Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Ulhas Naik, Dept. of Biological Sciences. Includes bibliographical references.

Factor Va is a plasma protein that plays an important role in the regulation of blood coagulation by serving as the essential cofactor in thrombin generation via the prothrombinase complex. The procofactor, factor V, exists in two whole blood pools with 75-80% found in plasma, and 20-25% stored in the α-granules of platelets. As compared to the plasma procofactor, platelet-derived factor V is physically and functionally distinct, and displays a more procoagulant phenotype. Despite these profound differences, platelet-derived factor V originates via endocytosis of the plasma-derived procofactor by megakaryocytes. Endocytosis is mediated by two receptors: an unidentified, specific factor V receptor, and low density lipoprotein (LDL) receptor related protein-1 (LRP-1), a ubiquitous receptor that plays a role in endocytosis of proteins targeted for lysosomal degradation. These observations represent a novel role for LRP-1 in endocytosis of a protein that is functionally modified, and not targeted for lysosomal degradation. The goal of this study is to define the factor V regions involved in its interactions with the unidentified factor V receptor and LRP-1 expressed on megakaryocytes to begin to elucidate the molecular mechanisms regulating formation of the unique platelet-derived cofactor. Epitope mapping studies were performed using anti-factor V monoclonal antibodies, E9 and anti-factor V #2. Previous observations indicated that these factor Va light chain antibodies inhibited endocytosis of factor V by megakaryocytes. However, subsequent analyses demonstrated that only E9 inhibited both factor V binding and endocytosis. Thus, it was used for these studies. Western blotting of factor V and Va suggested that E9 recognizes a conformation-dependent epitope, which precluded the use of conventional epitope mapping approaches used for linear epitopes. E9 had no effect on factor Va cofactor activity in a plasma-based clotting assay suggesting that it does not perturb factor Va’s interactions with the membrane surface or factor Xa. Cleavage of lipid-bound factor Va by factor Xa at Arg1765 was also not affected by the presence of E9 suggesting that the epitope is not directed against this cleavage site. When E9 was used to immunoprecipitate the factor Xa-generated light chain cleavage products, both the 48/46 and 30 kDa light chain fragments were captured. These observations were confirmed using a solid phase competition assay where factor Xa-cleaved factor Va inhibited binding of 125I-factor V to E9 as well as intact factor V or Va. Limited proteolysis of the factor Va light chain with trypsin or Asp-N, generated products that were no longer detectable in this assay. These combined observations suggest that the anti-factor V light chain antibody, E9, has an epitope that is conformation-dependent and extremely labile. Future directions and alternative approaches are discussed.

Megakaryocytes (MKs) are a rare population of haematopoietic cells, which produce platelets. Platelet production is a complex process that is tightly regulated at the transcriptional level by lineage specific transcription factors such as p45 NF-E2. Understanding how transcriptional regulators operate is imperative to advance our knowledge of disease pathophysiology and to propose novel treatment options. Therefore, the aims of this study were to: i) study the effects of p45 NF-E2 overexpression on various stages of megakaryopoiesis; (ii) elucidate the nuclear transport mechanisms of p45 NF-E2; and iii) determine the impact of a p45 NF-E2 modification called SUMOylation on thrombopoiesis. Exogenous p45 NF-E2 was overexpressed in haematopoietic cells in culture and various aspects of megakaryopoiesis were examined. Overexpression of p45 NF-E2 enhanced multiple stages of MK differentiation such as colony forming unit (CFU)-MK formation and terminal MK maturation. Most importantly, p45 NF-E2 overexpression resulted in significant increases in proplatelet and functional platelet production in vitro. This latter result was confirmed in vivo using lethally irradiated mice transplanted with cells that overexpressed p45 NF-E2. Unexpectedly, the enhancement of MK differentiation was at the expense of myeloid development and, for the first time, identified p45 NF-E2 as a negative regulator of myeloid differentiation. Secondly, we determined the nuclear localisation signal of p45-NF-E2 and the pathway responsible for nuclear import. We also investigated the importance of p45 NF-E2 nuclear import in thrombopoiesis. Finally, we showed that p45 NF-E2 is modified mainly by SUMO-2/3 in bone marrow cells and this process is involved in the transcriptional activation of MK-specific genes and platelet release. Taken together, these results suggest that enforced expression of p45 NF-E2 selectively enhances many aspects of MK differentiation including early and terminal MK maturation, proplatelet formation and platelet release. Equally important, this thesis also indicates that white blood cell differentiation may be inhibited by p45 overexpression, while molecular processes such as the nuclear import and SUMOylation of p45 NF-E2 are vital for thrombopoiesis. These observations will facilitate subsequent studies into the feasibility of manipulating p45 NF-E2 protein levels for the treatment of conditions such as thrombocytopaenia and other platelet disorders.

