ALBERT

Description: Rodents treated with 150 mg/kg of 5-fluorouracil (5-FU) exhibit a marked and prolonged rebound thrombocytosis, suggesting that feedback control of one or more megakaryocyte characteristics (size, polyploidy, or concentration) is altered. To determine the changes in megakaryocytes that lead to such a profound thrombocytosis, C3H mice were injected with 150 mg/kg 5-FU, and platelet and megakaryocyte responses were examined at frequent intervals from days 1 through 25. After 5-FU injection, all megakaryocyte indices decreased, as did platelet number. However, the decrease in platelets to one third of control was greater than the decreases in megakaryocyte indices, suggesting that thrombocytopoiesis was ineffective from days 3 through 7 post 5-FU. Megakaryocyte size began to recover on day 4, followed by polyploid DNA content on day 5, and megakaryocyte concentration and platelets at 7.5 days. Megakaryocyte size peaked on days 6 through 8 (1.25 x normal), followed by megakaryocyte polyploid DNA content on day 8, megakaryocyte concentration on days 9 through 12 (2 1/2 to 3x normal), and platelets on days 12 through 15 (2x normal). Platelet levels are thought to be important in the feedback regulation of megakaryocytes; however, only polyploid DNA content distributions showed a close inverse relationship to platelet counts during both the recovery and rebound thrombocytosis phases after 5-FU. In contrast, megakaryocyte size peaked before platelet recovery commenced, while megakaryocyte concentration increased in parallel with platelets from 7.5 to 10 days post 5-FU and continued to be maintained at 2 to 3 times normal through day 13, despite platelet levels that were more than twice normal. Both megakaryocyte size and polyploid DNA content distributions shifted toward lower values in response to the rebound thrombocytosis (DNA content on day 10 and size on days 12 and 13). Splenectomy did not substantially alter the pattern of post 5-FU rebound thrombocytosis or megakaryocyte response from that seen in intact mice, indicating that splenic megakaryocytes are not responsible for the prolonged thrombocytosis seen after this drug. In summary, the prolonged thrombocytosis after 5-FU administration results from failure to down-regulate the number of precursors entering the differentiating megakaryocyte compartment. These data indicate that megakaryocyte size and DNA content are responsive to different feedback controls than megakaryocyte concentration in this model system.

Description: Hereditary macrothrombocytopenia and prolonged bleeding times are associated with the recessive mouse pigment dilution gene gunmetal (gm). Other platelet abnormalities include a mild storage pool deficiency and abnormal expression of two low-molecular-weight guanosine triphosphate binding proteins. These studies were designed to further elucidate the cause of the macrothrombocytopenia. The life span of gunmetal mouse platelets was not significantly different from normal. However, rates of platelet synthesis, measured by sulfate incorporation, were decreased to 25% of normal values. Bone marrow transplantation of normal marrow cells corrected the thrombocytopenia. Furthermore, direct morphologic analysis of mature mutant marrow megakaryocytes by transmission electron microscopy showed reductions in the normal cytoplasmic demarcation membrane system, areas of abnormal membrane complexes, and an increased incidence of emperipolesis. Mutant platelets were relatively more heterogeneous in size and contained unusual elongated and striated inclusions. Mutant megakaryocyte numbers were increased threefold to fivefold over normal numbers in marrow and spleen. Thus, the efficiency of platelet production from gunmetal megakaryocytes is reduced by an order of magnitude. Mutant marrow had a greater proportion of 32N and a smaller proportion of 8N megakaryocytes. Collectively, the results indicate that the gunmetal gene acts intrinsically in megakaryocytes and that an abnormality in this gene causes significant qualitative and quantitative effects on platelet production.

