Search

Megakaryocytopoiesis was evaluated in Sl/Sldmice and their heterozygous and homozygous normal (+/+) littermates. Sl/Sldmice had a normal concentration of blood platelets which were of normal size. Numbers of megakaryocytes in bone marrow and spleen were reduced, but individual megakaryocytes were larger than normal.

Megakaryocytopoiesis was evaluated in genetically anemic mice of the W/Wvgenotype and was found to be abnormal. Concentration and size of blood platelets were normal. Megakaryocytes were decreased in number in tibial marrow and spleen, and the size of mature megakaryocytes was increased.

Mice of the W/Wvgenotype have an hereditary macrocytic anemia resulting from a diminished proliferative capacity of hemopoietic precursor cells. Platelet production was studied because W/Wvmice maintain a normal number of circulating platelets that are of normal size in spite of a diminished number of megakaryocytes. Using [75Se]selenomethionine and calculating the percentage of the injected dose incorporated by platelets, the platelet production rate of W/Wvmice was found to be equivalent to the production rate of their +/+ littermates. Using Na235SO4however, the platelet production rate, as measured by the same isotope incorporation method, was nearly twice the rate of the normal littermates. The increase in 35S incorporation appeared to be explained by a greater availability of isotope in the W/Wvas evidenced by the higher plasma levels of 35S that were concurrently found. This suggests that the W/Wvmouse has an aberrant metabolism of inorganic sulfate probably unrelated to platelet production. These results also demonstrate how failure to take plasma radioactivity levels into account can lead to conflicting and probably erroneous conclusions in animals with an inherent or induced metabolic abnormality.

Bone marrow tissue was cross-transplanted subcutaneously between S1/ Sldmice and their normal +/+ littermates. Megakaryocyte size was evaluated in bone marrow from donors, hosts, and regenerated implants. Macromegakaryocytosis was found in Sl/Sldmarrow in situand in all implants in Sl/Sldhosts. Megakaryocytes were of normal size in +/+ marrow and in all implants in +/+ hosts. The macrocytosis of megakaryocytes in Sl/Sldmice appeared to be due to systemic factors or host elements that were incorporated into the regenerated implants rather than to a stromal abnormality that persisted with graft regeneration after transplantation.

Megakaryocytopoiesis in the spleens of lethally irradiated mice transplanted with marrow cells was suppressed by platelet transfusions. In one group of experiments, animals were irradiated and transfused with bone marrow cells on day 0. They were then given either no treatment, platelets, platelet-poor plasma, or saline on days 0, 2, 4, 6, and 8, and then were sacrificed on day 10. Megakaryocytes per section in the spleens of mice receiving platelets were 24%-48% of the values in the groups given plasma, saline, or bone marrow only. The number of pure megakaryocyte colonies was also diminished by platelet hypertransfusion. Another experiment examined the effect of platelets or plasma administered on days 1 and 2 or days 6 and 7 after irradiation and bone marrow transfusion. Hypertransfusion on days 6 and 7 was as effective in suppressing megakaryocytopoiesis as hypertransfusion every other day for 10 days. Animals given platelets or plasma only on days 1 and 2 did not have any significant change in their megakaryocyte number. These results implied that committed megakaryocyte precursors were more sensitive to inhibition by increased platelet levels than pluripotential stem cells. Further experiments with plethoric animals indicated that different levels of erythropoietin did not account for the effects of platelet hypertransfusion. The findings could be explained by inhibition of cell proliferation or of differentiation of megakaryocyte precursors by increased platelet levels.

A single injection of hydroxyurea (OHU) produced transient megakaryocytopenia in mice. An increase in the average mean size of mature, stage III megakaryocytes coincided with their depopulation. This was due to a selective reduction in numbers of smaller cells. In contrast, the macromegakaryocytosis of immunothrombocytopenia showed substantial increases in numbers of larger cells and reductions in smaller. Further reduction in numbers of smaller cells occurred when OHU was given to mice with immunothrombocytopenia, and the megakaryocytopenia was somewhat more severe than that produced by OHU in normal mice. OHU produced mild thrombocytopenia in normal mice and compromised recovery of the platelet count from immunothrombocytopenia. The most likely explanation for the increase in mean megakaryocyte size in the hypomegakaryocytic state produced by OHU is that the temporary imbalance between a low rate of influx and a normal rate of maturation produced a shift of the age distribution of the cells due to a deficiency of immature cells.

