Megakaryocytes have a unique differentiation process in which they become polyploid through endomitosis and then undergo extensive 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. We have developed a procedure to purify guinea pig megakaryocytes, and perhaps more importantly, their immediate platelet-antigen expressing precursors, based on their ability to be specifically aggregated by bovine von Willebrand factor. In addition, we found that recognizable megakaryocytes can be fractionated into immature and mature populations based on differences in their buoyant densities. these methodologies provide the opportunity to examine differentiation events within the recognizable megakaryocyte compartment as well as in immediate megakaryocyte precursors. A major aim of this proposal is to define the repertoire of proteins turned on or off at specific points in megakaryocyte differentiation by studying synthesis and post-translational modifications of proteins in selected megakaryocyte and megakaryocyte precursor populations using two-dimensional gel electrophoretic analyses after specific labeling procedures. Our working hypothesis is that during megakaryocyte differentiation, changes occur at the macromolecular level which can be detected by available techniques. Guinea pig megakaryocytes will be used as a model in these studies. Another aspect of this proposal will be investigation of mechanisms involved in determination of platelet size during platelet formation through study of a rat strain in which we have discovered hereditary macrothrombocytopenia. This characteristic shows an autosomal recessive mode of inheritance which suggests that these rats have a qualitative or quantitative defect in a component necessary for proper subdivision of megakaryocyte cytoplasm into platelets. Studies are proposed to define this defect in platelet formation, physiologically and at the molecular level. These studies should yield basic insights into megakaryocyte differentiation and platelet formation as well as provide some understanding of megakaryocyte abnormalities in hereditary and acquired diseases.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL031598-07
Application #
3342825
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-07-01
Project End
1992-06-30
Budget Start
1990-07-01
Budget End
1991-06-30
Support Year
7
Fiscal Year
1990
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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