Description: C3H mice have higher average ploidy megakaryocytes than all other mouse strains tested, but the mode of inheritance of this anomaly is unknown. Therefore, to clarify the genetics of high ploidy megakaryocytes in C3H mice, we measured megakaryocyte DNA content from both male and female offspring from F1, as well as backcross matings. In all, offspring from seven different matings of mice were studied: (1) C57BL X C57BL (the first strain listed is the male parent in each case), (2) B6C3F1 (offspring from C57BL X C3H mating) X C57BL, (3) C57BL X B6C3F1, (4) C57BL X C3H, (5) C3H X B6C3F1, (6) B6C3F1 X C3H, and (7) C3H X C3H. The polyploid megakaryocyte DNA content distributions of the offspring from these matings show that C3H mice have higher percentages of high ploidy megakaryocytes than did all other mice. Also, male mice had significantly higher percentages of high ploidy (32N and 64N) megakaryocytes than did female mice for all matings, except backcross mating no. 6. The megakaryocyte DNA content for individual offspring of a given backcross appeared to form a single, continuous distribution, rather than segregate into two distinct groups, suggesting that the higher megakaryocyte DNA content of C3H mice is caused by involvement of multiple allelles. This conclusion is further supported by our finding that the frequency of high ploidy megakaryocytes among offspring of the various matings was related to the proportion of C3H genotype contributed by the parents, ie, average megakaryocyte DNA content increased linearly (r2 = .88 for male mice and .84 for female mice. P 〈 .0001) with increasing C3H gene dosage; the correlations for both male and female mice were essentially parallel (slope = 0.08 and 0.09, respectively). In addition, we found an effect of genomic imprinting on megakaryocyte DNA content in backcross offspring. The genetic imprinting was characterized by the female parent having a greater influence on the offspring's megakaryocyte DNA content than the male parent, ie, although the overall genetic makeup was the same, female offspring from backcross no. 6 (in which the female was C3H) had higher average megakaryocyte ploidy values than those from backcross no. 5 (in which the female was B6C3F1

Description: The modal DNA content of normal marrow megakaryocytes from species so far examined usually has been reported to be 16N. In this report we describe an exception in the C3H mouse whose megakaryocytes have a modal DNA content of 32N. Female C3H/HEN mice had an average DNA content distribution of 14% 8N, 37% 16N, 43% 32N, and 6% 64N. Male C3H/HEN mice had somewhat higher proportions of 32N and 64N megakaryocytes (average DNA content distribution of 12% 8N, 29% 16N, 47% 32N, and 12% 64N) than females. All 11 other mouse strains examined had 16N as the modal megakaryocyte DNA content, although the proportions in the various polyploid DNA classes showed some strain variation. Megakaryocyte size was similar among all 12 strains evaluated, and mean platelet volume (MPV) of C3H/HEN mice differed from only 1 of the other 4 strains analyzed. Platelet counts of C3H/HEN mice were similar to those of six, and slightly but significantly lower than those of five other mouse strains examined. Compared with megakaryocyte concentrations of other mouse strains studied, that of C3H/HEN mice was similar to seven, somewhat higher than one, and slightly lower than three strains. Offspring from reciprocal matings of C57BL/6 and C3H/HEN mice had megakaryocyte DNA distributions intermediate between those of the parent strains, suggesting that a higher gene dosage of some component is responsible for the right-shifted megakaryocyte DNA content distribution phenotype of C3H mice. The proportions of 32N and 64N megakaryocytes increased in C3H/HEN mice in response to acute thrombocytopenia, as did those of CBA/CAJ mice used as a comparative strain. In summary, megakaryocytes of the C3H mouse have a higher average DNA content but similar platelet count, MPV, and megakaryocyte size and concentration as those of most other mouse strains. These results suggest that the number of platelets produced per unit of C3H megakaryocyte DNA is less than that for other mice.