The heterogeneity among immature megakaryocytes has been examined by physical properties, cell cycle status, and responsiveness to thrombopoietic stimulatory factor. Three types of immature megakaryocytes exist that can be recognized by acetylcholinesterase staining, nuclear shape, high nucleus/cytoplasm ratio, and small size (8–18 µ) with respect to mature megakaryocytes (>18 µ).These three acetylcholinesterase-containing cell types are distinguished by their nuclear configuration: a round, indented, and lobed nucleus. The lobed cell type was found to overlap with and enhance detection of megakaryoblasts (stage I megakaryocytes). These cells had a sedimentation velocity range of 3.5–19.0 mm hr–1and a density range of 1.072–1.095 g cm–3. Separation of these three classes of immature megakaryocytes was achieved by equilibrium density centrifugation with modal buoyant densities of 1.079 g cm–3(round), 1.084 g cm–3(indented), and 1.089 g cm–3(lobed). In the presence of thrombopoietic stimulatory factor, the round nucleated cells, but not the indented or lobed nuclei morphology, were observed to develop into large mature megakaryocytes in 60-hr semisolid cell cultures. Development of two cell groups, or colonies of megakaryocytes, was not observed during this in vitro incubation period. In vivo treatment with hydroxyurea indicated that 57.5% ± 19% of the round nucleus form were actively synthesizing DNA. No reduction in the numbers of indented or lobed nucleus forms were observed following hydroxyurea treatment. The data in this report strongly support the concept that these three types of immature megakaryocytes reflect the early maturation stages occurring in megakaryocyte differentiation.

Thrombocytopoiesis was evaluated in T cell-deficient nu/ nu mice and in T cell-replete nu / + controls to determine if abnormalities would be associated with the deficiency of T cells. Mice were studied in the unperturbed steady state and after acute immunothrombocytopenia was induced by an injection of guinea pig antimouse platelet serum (APS). The state of thrombocytopoiesis was determined from platelet counts, megakaryocyte size, megakaryocyte num ber, and numbers of Meg-CFC. Splenic lymphocytes were evaluated by responses to the mitogens bacterial lipopoly-saccharide (LPS), phytohemagglutinin (PHA), and concana-valin A (Con A). Hematocrits, reticulocyte counts, leuko cyte counts, marrow cellularity, GM-CFC, and BFU-E also were measured. Steady state thrombocytopoiesis was identical in nu/nu and nu/+ mice. In response to an injection of APS, acute thrombocytopenia was followed by macromegakaryocytosis and rebound thrombocytosis in mice of both genotypes. Splenic Meg-CFC increased in nude mice after APS or an injection of normal guinea pig serum (NGpS), and splenic GM-CFC increased after APS. Neither Meg-CFC nor GM-CFC increased in the spleens of nu/ + mice, but they showed early transient increases in bone marrow that did not occur in nu/nu mice. Sporadic, but weak, mitogenic responses to PHA or Con A were occasionally observed with nu/nu spleen cells, but these did not correlate with the state of thrombocytopoiesis. The results demonstrated that platelet production was normal in nu/nu mice and that megakaryocytopoiesis and platelet production responded to the stimulus imposed by acute immunothrombocytopenia. Increases in megakaryocyte size and platelet production occurred independently of changes in numbers of Meg-CFC, GM-CFC, or BFU-E. A normal complement of T cells appears to be unnecessary for normal platelet production and its augmentation in response to the stimulus of acute immunothrombocytope nia in vivo. This is a US Government work. There are no restrictions on its use.

Megakaryocytopoiesis was evaluated in W/Wvmice and their normal +/+ littermates to analyze the mechanisms by which normal platelet production is maintained in W/Wvmice even though numbers of megakaryocytes are low. Relative sizes of megakaryocytes, and their nuclei and cytoplasm, were measured microscopically in bone marrow smears, and the ploidy of the same cells was measured by two-wavelength microspectrophotometry. Maturation rate of megakaryocytes was estimated after they were labeled with tritiated thymidine. W/Wvmegakaryocytes were macrocytic: average cell size was increased in each ploidy group. The increase in cytoplasmic area exceeded that of the nucleus. Further analysis of the predominant 16N ploidy group revealed that the increase in average cell size was due to depletion of cells of small size. Megakaryocytes matured more rapidly than normal in W/Wvmice. These results showed that megakaryocyte size and ploidy can be regulated separately. They suggest that alterations in cell growth and maturation may be mechanisms by which the organism can compensate for a deficiency in numbers of megakaryocytes, but they do not define the mechanism by which the deficiency may be sensed or by which the compensatory changes may be mediated.