Megakaryocytes (MKs) are a rare population of haematopoietic cells, which produce platelets. Platelet production is a complex process that is tightly regulated at the transcriptional level by lineage specific transcription factors such as p45 NF-E2. Understanding how transcriptional regulators operate is imperative to advance our knowledge of disease pathophysiology and to propose novel treatment options. Therefore, the aims of this study were to: i) study the effects of p45 NF-E2 overexpression on various stages of megakaryopoiesis; (ii) elucidate the nuclear transport mechanisms of p45 NF-E2; and iii) determine the impact of a p45 NF-E2 modification called SUMOylation on thrombopoiesis. Exogenous p45 NF-E2 was overexpressed in haematopoietic cells in culture and various aspects of megakaryopoiesis were examined. Overexpression of p45 NF-E2 enhanced multiple stages of MK differentiation such as colony forming unit (CFU)-MK formation and terminal MK maturation. Most importantly, p45 NF-E2 overexpression resulted in significant increases in proplatelet and functional platelet production in vitro. This latter result was confirmed in vivo using lethally irradiated mice transplanted with cells that overexpressed p45 NF-E2. Unexpectedly, the enhancement of MK differentiation was at the expense of myeloid development and, for the first time, identified p45 NF-E2 as a negative regulator of myeloid differentiation. Secondly, we determined the nuclear localisation signal of p45-NF-E2 and the pathway responsible for nuclear import. We also investigated the importance of p45 NF-E2 nuclear import in thrombopoiesis. Finally, we showed that p45 NF-E2 is modified mainly by SUMO-2/3 in bone marrow cells and this process is involved in the transcriptional activation of MK-specific genes and platelet release. Taken together, these results suggest that enforced expression of p45 NF-E2 selectively enhances many aspects of MK differentiation including early and terminal MK maturation, proplatelet formation and platelet release. Equally important, this thesis also indicates that white blood cell differentiation may be inhibited by p45 overexpression, while molecular processes such as the nuclear import and SUMOylation of p45 NF-E2 are vital for thrombopoiesis. These observations will facilitate subsequent studies into the feasibility of manipulating p45 NF-E2 protein levels for the treatment of conditions such as thrombocytopaenia and other platelet disorders.

Integrins are cell surface receptors mainly involved with cell-cell adhesion and cell adhesion to extracellular matrix (ECM) proteins. Integrin signaling participates in many cellular pathways and has multiple effects on cell behavior. The role of integrins on megakaryocytes (MKs) has been extensively studied, and integrins have been proven to be vital in regulating MK development and functions. MKs are large platelet-producing cells primarily residing in the bone marrow (BM). The interactions between MK and ECM proteins in the BM via integrins are thought to play an important role in maintaining normal thrombopoiesis, and deregulation of integrin signaling may lead to impaired MK maturation, MK migration, and proplatelet formation. Integrins also take part in the cell-cell adhesion of MK to other cells in the BM, such as osteoblasts and fibroblasts. MK adhesion is proved to stimulate the expansion of fibroblasts and osteoblasts, which may have strong implications for treating bone marrow fibrosis (BMF) and osteoporosis. In this review, we introduce different types of integrins expressed on MK and discuss their roles during MK differentiation and maturation from hematopoietic stem cells (HSCs) as well as their functions during thrombopoiesis. We also focus on surveying the interactions of MK with other BM cells via integrin signaling and examining the functions of MK integrins in regulating BM homeostasis. Finally, we explain diseases caused by defects associated with MK integrins and explore potential therapeutic treatments.

