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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.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but extremely dangerous side effect that has been reported for several adenoviral (Ad)-vectored COVID-19 vaccines. VITT pathology had been linked to production of antibodies that recognize platelet factor 4 (PF4), an endogenous chemokine. In this work we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact-mass MS measurements indicate that a significant fraction of this ensemble is comprised of antibodies representing a limited number of clones. MS analysis of large antibody fragments (the light chain, as well as the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire, and reveals the presence of a fully mature complex biantennary N-glycan within its Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS analysis were used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to IgG2 subclass and verify that the light chain belongs to the λ-type. Incorporation of enzymatic de- N -glycosylation into the peptide mapping routine allows the N -glycan in the Fab region of the antibody to be localized to the framework 3 region of the V H domain. This novel N -glycosylation site (absent in the germline sequence) is a result of a single mutation giving rise to an NDT motif in the antibody sequence. Peptide mapping also provides a wealth of information on lower-abundance proteolytic fragments derived from the polyclonal component of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1 through IgG4) and both types of the light chain (λ and κ). The structural information reported in this work will be indispensable for understanding the molecular mechanism of VITT pathogenesis.