Both immunoreactive erythropoietin (Ep) and biologically active Ep were measured in 23 samples of human serum and 21 concentrates of human urine. Immunoreactive Ep was measured by radioimmunoassay (RIA). Biological activity was determined in the plethoric mouse bioassay in which 59Fe incorporation was converted to units of Ep from standard reference curves. Low values for Ep were determined from standard curves plotted as probits to improve sensitivity for levels of Ep as low as 30 mU/ml. Ep levels in 35 samples ranged between 30 and 1000 mU/ml by both assays: in 9 samples Ep was 15.2-37.5 mU/ml by RIA but was not detectable by bioassay. Analysis of the data for the 35 samples in which Ep could be measured by both assays showed a strong correlation between the values obtained by the two assays. These results indicate that the RIA used in these experiments detects biologically active Ep in human serum and urine when it is present in amounts only moderately higher than normal. The ultrafiltration method used for preparation of urine samples was effective in concentrating Ep in some urines, but the results were too erratic and nonquantitative to permit its use as a method for quantifying human urinary Ep excretion.

Labeling of rat megakaryocytes was observed after a single intravenous injection of tritiated thymidine, and the following conclusions were made. 1. Morphologic appearance of megakaryocytes corresponded to their stage of maturation. 2. Only the youngest recognizable megakaryocytes (stage I) appeared to have a DNA synthetic period. 3. Megakaryocytes did not undergo permanent cellular division. 4. There was probably some degree of ineffective megakaryocytopoiesis. 5. The average total maturation time of rat megakaryocytes was estimated to be 43-75 hours wih 8-14 hours in stage I, 11-19 hours in stage II, and 24-42 hours in stage III. 6. There was continuous influx into the megakaryocyte compartment from a precursor compartment. The immediate precursor cell was morphologically unrecognizable, but it appeared to have a generation time of ∼16 hours, a DNA synthesis period of ∼10 hours and a post-synthetic maturation time of ∼2 hours. 7. Reutilization of tritium probably accounted for maintenance of a high degree of labeling of megakaryocytes for a period of 1.5-4 days.

Increased production of platelets was apparent during recovery from acute thrombocytopenia in rats. Preceding this there was acceleration of maturation of megakaryocytes as determined from the pattern of labeling with 3HTdR. Turnover of an increased number of megakaryocytes was indicated by maintenance of normal megakaryocyte differential counts in the presence of accelerated maturation.

Thrombocytopenia was produced in rats by exchange transfusion with platelet-poor homologous blood or heterologous antiserum. The subsequent rapid rise in platelet counts to thrombocytotic levels suggested accelerated platelet production. During this recovery period macromegakaryocytes were present in the bone marrow. The findings suggested that thrombocytopenia affected megakaryocytopoiesis at the precursor cell level with resultant macrocytosis.

Viability of rabbit platelets stored for 1 to 6 days in different media at 4 C. was studied using the Cr51-platelet survival technic. Platelets stored for 24 hours in Triton-saline or DAS-gelatin had practically no viability. When platelets were stored for 24 hours in diluted plasma, significant degrees of viability were found, but this decreased rapidly in the successive 48 hours. The viability of platelets stored in diluted plasma containing inosine, adenine, glucose and phosphate buffer was maintained at significant levels over a six day period. The data presented here suggest that viability and, consequently, the hemostatic properties of platelets stored at 4 C. can be maintained over relatively long periods of time provided that loss of the ATP reserve of the cells can be prevented.

Five cases of chronic lymphocytic leukemia complicated by thrombocytopenic purpura are presented. They differed from the usual cases with this complication in that megakaryocytes were plentiful in spite of leukemic involvement of the bone marrow. Hypersplenism did not appear to be a factor. The evidence suggests that the thrombocytopenia was of an autoimmune nature, due to antiplatelet antibodies. Brief platelet survival times, the presence of a platelet agglutinin, staining of megakaryocytes by fluorescein-labeled anti-human globulin, and responses to corticosteroids were demonstrated.

1. When whole blood was incubated in vitro with S-35 L-cystine and L-methionine, the blood cells became radioactive. 2. Preincubation of whole blood from normals and from patients susceptible to agranulocytosis with chlorpromazine showed no effect upon uptake of S-35 L-cystine and L-methionine by leukocytes. 3. The in vivo administration of S-35 L-cystine was followed by the appearance of radioactive leukocytes. Peak radioactivity occurred in leukocytes in 5 to 12 days. 4. Pretreatment of test subjects with large doses of chlorpromazine did not block the uptake of S-35 L-cystine by leukocytes in vivo. Leukocytes of women showed an increase in the incorporation of S-35 L-cystine, in vivo. Studies performed in vivo on two persons during recovery from agranulocytosis showed enhanced uptake of L-cystine in one and a normal uptake in the other.