Immune thrombocytopenia (ITP) is a heterogeneous immune-mediated blood disorder with multiple pathologies that cause thrombocytopenia. The primary source of this thrombocytopenia is platelet destruction by antiplatelet autoantibodies. Although several treatment options are available for ITP, they are often transient, and responses can be difficult to predict. Different studies show ITP plasma and autoantibodies can also inhibit platelet production, but the mechanism and its impact in causing thrombocytopenia remains unknown. By identifying the different mechanisms causing ITP thrombocytopenia, it may be possible to identify more effective and patient specific treatment options, as well as identify patients who could be at an increased risk of bleeding. To study the antibody mediated inhibition of platelet production, I developed a peripheral blood based megakaryopoiesis assay that used the patient’s own hematopoietic stem and progenitor cells (HSPC) as a starting cell source to grow megakaryocytes. I demonstrate this assay can use a small amount of peripheral blood to grow mature megakaryocytes that are capable of thrombopoiesis. Using this assay, I investigated the effect patient plasma had on platelet production. As such, this study is the first autologous investigation of the effect ITP plasma has on platelet production. I found no inhibition of megakaryopoiesis, but did find an effect on thrombopoiesis, indicating that the plasma is affecting the end stages of platelet production. Secondary observations also show that some ITP HSPC have an enhanced megakaryopoiesis potential, generating more mature megakaryocytes than what was observed with healthy donors. While screening monoclonal antiplatelet antibodies, I discovered an anti-GPIb antibody that inhibited megakaryocyte maturation and found this affect was also present with the Fab antibody fragment. From my research I have developed several tools that can be used to investigate impaired platelet production in ITP and further our understanding of this pathology. / Thesis / Doctor of Philosophy (PhD) / Immune thrombocytopenia (ITP) is an immune mediated blood disorder where autoantibodies target and destroy platelets, cells that are crucial for preventing blood loss. Evidence from different studies show that ITP autoantibodies are also affecting the cells producing platelets (megakaryocytes), but the mechanism of this effect remains unknown. To study antibody mediated inhibition of megakaryopoiesis, I developed a peripheral blood based megakaryopoiesis assay that used the patient’s own cells as a starting source to grow megakaryocytes. With this assay, I investigated the effect patient plasma had on platelet production. I found no inhibition of megakaryocyte growth but did find an effect on their ability to produce platelets. I also found a model antibody that affected the maturity of the megakaryocytes during their development. These tools can now be used to further investigate impaired platelet production in ITP patients and determine the impact this inhibition has on ITP pathology.

