Rose Clift

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.

Effect of weaning and dietary galactose supplementation on digesta glycoproteins in pigs.

Changes in pig digesta mucin and glycoprotein content at weaning and with the inclusion of galactose in the postweaning diet were studied. Mucus was collected from ileal contents of cannulated pigs pre- and postweaning, and glycoproteins were analysed based on staining patterns and size exclusion chromatography. An increased concentration of intestinal mucin in lumen contents was observed in newly weaned pigs compared to the same pigs prior to weaning. Analysis of O-linked oligosaccharides indicated changes in mucin structure from pre- to postweaning. Supplementing the diet with galactose resulted in modification of mucin in postweaned pigs compared to a control diet and may limit microbial degradation of mucin. These data indicate that weaning and the subsequent transition to a grain-based diet result in changes to digesta mucin and endogenous glycoproteins; and dietary galactose may play a role in the final composition and quantity of these compounds.

Effects of Hypoxia on Thrombocytopoiesis and Thrombopoietin Production of Mice

Summary Mice were placed in hypoxia chambers for various lengths of time prior to and after injection of platelet specific antiserum to determine the cause of thrombocytopenia in hypoxic mice. The results indicate that hypoxia decreased platelet production by action on a precursor cell or a primitive population of megakaryocytes without altering the ability of mice to produce thrombo-poietin. Hypoxia may cause thrombocytopenia by stem-cell competition between the erythrocytic and megakaryocytic cell lines.

Effects of hypoxia on the small acetylcholinesterase-positive megakaryocyte precursor in bone marrow of mice.

Abstract The number of small acetylcholinesterase-positive (SAChE+) cells in the marrow of hypoxic mice was measured. Mice were exposed to 6-7% O2 levels by enclosure in cages covered with dimethyl-silicone rubber membranes for 1-14 days. The mice showed a linear increase in packed cell volumes with time in the hypoxic atmosphere, but platelet counts showed a characteristic biphasic response, i.e., increased platelet counts were observed after 1-3 days of hypoxia, and significantly (P < 0.05-P < 0.0005) decreased platelet counts were observed thereafter (6-14 days). The total number of megakaryocytes in the marrow of hypoxic mice decreased significantly (P < 0.005) with time. In agreement with the data on platelet counts, hypoxia caused the total number of SAChE+ cells in the marrow of mice to be biphasic. At Day 2 there was a significant increase (P < 0.05) in the total number of SAChE+ cells/mm3 of bone marrow; however, by days 10-14 the numbers had decreased markedly (P < 0.005). These data indicate that hypoxia decreases platelet production by action on a precursor cell to the SAChE+ cell. The hypoxia-induced thrombocytopenia is probably caused by stem-cell competition between the erythrocytic and megakaryocytic cell lines.

Recovery of thrombopoietin during purification

A thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) was previously purified by a six-step purification procedure. However, the exact quantity of TSF that was recovered, through the various purification procedures, was unknown because of the absence of a method for establishing a unit of measure of TSF. In the present work dose-response relationships on both the crude TSF preparations and on the more highly purified TSF were determined. TSF units were calculated from the dose-response curves. A unit of TSF is defined as the amount of material (mg) that is required to increase the percentages 35S incorporation into platelets of immunothrombocythemic mice by 50% above the baseline. The results of determining the TSF units on the crude TSF preparation indicated that 0.11 unit (U) of TSF/mg protein was present. Results showed that the specific activity of TSF can be increased to about 3.6 U/mg by a single purification procedure using Sephadex G-75 column chromatography. Increased specific activities were obtained by additional purification steps, i.e., DEAE-cellulose column chromatography, SE-HPLC, DEAE-HPLC, and SDS-PAGE. The purified product appears to have a specific activity of about 11,000 U/mg of protein with 0.00003% of the protein and 1.1% of the TSF recovered from the starting material. Establishing a unit of measure for TSF will allow calculations of its degree of purity, provide a method for quantitation of recoveries of activities after various purification procedures, and allow comparisons of results from different experiments and different laboratories.

A Comparison of [35S]Sodium Sulfate and [75Se]Selenomethionine as Platelet Labels for the Assay of Thrombopoietin

Summary Platelet production rates were measured in thrombopoietin (TSF) assay mice by use of both [35S]sodium sulfate and [75Se]selenomethionine (75SeM) as platelet labels. Although significantly higher values were found using Na2 35SO4 than 75SeM, the incorporation patterns for both isotopes showed similarities in dose-response experiments. The authors thank Marilyn Cottrell for expert technical assistance and Pat Taylor for stenographic aid.

Mechanism of Thrombocytopenia Induced in Mice by Anti-Platelet Serum

Although anti-platelet serum is frequently used to produce thrombocytopenia, the mechanism of how platelets are removed from the circulation is poorly understood. In vitro studies indicated that anti-platelet serum caused platelet aggregation, whereas normal rabbit serum did not. The aggregation was dose related; at the lower dose, aggregated platelets were shown to deaggregate and platelets released from the aggregates responded normally to ADP but not to collagen. In mice whose platelets were previously labeled with Na235SO4, anti-platelet serum caused an increase in the amount of 35S mucopolysaccharides (MPS) recovered from the kidneys. No increase in radioactivity was found in the lungs, livers, or spleens of similarly treated mice. The data suggest that platelet specific anti-serum causes platelets to aggregate leading to ADP (and consequently 35S-MPS) release; the accumulation of 35S in kidneys may be due to removal of MPS and/or platelets from the circulation.

Canine cyclic hematopoiesis: platelet size and thrombopoietin level in relation to platelet count.

Summary Platelet size, platelet count, and thrombopoietin levels in plasma fractions were determined in cyclic hemato-poietic (CH) dogs. Results show that platelet sizes varied inversely with platelet counts of CH dogs; significantly elevated levels of thrombopoietin were found in fractions of plasma from CH dogs at the beginning of active thrombopoiesis. These dogs serve as a useful model for studying cycling thrombopoiesis in man. The authors thank Janet Jolly, Marilyn Cottrell, and Bill Wolfenbarger for technical assistance, Patricia Wayne for art and stenographic aid, and Dr. T. T. Odell, Jr., for helpful suggestions.