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.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/26125 |
| Date | January 2020 |
| Creators | Ivetic, Nikola |
| Contributors | Nazi, Ishac, Arnold, Donald, Kelton, John, Biochemistry and Biomedical Sciences |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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