The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of transcriptional complexes consisting of cellspecific and ubiquitous transcription factors. Understanding the molecular events associated with normal megakaryocyte (Mk) differentiation is an issue of central importance to haematology. The aims of this study were therefore to: (i) define the transcription factors responsible for regulating the expression of Mkspecific genes such as Glycoprotein IX, (ii) identify the protein partners of such important Mk-regulatory transcription factors and (iii) examine the mechanisms utilised by these factors to regulate gene expression. First, the regulatory elements in the GPIX promoter required for basal and inducible expression were examined in megakaryoblastic Dami cells stimulated to undergo differentiation. The resulting data suggested that an Ets site in the GPIX promoter binding the Ets-family member Fli-1 was crucial in regulating both constitutive and inducible GPIX expression. Second, a two-hybrid screen of a K-562 cDNA library was used to identify transcription factors that interacted with Fli-1 and were potential regulators of Mk development. Results of this screen identified a novel protein-protein interaction with GATA-1, a previously well-characterised zinc finger transcription factor also implicated in erythroid and Mk development. Mapping of the domains required for the interaction show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. The biological significance of the Fli-1/GATA-1 interaction was demonstrated in transient transfection assays, which resulted in synergistic activation of Mkspecific promoters. Analysis of Fli-1 and GATA-1 expression in a series of erythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli- 1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expression of Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) induces endogenous GPIX expression. Quantitative mobility shift assays reveal that Fli- 1 and GATA-1 exhibit cooperative DNA-binding in which the binding of GATA-1 to DNA is increased approximately 26 fold in the presence of Fli-1. This data provides a mechanism for the observed transcriptional synergy. In conclusion, this work suggests that Fli-1 and GATA-1 work together through protein-protein interaction and cooperative DNA-binding to activate the expression of genes associated with the terminal differentiation of Mks.

Thrombocytopenia is one of the most common hematologic presentations of active human cytomegalovirus (HCMV) infection, especially in recipients of allogeneic hematopoietic stem cell transplantations and newborns of congenital HCMV infection. However, mechanisms of HCMV-induced thrombocytopenia have not been well understood. The precursor of circulating platelets – megakaryocyte, is derived from hematopoietic stem/progenitor cell in bone marrow. We postulate that inhibition to megakaryocytic development is the major pathogenesis of HCMV-induced thrombocytopenia. Megakaryocytic cells as well as supportive microenvironment in bone marrow are major targets of HCMV infection. Presented study mainly focused on the impacts of HCMV to megakaryocytic cells and multipotent mesenchymal stromal cells (MSCs) - the precursor of bone marrow stromal cells. Based on a megakaryocytic cell model challenged by HCMV in vitro, inhibited megakaryocytic endomitosis, proliferation, and cellular expression were respectively demonstrated as decreased polyploidy population, decreased colony formation, and reduced c-Mpl (thrombopoietin receptor) expressing cells. Evoked apoptosis of megakaryocytic cells was also evidenced with increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation after HCMV infection. Involvement of mitochondrial-mediated intrinsic apoptosis was further shown as losing JC-1 fluorescent signal in infected megakaryocytic cells. These results suggest that inhibition induced by HCMV is exerted through multiple processes directly affecting the megakaryopoietic development. Functional failure of bone marrow microenvironment was demonstrated in bone marrow derived MSCs infected by HCMV in vitro. Suppressed cytokine production, impaired cellular migration, and hindered differentiation of HCMV-infected MSCs were respectively demonstrated by lowered level of stromal cell-derived factor 1 in culture medium, decreased number of cells passed through a porous membrane in a transwell culture, and reduced differentiated cells in either adipogenic or osteogenic induction cultures. Alongside with these changes, HCMV-induced programmed cell death further contributed to the supportive failure. Autophagic cell death in infected MSCs was demonstrated as massive accumulation of vacuoles with double membrane structure and LC-3b II molecules followed by viability loss. De novo apoptosis was also observed as another process of programmed cell death, shown as increased phosphatidylserine exposure on cell surface and intracellular caspase-3 activation of infected MSCs. Increased programmed cell death appeared to be associated with extensive HCMV replication in MSCs, which was featured with typical cytopathic morphology, expression of viral tegument protein pp65, and massive accumulation of various viral particles including mature virions. Sustained activation of extracellular signal-regulated kinases likely represented a signal transduction network connecting viral expression or replication with programmed cell death. In a “MSCs-dependent” megakaryopoiesis