombopoietin Utilizing Mice in Rebound Thrombocytosis

Summary A bioassay for the detection of the thrombopoietic stimulating factor (TSF or thrombopoietin) that utilizes thrombo–cythemic mice has been presented. A single injection of anti–mouse platelet serum (AMPS) to mice produced a characteristic thrombocytopenia that was followed by rebound thrombocytosis. Two to three days later (7–8 days after the AMPS injection), a significantly (p < 0.05) depressed thrombo–poiesis existed in response to the thrombocytosis. Normal and thrombocythemic mice were injected with normal sheep sera or sera from platelet–depleted sheep and Na2 35SO4 incorporation into platelets was measured. A greater difference in 35S uptake existed between AMPS–treated control mice (mice injected with normal sheep serum) and AMPS–treated stimulated mice (mice injected with serum from platelet–depleted sheep) than in normal mice treated with the same sera. Also, TSF in sheep sera was shown to lose activity after storage at −20°. The author is grateful to Mrs. Marilyn Cottrell and Miss Rose Clift for technical assistance and to Mrs. Ann Beardsley for her art and stenographic services. Appreciation is also extended to Mr. George Creswell for help in bleeding and maintaining the sheep, to Mr. James Dyer for help in performing the mouse assays, and to Dr. T. T. Odell, Jr. and Dr. Shirley Ebbe for their many helpful suggestions.

Acute megakaryoblastic leukaemia with 3q inversion and elevated thrombopoietin (TSF): an autocrine role for TSF?

Summary. A patient with acute megakaryoblastic leukaemia is described in whom exactly the same paracentric inversion of 3q was detected as in three previously documented cases. The patients serum thrombopoietin (TSF) was significantly raised. Based on these findings we postulate a role for a gene (? oncogene) on chromosome 3q in thrombopoietin production. Abnormalities of 3q may assist in delineating a subgroup of acute nonlymphocytic leukaemia, namely acute megakaryoblastic leukaemia.

A Comparison of Platelet Size, Platelet Count, and Platelet 35S Incorporation as Assays for Thrombopoietin

SUMMARY. Average platelet size, platelet count, and 35S‐incorporation into platelets were compared as methods for the measurement of thrombopoietin‐stimulated thrombopoiesis. In mice injected with rabbit anti‐mouse platelet serum (RAMPS) average platelet size was shown to be increased as mice were recovering from thrombocytopenia. Also, 35S‐measurements on platelets of these mice showed significant increases in cpm/average platelet 2–4 days after RAMPS treatment. Significant increases in 35S‐incorporation into the total circulating mass of platelets were found on days 3–4. In normal mice or mice in rebound‐thrombocytosis injected with thrombopoietin, platelet size remained unchanged, whereas the platelet count and 35S‐incorporation into platelets were shown to be significantly increased. Moreover, a dose‐response experiment in mice pretreated with RAMPS showed a slight increase in platelet count as the dose of TSF was increased, but platelet sizes were unaltered. The %35S‐incorporation into platelets showed a significant linear dose‐response, i.e. as the dose of thrombopoietin was increased, an increase in %35S‐incorporation into platelets was observed. These data indicated that of the three indirect measurements of thrombopoietin, the %35S‐incorporation into mouse platelets was the most sensitive, followed by platelet counting; the least sensitive measurement of thrombopoiesis was change in platelet size.

Hepatoblastoma, thrombocytosis, and increased thrombopoietin

Two children with hepatoblastoma, marked thrombocytosis, and extremely elevated alpha fetoproteins are reported. It is not clear from review of the literature whether hepatoblastomas or hepatocarcinomas are associated with thrombocytosis. Elevated thrombopoietin levels were found in liver tumor extract from one patient.