Description: The mechanisms that determine and regulate platelet size are unknown. By phase microscopy, we observed that Wistar Furth (WF) rats had macrothrombocytopenia. In this study, we have characterized and compared platelets and megakaryocytes of WF rats with those of Wistar, Long-Evans hooded (LE), and Sprague-Dawley rats. In addition, we have examined the mode of inheritance of this WF rat platelet abnormality. The average platelet count of WF rats was only one-third that of the other three rat strains. In contrast, the mean platelet volume (MPV) of adult WF rats was twice that of the other rat strains; however, the average megakaryocyte diameter and DNA content distribution of WF rats were not significantly different from those of LE rats. The average megakaryocyte concentration was 30% lower in the WF strain compared with that of LE rats. Mazelike membrane formations were observed in WF platelets and megakaryocytes by electron microscopy. Reciprocal crosses of WF and LE rats resulted in offspring with MPVs and platelet counts like those of LE rats, indicating that the macrothrombocytopenic trait is recessive in its inheritance. Reciprocal marrow transplants between the WF and LE strains resulted in MPVs like those of the donor strain, demonstrating that the macrothrombocytopenia is an intrinsic marrow abnormality of the WF strain. Splenectomy did not alter the MPV of WF rats. The response of WF megakaryocytes and platelets to severe, acute thrombocytopenia was similar to that of LE rats except that the shift to higher megakaryocyte DNA contents was muted and platelet recovery was slower in the WF rats. In summary, the WF rat has a hereditary macrothrombocytopenia that is recessive in nature and not due to differences in megakaryocyte size or DNA content. These results suggest that the macrothrombocytopenia of WF rats results from the formation of fewer platelets per megakaryocyte, possibly resulting from a qualitative or quantitative defect in some component necessary for proper subdivision of megakaryocyte cytoplasm into platelets.

Description: The ploidy distribution of megakaryocytes shifts in response to platelet demand and thus provides a sensitive index of megakaryocytopoiesis. Flow cytometry (FCM) is a potentially valuable method for rapid determination of ploidy distributions of megakaryocyte populations; however, because megakaryocytes constitute only a very small proportion of the cells in unfractionated marrow, other rare events, such as cell clumping, complicate FCM analysis. We describe the measurement of cellular DNA distributions of megakaryocytes by two- color FCM in unfixed, unfractionated marrow--a method based on the resistance of megakaryocytes to hypotonic lysis in the cold for at least 2 days. Specific platelet antiserum was used to label megakaryocytes by indirect immunofluorescence with fluorescein (green fluorescence), and DNA was stained with propidium iodide (red fluorescence) in hypotonic citrate solution. The ploidy distribution of megakaryocytes was selectively determined with two-color, green-gated FCM, with which the red and green fluorescence of all cells is analyzed, but only the red fluorescence (DNA content) of cells that specifically bound the platelet antibody is recorded. We demonstrate that this method can readily detect changes in megakaryocyte DNA distributions due to experimental thrombocytopenia or platelet hypertransfusion and, therefore, should be useful for both experimental and clinical investigations of megakaryocytopoiesis.

Description: To gain insight into the regulation of megakaryocyte precursors in vivo, we assayed (in vitro) megakaryocyte growth-promoting activity (Meg-GPA) in plasma of rats in which both marrow hypoplasia and thrombocytopenia had been induced by irradiation. Rats received whole body irradiation of 834 rad from a 137Cs source. Plasma was collected at intervals of hours to days, up through day 21 postirradiation, and was tested, at a concentration of 30%, for Meg-GPA on bone marrow cells cultured in 1.1% methylcellulose with 5 X 10(-5) M 2-mercaptoethanol. With normal rat plasma, no megakaryocyte colonies (defined as greater than or equal to 4 megakaryocytes) were seen and only a few single megakaryocytes and clusters (defined as 2 or 3 megakaryocytes) were formed. Two peaks of plasma Meg-GPA were observed after irradiation. The first appeared at 12 hr, before any decrease in marrow megakaryocyte concentration or platelet count. The second occurred on days 10–14 after irradiation, after the nadir in megakaryocyte concentration and while platelet counts were at their lowest levels. A dose-response study of plasma concentration and megakaryocyte growth, using plasma collected 11 days postirradiation, demonstrated that patterns of megakaryocyte growth were related to plasma concentration; formation of single megakaryocytes was optimal over a range of 20%-30% plasma concentration, while cluster and colony formation were optimal at a plasma concentration of 30%. All forms of megakaryocyte growth were decreased with 40% plasma. There was a linear relationship between the number of bone marrow cells plated and growth of single cells, clusters, and colonies using a concentration of 30% plasma collected 11 days after irradiation. We conclude that irradiation causes time- related increases in circulating megakaryocyte growth-promoting activity. We suggest that the irradiated rat is a good model for studying the relationships between Meg-GPA and megakaryocyte and platelet concentration in vivo.