model, HCMV-infected MSCs failed to support survival and maintenance of megakaryocytic cells. Taken together, these results suggest that active HCMV expression or replication inhibits multiple cellular functions and induces multiple processes of programmed cell death of MSCs. Such inhibition compromises supportive functions of bone marrow microenvironment, and subsequently reduces platelet production in an indirect manner. In summary, HCMV suppresses cellular function and induced apoptosis on both megakaryocytic cells and their supportive cells, MSCs. Therefore, the inhibitory effects of HCMV on megakaryopoiesis are operated via both direct and indirect mechanisms. / published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of transcriptional complexes consisting of cellspecific and ubiquitous transcription factors. Understanding the molecular events associated with normal megakaryocyte (Mk) differentiation is an issue of central importance to haematology. The aims of this study were therefore to: (i) define the transcription factors responsible for regulating the expression of Mkspecific genes such as Glycoprotein IX, (ii) identify the protein partners of such important Mk-regulatory transcription factors and (iii) examine the mechanisms utilised by these factors to regulate gene expression. First, the regulatory elements in the GPIX promoter required for basal and inducible expression were examined in megakaryoblastic Dami cells stimulated to undergo differentiation. The resulting data suggested that an Ets site in the GPIX promoter binding the Ets-family member Fli-1 was crucial in regulating both constitutive and inducible GPIX expression. Second, a two-hybrid screen of a K-562 cDNA library was used to identify transcription factors that interacted with Fli-1 and were potential regulators of Mk development. Results of this screen identified a novel protein-protein interaction with GATA-1, a previously well-characterised zinc finger transcription factor also implicated in erythroid and Mk development. Mapping of the domains required for the interaction show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. The biological significance of the Fli-1/GATA-1 interaction was demonstrated in transient transfection assays, which resulted in synergistic activation of Mkspecific promoters. Analysis of Fli-1 and GATA-1 expression in a series of erythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli- 1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expression of Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) induces endogenous GPIX expression. Quantitative mobility shift assays reveal that Fli- 1 and GATA-1 exhibit cooperative DNA-binding in which the binding of GATA-1 to DNA is increased approximately 26 fold in the presence of Fli-1. This data provides a mechanism for the observed transcriptional synergy. In conclusion, this work suggests that Fli-1 and GATA-1 work together through protein-protein interaction and cooperative DNA-binding to activate the expression of genes associated with the terminal differentiation of Mks.

The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of transcriptional complexes consisting of cellspecific and ubiquitous transcription factors. Understanding the molecular events associated with normal megakaryocyte (Mk) differentiation is an issue of central importance to haematology. The aims of this study were therefore to: (i) define the transcription factors responsible for regulating the expression of Mkspecific genes such as Glycoprotein IX, (ii) identify the protein partners of such important Mk-regulatory transcription factors and (iii) examine the mechanisms utilised by these factors to regulate gene expression. First, the regulatory elements in the GPIX promoter required for basal and inducible expression were examined in megakaryoblastic Dami cells stimulated to undergo differentiation. The resulting data suggested that an Ets site in the GPIX promoter binding the Ets-family member Fli-1 was crucial in regulating both constitutive and inducible GPIX expression. Second, a two-hybrid screen of a K-562 cDNA library was used to identify transcription factors that interacted with Fli-1 and were potential regulators of Mk development. Results of this screen identified a novel protein-protein interaction with GATA-1, a previously well-characterised zinc finger transcription factor also implicated in erythroid and Mk development. Mapping of the domains required for the interaction show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. The biological significance of the Fli-1/GATA-1 interaction was demonstrated in transient transfection assays, which resulted in synergistic activation of Mkspecific promoters. Analysis of Fli-1 and GATA-1 expression in a series of erythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli- 1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expression of Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) induces endogenous GPIX expression. Quantitative mobility shift assays reveal that Fli- 1 and GATA-1 exhibit cooperative DNA-binding in which the binding of GATA-1 to DNA is increased approximately 26 fold in the presence of Fli-1. This data provides a mechanism for the observed transcriptional synergy. In conclusion, this work suggests that Fli-1 and GATA-1 work together through protein-protein interaction and cooperative DNA-binding to activate the expression of genes associated with the terminal differentiation of Mks.