Megakaryocytes have a unique differentiation process in which they become polyploid through endomitosis and then undergo extensive cytoplasmic growth and maturation leading to the formation of several thousand platelets per cell. Although the cell kinetics of this process have been studied in detail, little is known about differentiation at the macromolecular level. Recent advances in methodology such as isolation and purification of megakaryocytes in large quantities, analysis of their cell contents by flow cytometry and assays of megakaryocyte precursors in vitro now allow the study of megakaryocyte differentiation in more detail. By modifying a technique (density fractionation) used in megakaryocyte purification, we have been able to fractionate megakaryocytes into immature and mature populations. This separation will permit us to examine differentiation within the recongnizable megakaryocyte compartment. A major aim of this proposal is to compare the labeling patterns of proteins of these two megakaryocyte populations by SDS slab gel electrophoresis and flow cytometry after specific protein labeling in normal and altered states of megakaryocytopoiesis. Our working hypothesis is that during megakaryocyte differentiation, changes occur at the macromolecular level which can be detected by available techniques. Rat megakaryocytes will be used as a model for these studies. Similar studies will be done on human megakaryocytes if sufficient numbers can be obtained by marrow aspiration. Another aspect of this proposal concerns the precursors that form single megakaryocytes in vitro. We hypothesize that these cells are the immediate precursors of megakaryocytes and are more responsive to changes in platelet demand than are colony-forming cells. To test this hypothesis we will determine whether the number of these precursors is altered early after induction of acute thrombocytopenia in rats as well as examine their biological and physical characteristics. Finally, we propose to use the irradiated rat as a model for determining the relationship between megakaryocyte colony-stimulating activity produced in vivo and platelet and megakaryocyte concentration. These studies should provide basic insights into differentiation and regulation of megakaryocytopoiesis as well as provide some understanding of megakaryocyte abnormalities in diseases such as idiopathic thrombocytopenic purpura and chronic myelocytic leukemia.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL031598-02
Application #
3342821
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-07-01
Project End
1987-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
St. Jude Children's Research Hospital
Department
Type
DUNS #
067717892
City
Memphis
State
TN
Country
United States
Zip Code
38105
Stenberg, P E; Beckstead, J H; Jackson, C W (1998) Wistar Furth rat megakaryocytes lack dense compartments and intercellular plaques, membranous structures rich in cytoskeletal proteins. Cell Adhes Commun 5:397-407
Stenberg, P E; McDonald, T P; Jackson, C W (1995) Disruption of microtubules in vivo by vincristine induces large membrane complexes and other cytoplasmic abnormalities in megakaryocytes and platelets of normal rats like those in human and Wistar Furth rat hereditary macrothrombocytopenias. J Cell Physiol 162:86-102
Pestina, T I; Jackson, C W; Stenberg, P E (1995) Abnormal subcellular distribution of myosin and talin in Wistar Furth rat platelets. Blood 85:2436-46
Sullivan, P S; Jackson, C W; McDonald, T P (1995) Castration decreases thrombocytopoiesis and testosterone restores platelet production in castrated BALB/c mice: evidence that testosterone acts on a bipotential hematopoietic precursor cell. J Lab Clin Med 125:326-33
McDonald, T P; Jackson, C W (1994) Mode of inheritance of the higher degree of megakaryocyte polyploidization in C3H mice. I. Evidence for a role of genomic imprinting in megakaryocyte polyploidy determination. Blood 83:1493-8
McDonald, T P; Jackson, C W (1994) The role of genotype, genomic imprinting, and sex hormones in platelet and megakaryocyte production. Exp Hematol 22:959-66
Jackson, C W; Steward, S A; Ashmun, R A et al. (1992) Megakaryocytopoiesis and platelet production are stimulated during late pregnancy and early postpartum in the rat. Blood 79:1672-8
McDonald, T P; Cottrell, M B; Clift, R E et al. (1992) Effects of hypoxia on megakaryocyte size and number of C3H and BALB/c mice. Proc Soc Exp Biol Med 199:287-90
McDonald, T P; Swearingen, C J; Cottrell, M B et al. (1992) Sex- and strain-related differences in megakaryocytopoiesis and platelet production in C3H and BALB/c mice. J Lab Clin Med 120:168-73
McDonald, T P; Cottrell, M B; Steward, S A et al. (1992) Comparison of platelet production in two strains of mice with different modal megakaryocyte DNA ploidies after exposure to hypoxia. Exp Hematol 20:51-6

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