Marilyn Cottrell

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.

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.

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.

Effect of Nicotine on Clot Retraction of Rat Blood Platelets

Clot retraction in platelet-plasma preparations was enhanced by nicotine treatment of rats. Platelets from nicotine-treated rats retracted to a greater degree if incubated in plasma from nicotine treated rats than if incubated in control plasma. Furthermore, platelets from nontreated rats retracted to a greater degree if they were incubated in plasma of nicotine-treated rats, than in plasma of nontreated rats. The results indicate that the effects of nicotine are not directly on platelets. It is concluded that the enhanced clot retraction observed in the blood of nicotine-treated animals was due to a plasma factor and not to changes in the platelets themselves.

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.

Thrombopoietin Production in Mice Treated with Acetylsalicylic Acid

Abstract Recent work revealed that mice in which platelet function was inhibited by acetylsalicylic acid (ASA) treatment showed evidence of increased platelet production. It was proposed that poorly functioning platelets gave rise to elevated thrombocytopoiesis by causing the release and action of thrombopoietin. However, direct evidence is lacking. Therefore, in the work reported here, plasma from mice treated with ASA was injected into normal recipient mice in an attempt to document the existence of the humoral factor. Compared with control mice given normal plasma, the injection of mice with plasma from ASA-treated mice resulted in increased thrombocytopoiesis, as evidenced by significant increases in the percentage of 35S incorporation into platelets, larger platelet size, and elevated megakaryocyte precursor cells (the small acetylcholinesterase-positive cell). For a positive control, additional mice were treated with plasma from animals made thrombocytopenic by an injection of antiplatelet serum. These mice also showed significant increases in thrombocytopoiesis. The results support the hypothesis that platelet production in ASA-treated mice is elevated by release and action of thrombopoietin.

Stimulation of megakaryocytic spleen colonies in mice by thrombopoietin.

Abstract Kidney cell culture medium that was shown to stimulate thrombocytopoiesis in TSF assay mice was injected into lethally-irradiated, bone marrow transplanted mice. Results showed that the injection of TSF-rich material caused an increase in the number of megakaryocytic colonies when compared to control mice. The numbers of surface colonies and colonies/spleen section were not altered by TSF treatment. Erythroid, granulocytic, and undifferentiated colonies were not elevated by TSF injection. These data support the hypothesis that kidney cells in culture produce a humoral factor that controls the regulation of platelet and megakaryocyte production.