Rat platelets were labeled with tritiated diisopropylfluorophosphate (3H-DFP) during recovery from acute thrombocytopenia. The results indicated that there was significant labeling of megakaryocytes by 3H-DFP which, in the presence of an increased rate of platelet production, resulted in maintenance of relatively constant values for platelet-bound radioactivity during the period of maximum platelet production and reactive thrombocytosis. Significant random loss of platelets was apparent, and, when a cohort of young platelets was transfused to normal recipients, they were destroyed at a normal rate.

Platelet depletion in mice, by injection of heterologous anti-platelet serum (APS), resulted in apparent stimulation of megakaryocytopoiesis as evidenced by a subsequent over-compensation of the level of circulating platelets. Injection of APS immediately before or after exposure to sublethal radiation or a day after exposure appeared to modify the response to radiation. Radiation-induced thrombocytopenia was neither as severe nor as prolonged in animals so treated as in irradiated controls. More chronic thrombocytopenia at the time of irradiation, however, appeared to increase the radiosensitivity of the megakaryocytic system.

The interaction of human blood platelets with influenza virus (PR-8) was studied in vitro and in vivo. It was found that "live" influenza virus was rapidly adsorbed onto human blood platelets at 4 C. and completely eluted at 37 C. "Dead" virus was adsorbed at 4 C. but not eluted at 37 C. unless the platelets were treated with RDE (receptor destroying enzyme). Adorption of virus also occurred at tem peratures above 4 C. (from 20 to 37 C.). However, while adsorption was maintained throughout incubation at 4 C., slow elution occurred after 30 to 90 minutes incubation at 26 to 37 C. Storage of the platelets for lengthy intervals at 4 C. or coating of the platelets with macromolecules did not interfere with virus adsorption. After one cycle of adsorption-elution, blood platelets could not adsorb virus again. Treatment with RDE greatly reduced virus adsorption. During the process of virus adsorption, prominent platelet clumping occurred. During elution, clumping remained unchanged, and gross alterations in morphology of the platelets were observed. In the process of virus adsorption-elution, large numbers of platelets were lysed. Comparative experiments were performed simultaneously with human red blood cells (RBC) and identical results were obtained as with blood platelets. However, the extent of adsorption of live virus was equal for platelets and RBC only when the relationship between platelet number and RBC number in the preparations used was 6:1. This suggested a direct proportion between the surface area of both platelet and RBC and the number of available virus receptors. Virus suspensions infused into rabbits produced a sharp and sustained drop of the platelet count. Survival of radioactively labeled platelets treated with virus prior to infusion was markedly shortened with live virus and was only slightly reduced with dead virus. It is suggested from these experiments that blood platelets, as other blood cells, may serve as carriers of viruses in the circulation and that in this process the platelets are damaged and partially destroyed.

The occurrence of platelet isoimmunity was studied in rabbits sensitized by 3 to 12 transfusions of homologous blood. Platelet isoimmunity in the transfused animals was detected and measured by two different technics: (1) determination of the survival of homologous platelets labeled in vivo with P32 and infused in the sensitized recipient rabbits; and (2) study of platelet agglutinins in the serum of the multitransfused animals. It was found that: 1. After 3 to 12 blood transfusions, platelet sensitization, as determined by reduction in survival of homologous platelets in the transfused rabbits, occurred in most animals. 2. Survival of homologous specific platelets, i.e., platelets collected from the sensitizing donor and infused in the respective recipient, usually had lower values than the survival of homologous non-specific platelets. 3. Higher degrees of depression in survival of homologous platelets were found in the rabbits which had received larger numbers of blood transfusions. 4. Platelet isoagglutinins could be found only in three of the five rabbits which had been sensitized with 12 blood transfusions and the test was strongly positive in only one rabbit. 5. Repeat studies performed 2½ months after the transfusions demonstrated persistence of platelet isosensitization, while after 15 months isosensitization had greatly decreased; although in one rabbit a good degree of depression in homologous platelet survival wa still present after this time interval. These studies mainly demonstrate the high frequency of platelet isoimmunity in multitransfused rabbits and the inadequacy of the agglutinin test in the detection of even relatively severe degrees of platelet isosensitization.