Thyroxine Suppresses Thrombocytopoiesis and Stimulates Erythropoiesis in Mice

Abstract Thyroxine has been shown in vitro to stimulate erythropoiesis by two mechanisms: a direct, β2-adrenergic receptor-mediated stimulation of red cell precursors, and an indirect, erythropoietin-mediated mechanism. Clinical reports have suggested that excess thyroxine also exerts depressive effects on thrombocytopoiesis, but the most sensitive methods of assessing platelet production, i.e., percentage of 35S incorporation into platelets and determination of megakaryocyte size and number, are not appropriate for analysis of platelet production in human patients. The purpose of this study was to use a mouse model to investigate the effects of the hyperthyroid state on erythropoiesis and thrombocytopoiesis, and to assess in vivo the two mechanisms by which thyroxine has been described to stimulate erythropoiesis in vitro. We found that thyroxine administration significantly depressed platelet production and stimulated erythropoiesis in mice. Both the D- and L-isomers of thyroxine in appropriate doses produced this depression of thrombocytopoiesis, and the effect was dose dependent for both isomers. Daily administration of thyroxine:increased blood volume; decreased the peripheral platelet count, total circulating platelet count and mass, percentage of 35S incorporation into platelets, and megakaryocyte number and size; and concurrently increased indices of red cell production (packed cell volume, red blood cell count, total circulating red blood cell count and mass, and reticulocyte count). Additionally, propranolol, a nonspecific β-blocker, partially reversed the suppression of platelet production by L-thyroxine, lending credence to the assertion that the direct, β2-adrenergic receptor-mediated stimulation of the erythroid cell line by thyroxine reported to exist in vitro may also be important in vivo. [P.S.E.B.M. 1992, Vol 201]

Monoclonal Antibodies to Human Urinary Thrombopoietin

Abstract Monoclonal antibodies (MA) to a thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) were obtained from hybridomas derived from the fusion of P3 × 63/Ag 8 cells and spleen cells from TSF-immunized BALB/c mice. The immunizing protein was a partially purified TSF-rich preparation from the urine of a thrombocytopenic patient, and was shown to stimulate platelet production in rebound-thrombocytotic mice; i.e., platelet counts of recipient mice were increased to 133% of control and the values for percentage 35S incorporation into platelets were elevated to 225% of control. Media from several hybrid cultures were tested in a microantibody detection technique that measured the binding of MA to a 125I-purified TSF preparation from human embryonic kidney (HEK) cells. The immune complex was precipitated by the addition of goat anti-mouse IgG serum and centrifugation. One clone gave 25% binding of 125I-TSF after a sevenfold dilution of the medium. This cell line was recloned and four of the subclones produced MA that gave even greater binding capacities. Hybridized cells were injected into “pristane-primed” mice and the antibodies produced in the ascites fluid were also shown to bind the 125I-TSF. Compared to the results of normal mouse serum, ascites fluid containing MA was shown to bind the unlabeled TSF from HEK cells. The TSF activity was significantly reduced in the supernatant fluid after precipitating the TSF-anti-TSF immune complex by a second antibody when tested in an immunothrombocythemic mouse assay. After SDS-PAGE, the precipitate from this TSF-MA conjugate showed that the antiserum bound a single 32,000 mol wt component, indicating the monospecificity of the MA. MA directed toward human TSF will allow studies that were not previously possible.

Stimulation of mouse megakaryocyte endomitosis by plasma from thrombocytopenic rats.

Summary Injection of plasma from thrombocytopenic donor rats resulted in an increase in the endomitotic index of megakaryocytes of recipient mice 32 hr after the initial treatment with plasma. The results suggested a dose-response relationship between the amount of plasma administered and the degree of stimulation of megakaryocytopoiesis. These findings demonstrate that an agent capable of stimulating megakaryocytopoiesis is released in response to thrombocytopenia and that this factor can be successfully transferred between species. They also substantiate the assumption that the increase in peripheral platelet numbers and in platelet labeling after administration of presumptive TSF occurs via stimulation of megakaryocytopoiesis.

