Antiphospholipid syndrome (APS) is an autoimmune disorder defined as the persistent presence of antiphospholipid antibodies (aPL) associated with recurrent thrombotic events and/or fetal loss. The mechanisms for fetal loss in this syndrome have not yet been clearly explained, although several hypotheses based on experimental data have been put forward. It has been shown that proliferation of human umbilical vein endothelial cells (HUVECs) decreased significantly in cultures that contained sera positive for anticardiolipin antibody activity collected from patients with recurrent fetal loss. We explored the effect of ACAs on Vascular Endothelial Growth factor- A (VEGF-A) secretion of cultured HUVECs. VEGF appears to be the most endothelial cell-specific and unequivocal angiogenic factor. In vitro, VEGF causes endothelial cell proliferation and migration; in vivo, it is potently angiogenic and causes vascular permeability. We determined the effect of ACA IgG 40μg/ml and 80μg/ml on VEGF secretion using ELISA. The results were measured in mean ± SD and expressed in pg of VEGF/5 x 10⁴ cells. P ≤ 0.05 considered significant as compared to control. The results showed that ACA increases VEGF-A secretion and/or may bind at VEGF-A binding sites. This finding may be useful for finding therapies for patients with recurrent miscarriages. / Access to thesis permanently restricted to Ball State community only. / Department of Physiology and Health Science

An outline of the work contained in this thesis is presented. The first chapter is a critical review of the literature pertaining to the pathophysiological mechanisms operational with regards to the antiphospholipid syndrome (APS). The syndrome is characterised by venous and arterial thrombosis, and recurrent fetal loss, in association with the persistent presence of antibodies targeting the main autoantigen beta 2-glycoprotein I (β2GPI). The second chapter reviews the literature delineating the diverse physiological functions of β2GPI, and then relates them to its role in our current understanding of the pathophysiology of APS. The third chapter presents a critical review of the evidence base for the diagnosis and management of APS. The fourth chapter describes the interaction between β2GPI and the glycoprotein Ib alpha (GPIbα) subunit of the platelet receptor GPIb-IX-V. GPIbα is an important platelet adhesion receptor, which mediates multiple additional functions on the platelet surface, including binding coagulation factor XI (FXI). The implication of the interaction between β2GPI and GPIbα on platelet activation and the release of thromboxane in the presence of anti-β2GPI antibodies is explored, as well as the intracellular pathways via which this activation occurs. The relevance of these findings to understanding APS pathogenesis, in particular thrombosis, is discussed. The fifth chapter delineates the interaction between the fifth domain of β2GPI and FXI and its activated form factor XIa (FXIa). The ability of FXIa to cleave β2GPI between lysine (Lys) 317 and threonine (Thr) 318, and modulate its function is reported. The sixth chapter describes the ability of β2GPI to inhibit FXIa autoproteolytic hydrolysis at the specific FXIa residues arginine (Arg) 507, Arg532 and Lys539. This interaction with β2GPI stabilizes FXIa activity over time, and leads to enhanced FXIa mediated fibrin formation. This is a novel physiological function of β2GPI with important implications. Recent epidemiological studies by others have emphasized the critical role of FXIa in pathological thrombus propagation. The seventh chapter defines the relevance of the FXIa residues Arg507, Arg532 and Lys539 to FXIa mediated inactivation by the main FXIa inhibitor Protease Nexin 2 (PN2), and by Antithrombin III (ATIII). Insights into future directions for research are presented and discussed within each individual chapter.

An outline of the work contained in this thesis is presented. The first chapter is a critical review of the literature pertaining to the pathophysiological mechanisms operational with regards to the antiphospholipid syndrome (APS). The syndrome is characterised by venous and arterial thrombosis, and recurrent fetal loss, in association with the persistent presence of antibodies targeting the main autoantigen beta 2-glycoprotein I (β2GPI). The second chapter reviews the literature delineating the diverse physiological functions of β2GPI, and then relates them to its role in our current understanding of the pathophysiology of APS. The third chapter presents a critical review of the evidence base for the diagnosis and management of APS. The fourth chapter describes the interaction between β2GPI and the glycoprotein Ib alpha (GPIbα) subunit of the platelet receptor GPIb-IX-V. GPIbα is an important platelet adhesion receptor, which mediates multiple additional functions on the platelet surface, including binding coagulation factor XI (FXI). The implication of the interaction between β2GPI and GPIbα on platelet activation and the release of thromboxane in the presence of anti-β2GPI antibodies is explored, as well as the intracellular pathways via which this activation occurs. The relevance of these findings to understanding APS pathogenesis, in particular thrombosis, is discussed. The fifth chapter delineates the interaction between the fifth domain of β2GPI and FXI and its activated form factor XIa (FXIa). The ability of FXIa to cleave β2GPI between lysine (Lys) 317 and threonine (Thr) 318, and modulate its function is reported. The sixth chapter describes the ability of β2GPI to inhibit FXIa autoproteolytic hydrolysis at the specific FXIa residues arginine (Arg) 507, Arg532 and Lys539. This interaction with β2GPI stabilizes FXIa activity over time, and leads to enhanced FXIa mediated fibrin formation. This is a novel physiological function of β2GPI with important implications. Recent epidemiological studies by others have emphasized the critical role of FXIa in pathological thrombus propagation. The seventh chapter defines the relevance of the FXIa residues Arg507, Arg532 and Lys539 to FXIa mediated inactivation by the main FXIa inhibitor Protease Nexin 2 (PN2), and by Antithrombin III (ATIII). Insights into future directions for research are presented and discussed within each individual chapter.

We report on 2 cases of chronic relapsing thrombotic thrombocytopenic purpura, in which anti-phospholipid antibodies were also found. The first patient was felt to have the antiphospholipid antibody syndrome, while the second patient had anti-phospholipid antibodies without clinical manifestations of the anti-phospholipid antibody syndrome. We discuss chronic relapsing thrombotic thrombocytopenic purpura and the anti-phospholipid antibody syndrome. Furthermore, we introduce the possibility of an association between chronic relapsing thrombotic thrombocytopenic purpura and the presence of anti-phospholipid antibodies.

Group A streptococcal disorders can result from autoantibodies generated against M proteins. These autoantibodies cross react with the basal ganglia resulting in movement disorders. Previously, we demonstrated binding of streptococcal mAb10F5, with CPu and phospholipids. To determine if mAb10F5 binding to basal ganglia and phospholipids is due to virulence of the antibody or antibody subtype, rats were injected with control IgG2 antibodies and euthanized after 24, 48, or 72 hours. Brains were harvested and immunofluorescence was used to analyze brain slices. Control IgG2 rats showed significantly less fluorescence in the CPu than mAb10F5 injected rats at every time point. These findings reaffirm 10F5 is an anti-basal ganglia antibody. To evaluate mechanism of antibody entry, mAb10F5 was examined for anti-phospholipid activity. MAb10F5 displayed greater affinity to phospholipids when compared to IgG2 controls. Our findings support mAb10F5 is an anti-basal ganglia and anti-phospholipid antibody due to its own virulence. / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science