Description: Hypertransfusion can enhance myeloid recovery after bone marrow depletion, but its influence on thrombopoietic recovery has not been previously defined. We have studied the pattern of platelet and megakaryocyte recovery in mice hypertransfused after receiving 350 rad whole body irradiation. The platelet counts of the hypertransfused group showing an initial fall due to hemodilution in the expanded blood volume and then fell to a lower nadir than that of the control mice. The rate of platelet recovery was more rapid in the hypertransfused mice. Bone marrow megakaryocyte concentrations in both groups showed a degenerative phase, abortive rise, and regenerative phase. The decrease in megakaryocytes was the same in both groups. The hypertransfused mice showed a greater abortive rise in megakaryocyte concentration preceded by the appearance of a greater number of large megakaryocytes in the bone marrow. However, the most striking effect of hypertransfusion was on megakaryocyte recovery. Although the time of onset of recovery was not different, the rate of recovery was approximately twice as rapid in the hypertransfused group. Administration of daily erythropoietin to hypertransfused mice abolished this more rapid recovery. Thus, the presence of a simultaneous demand for erythroid precursors does affect the rate of megakaryocyte regeneration. Just as the more rapid recovery of granulopoiesis following hypertransfusion may be clinically beneficial, the more rapid reconstitution of thrombopoiesis may also offer clinical advantage in some circcumstances.

Description: Rats were made acutely thrombocytopenic by injection of antiplatelet serum. Marrow sections and squash preparations were made at intervals during 120 hr. Determinations were made of mitotic index, stage of maturation, ploidy level, and cell size of megakaryocytes; number and size of platelets were measured. Increased endomitosis among megakaryocytes was followed by an increase in the proportion of immature megakaryocytes, a greater average ploidy level of recognized megakaryocytes, and larger megakaryocytes. Maximum changes in these several parameters occurred between 32 and 72 hr after induction of thrombocytopenia. By 120 hr all megakarocyte parameters were near normal. For about 3 days, beginning at about 36 hr, platelet numbers increased rapidly. Average platelet size rose and returned to normal within about 60 hr. Changes in ploidy and size of megakaryocytes were measured in the immature and mature maturation stages. The results suggest that the initial stimulus in response to acute thrombocytopenia acts primarily on diploid precursors, programming them to mature into a population of megakaryocytes with an average ploidy approximately one level greater than in normal rats and a proportionate increase in cell size. The larger megakaryocytes presumably produce more platelets, accounting for a major part of the increased rate of platelet production. Since the changes in megakaryocytes begin to reverse before circulating platelet numbers have reached the normal level, reversal of the stimulus appears to be initiated by some change other than platelet mass.

Description: Small megakaryocytes are frequently seen in patients with acute nonlymphocytic leukemia (ANLL). In this study, median megakaryocyte diameters were determined in marrow biopsy specimens of 32 children at diagnosis of ANLL and related to platelet count and chemotherapeutic response. The association between median megakaryocyte size and time-to- failure was striking. Seven of 9 patients with median megakaryocyte diameters greater than 20 microns remain in continuous complete remission for more than 3 yr, whereas 20 of 23 patients with smaller median megakaryocyte diameters failed therapy within 15 mo (p = 0.002). By Cox-regression analysis, megakaryocyte size had independent prognostic value (p less than 0.001), surpassing that of spleen size, the only other feature having significant association with time-to- failure. Megakaryocyte size at diagnosis may be useful for predicting the likelihood of prolonged complete remission in ANLL.