Effects of Hypoxia on Megakaryocyte Size and Number of C3H and BALB/c Mice

Abstract In an effort to explain the different platelet production capabilities of both normal and hypoxic male and female C3H and BALB/c mice, megakaryocyte size and number were determined utilizing bone marrow from both normal and hypoxic mice. The results indicate that normal BALB/c female mice have increased numbers of megakaryocytes, but of smaller size compared with either BALB/c male mice or to both sexes of C3H mice. An inverse relationship between the size and number of megakaryocytes was found in both normal and hypoxic mice; therefore, to evaluate total megakaryocyte characteristics, we calculated total megakaryocyte masses (TMM). With hypoxia, megakaryocyte number decreased, whereas megakaryocyte size increased. Despite the increase in megakaryocyte size, hypoxia caused a significant decrease in TMM (P < 0.005) in all mice, but female C3H mice had higher TMM (P < 0.05) than did female BALB/c mice. These data show that hypoxia decreases TMM in mice, and that the effect is greater in C3H mice than in BALB/c mice.

Immunological Studies of the Renal Erythropoietic Factor (Erythrogenin)

An immunological study of the renal erythropoietic factor (REF or erythrogenin) has been described. The experiments indicate that the erythropoietic activity of REF, as assayed in the polycythaemic mouse, is neutralized in vitro by addition of serum obtained from a rabbit previously immunized with a REF preparation. These anti‐REF sera had no effect on the biological activity of ESF; however, a depression in erythropoiesis was observed after injection of anti‐REF into normal mice. It is concluded that the injection of an antibody developed against the REF into normal mice interrupts normal erythropoiesis by reducing REF levels; the reduced REF levels lead to decreased amounts of ESF.

ASSAY AND SITE OF PRODUCTION OF THROMBOPOIETIN

A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) controls platelet production by acting on precursor or immature cells to cause differentiation in to megakaryocytes, leading to increased platelet production. Recent developments in the techniques used for the assay of TSF have permitted studies on the site of production of the humoral factor. Several sites for the production of TSF have been proposed, including the spleen, liver and kidney. Although the precise site of TSF production is unknown, thrombocytopenia has been reported in up to 50% of patients with chronic renal disease (Rath et al, 1957). Additional recent work points to the kidney as at least one site of production of the hormone. Arraysfor thrombopoietin. The methods for assay of TSF have utilized changes in peripheral platelet counts (Spector, 1961; Ode11 et al, 1961; Krizsa, 1971), changes in platelet sizes (Weintraub & Karpatkin, 1974; McDonald, 1980), an immunologic measurement of thrombopoietin (McDonald, 1973a), measurement of megakaryocytopoiesis (Harker, 1968; Ebbe et al, 1968; Odell, 1973; Nakeff et a!, 1975; Nakeff, 1976), determination of the number of the small acetylcholinesterase positive (SAChE +) cells (Kalmaz, G.D. & McDonald, T.P., unpublished), and alterations in platelet labelling with radioisotopes (Harker, 1968, 1970; Penington, 1969, 1970; Evatt & Levin, 1969; Shreiner & Levin, 1970; Cooper et al, 1970; McDonald, 1973a, b). In some of these studies, the TSF recipient animals have been pretreated, i.e. splenectomized (Odell et al, 1961), transfused with platelets (Evatt & Levin, 1969; Schreiner & Levin, 1970; Cooper et al, 1970; McDonald et al, 1976), or injected with platelet specific antisera (Penington, 1969; McDonald, 1973b) in an attempt to increase their sensitivity to TSF. Of these several attempts to find the optimum conditions for the assay of TSF, mice in rebound-thrombocytosis appear to be more sensitive to exogenous TSF preparations than are normal mice (see review by McDonald, 1977a). Recent studies have improved these early assay techniques and have shown that the route of administration ofTSF did not seem to be important since TSF injected either subcutaneously or intraperitoneally gave essentially the same result; however, multiple injections were more effective than single injections (McDonald, 1977b). The time of measurement of 35S incorporation into platelets and selection of the mouse strain determines, to a large degree, the sensitivity of the TSF assay. Sex of the mice does not appear to be important; similar responses were found in both male and female mice (McDonald et al, 1979). In dose-response experiments, 35S-sodi~m sulphate gave significantly greater values at the higher TSF doses than did 75Se-selenomethionine, but either isotope can be used to measure platelet production rates in mice stimulated with TSF (Clift & McDonald, 1979). Site of TSF production. In an early study, Krizsa (1971) concluded that the kidney was