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This thesis investigates immune-mediated thrombocytopenia: the natural history of immune thrombocytopenia (ITP), a novel treatment strategy for ITP, and the diagnosis of heparin-induced thrombocytopenia (HIT). Retrospective study New international consensus guidelines on the diagnosis of ITP have recently been proposed (Rodeghiero et al, Blood 2009 113:2386-2393). Previous estimates of prevalence and the natural history of ITP may no longer remain relevant. We reviewed 129 patients who met revised criteria for the diagnosis of ITP. 49% of newly diagnosed primary ITP patients did not need treatment at presentation and remained free from grade III/IV bleeding or the need for future treatment. In contrast, patients who required treatment at presentation but were still thrombocytopenic at 1 month, were significantly more likely to remain refractory to medical therapies and experience grade III/IV bleeding. Prospective study We investigated the use of a novel combination of conventional therapies in ITP given over 4 weeks: 20 patients were enrolled onto phase IIb study of oral dexamethasone 40mg day 1-4, oral cyclosporine 2.5-3mg/kg/daily day 1-28 and intravenous low-dose rituximab 100mg day 7, 14, 21 and 28. There were no therapy-related serious adverse side effects and 6 month response rate was 60%. This study highlights the possibility of achieving an enduring remission for some patients from as little as 4 weeks of therapy. Diagnosis of HIT study Antibodies against Platelet factor (PF)4/heparin complexes are found in patients who have heparin-induced thrombocytopenia (HIT) and in asymptomatic patients undergoing cardiac surgery. The clinical significance of these antibodies in the latter group remains uncertain. We examined the incidence and time course of anti-PF4/heparin antibodies in patients undergoing cardiac surgery using a commercially available IgG specific ELISA kit in common clinical use. After identifying serum containing anti-PF4/heparin antibodies, we assessed their reactivity against a panel of 16 mutant(m)PF4 proteins and native PF4 (nPF4) purified in-house to determine if any differences exist with clinically pathogenic HIT samples. 24% of cardiac surgery patients are positive on commercial HIT screening versus only 8% by nPF4. We postulate that some patients identified by commercial HIT screening may be cross-reacting to PF4/nucleic acid complexes.

Human thrombopoietin (TPO) is the primary regulator of megakaryopoiesis. It is known that TPO levels in blood are regulated by a mechanism involving its uptake by platelet TPO receptors (c-MPL) and its subsequent intraplatelet destruction. However, this mechanism does not fully explain TPO regulation and there is evidence of an additional regulatory mechanism such as a negative feedback transcriptional mechanism at the level of the bone marrow stroma. This thesis focuses on further investigation of this transcriptional regulation. The aims of the investigation are to: i) characterize the potential repressor(s) of thrombopoietin (TPO) gene transcription; and ii) define the platelet factor 4 (PF4)-mediated pathway that causes suppression of TPO gene expression. A potential TPO gene repressor, ZEB1, was firstly studied. Promoter analysis showed that there was a small region that greatly reduced TPO promoter activity and this region carries a cluster of four conserved ZEB1 binding motifs. However further analysis revealed that repression of TPO promoter activity did not depend on this ZEB1 cluster, suggesting that ZEB1 is unlikely to be the repressor. This promoter region also contains binding motifs for MZF1. Electrophoretic mobility shift assay (EMSA) showed the binding of MZF1 to the putative binding motif in this region. siRNA knockdown of MZF1 expression reversed the reduced TPO promoter activity. A significant increase of TPO expression was also observed in MZF1 siRNA transfected OP9 cells (surrogate for bone marrow stromal cells). The signaling pathway that mediates PF4-induced repression of TPO expression was then investigated. I found that PF4 is the key factor in the serum that suppresses TPO expression. This suppression is dose-dependent and further experiments revealed that this suppression was through the interaction with LRP1 expressed on OP9 cells. PF4/LRP1 interaction triggered PI3K/Akt signaling pathway with phosphorylation of Akt. I suggest that PI3K/Akt activation possibly then results in MZF1 activation which then likely migrates into the nucleus and mediates repression of TPO transcription. In conclusion, the current study identifies a novel intronic transcription repressor (MZF1) of human TPO expression. TPO repression is mediated by PF4 which binds to LRP1, and activates PI3K/Akt signal transduction pathway.

Immune heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy and is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. Early diagnosis of HIT is important to reduce morbidity and mortality. I developed an enzyme immunoassay that detects the binding of HIT IgG to PF4-heparin in the fluid phase. This required techniques to purify and biotinylate PF4. The fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. I was able to detect anti-PF4-heparin IgG in 93% of HIT patients. I also investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 88% of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration. While HIT patients possess antibodies to PF4-heparin, I observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, I provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd=7-30nM) and polystyrene adsorbed PF4 alone (Kd=20-70nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. I conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and thus most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (sub-optimal) promotes recognition of this epitope. Under conditions that are more physiological and sensitive than previous studies, I observed that affinity-purified HIT IgG will cause platelet aggregation upon the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. I quantitated the binding of affinity-purified HIT 125I-IgG to platelets as they activate in a plasma milieu. Binding of the HIT IgG was dependent upon heparin and some degree of platelet activation. Blocking the platelet Fc receptor-II with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. I conclude that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to induce platelet aggregation. The Fab region of HIT IgG binds to PF4-heparin that is on the surface of activated platelets. I propose that only then does the Fc portion of the bound IgG activate other platelets via the Fc receptor. My data support a dynamic model of platelet activation where released PF4 enhances further antibody binding and more release.