Kurzfassung

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare haematologic disease caused by somatic mutations in the phosphatidylinositol glycan class A (PIG-A) gene, which leads to impaired expression of glycosylphophatidyl inositol (GPI)–linked proteins on the cell surface of bone marrow stem cell derived leukocytes, red blood cells and platelets [1]. PNH presents with the clinical triad of coombs-negative hemolysis, hypercoagulability and cytopenias [2], [3]. Among the deficient proteins, the lack...Paroxysmal nocturnal hemoglobinuria (PNH) is a rare haematologic disease caused by somatic mutations in the phosphatidylinositol glycan class A (PIG-A) gene, which leads to impaired expression of glycosylphophatidyl inositol (GPI)–linked proteins on the cell surface of bone marrow stem cell derived leukocytes, red blood cells and platelets [1]. PNH presents with the clinical triad of coombs-negative hemolysis, hypercoagulability and cytopenias [2], [3]. Among the deficient proteins, the lack of the complement regulatory proteins CD55 and CD59 results in an increased sensitivity to complement mediated lysis of PNH cells leading to intravascular hemolysis and platelet activation. While recent publications highlight the role of complement inhibition by CD55 and CD59 an erythrocytes and platelets in the pathophysiology of PNH [4], [5], there is only inconsistent information about the functional consequences of the lack of GPI-linked proteins on neutrophil functions, one report describing increased phagocytic activity along with a reduced oxidative burst [6] while others find an increased oxidative burst activity [7]. Importantly, there are no studies available addressing the role of cellular interactions in the pathophysiology of PNH. Since polymorphonuclear neutrophils (PMNs) play an important role in the early phase of the host defence against microbial pathogens by clearing bacterial and fungal infections [8], [9], PMNs interact with multiple components of the innate immune system, e.g. via specific receptors of complement. In addition, the deficiency of GPI-linked surface proteins is the most sensitive measure for the presence of a PNH clone [10], [11]. We hypothesize that PNH-related clinical complications or conditions such as thrombosis or hyper-inflammation are not only driven by cells of the PNH clone as previously demonstrated by others, but that PMNs derived from the PNH stem cells interact with platelets in a pathologic manner based on the lack of GPI-linked receptors. This in turn is then responsible for an inflammatory phenotype accompanied with complement activation and an increased risk for thrombosis. Therefore, the aim of this project is to elucidate the underlying mechanisms of hyper-inflammation and thrombosis in patients with PNH.
            The Radsak laboratory is specialized in the analysis of PMN functions and cellular interactions within the innate immune response [12]-[14]. Hence we propose to analyze PMN functionality and PMN interactions with platelets in patients with PNH, aplastic anemias (AA) and myelodysplastic syndromes (MDS). AA and MDS patients harbour an increased risk of developing a PNH and are screened for the presence of a PNH clone on a regular basis [15], [16]. Therefore, AA and MDS patients with a detectable PNH clone will be included into the study. For comparison, we will analyze a cohort of healthy volunteer donors in parallel. To define the optimal setting for the ex vivo analyses in patients (sensitivity, specificity, variability of functional assays), we will mimic PNH in healthy volunteer PMNs by pre-treating the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) to generate PNH-like cells [17]. Various mixtures of PMNuntreated:PMNPI-PLC treated will be employed in our PMN effector assays to establish reliable activation parameters. We will repetitively analyze up to 20 patients over 12 months in three month intervals to further confirm the assay reliability in individual patients. For functional testings, the patients will donate 20 ml of blood for the following assays that we have established in the laboratory for the analysis of PMN effector functions from whole blood for MDS patients [18]. We will analyze the phenotype of PMNs of PNH, AA and MDS patients with a PNH clone by flow cytometry discriminating wild-type and GPI-deficient cells by FLAER staining [11]. In addition, the oxidative burst activity, phagocytosis of polystyrene beads, degranulation of specific and gelatinase granules, L-selectin shedding and apoptosis of PMN upon stimulation with various agonists (TLR2, TLR4, GM-CSF or cycloheximide) will be determined. Beyond this, we will detect PMN/platelet aggregates (as established in the lab) and simultaneously analyze the platelet activation phenotype (by CD40L and CD62P expression). These studies will be complemented by platelet function testing (PFA-100) and the detection of soluble indicators of thrombotic or inflammatory activity: thrombin-antithrombin (TAT) complexes, soluble CD62P, D-dimer, antithrombin, fibrinogen, IL-1, IL-6, TNF-alpha, TGF-beta, soluble TREM-1, CRP, procalcitonin. These parameters will be correlated with clinical parameters such as treatment with complement inhibitors, history or current thrombosis and infections.
Our results will provide novel insights for a better understanding of the pathophysiology of PNH and related complications. This may lead to the better characterization of patients at risk that will benefit from complement inhibition before developing thrombosis.
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