Antiphospholipid syndrome (APS) is an autoimmune disease in which antiphospholipid antibodies are present in the plasma of patients. Catastrophic antiphospholipid syndrome (cAPS) is a rare, life-threatening form of antiphospholipid syndrome that occurs as a result of a trigger in patients positive for antiphospholipid antibodies. Interactions between antibodies and membrane-binding proteins are important for the development of the disease, but the mechanisms are not yet fully understood.
The aim of the master's thesis was to optimize the physiological conditions of binding of antibodies to anti-β2GPI in such a way that it can be observed under an atomic force microscope.
In the serum of a patient with catastrophic antiphospholipid syndrome, antibodies to β2-glycoprotein I were isolated in several steps according to the principle of affinity chromatography. The antibodies obtained were evaluated with anti-β2GPI IgG ELISA.
At the atomic force microscope, phospholipid vesicles, annexin A5, and the antigen-antibody complex (β2-glycoprotein I, antibodies against β2-glycoprotein I) were sequentially bound to mica. Initially, we observed the formation of a planar phospholipid bilayer. If more than 50% of the surface was obtained, annexin A5 was bonded to it and the formation of the crystal coat was observed and analyzed. In the final phase, binding of antibodies to β2-glycoprotein I to the annexin crystal coat was observed and their possible pathological potential was assessed.
Upon complete crystallization of annexin A5 on the phospholipid bilayer, no binding of β2-glycoprotein I, antibodies against β2-glycoprotein I, or the antigen-antibody complex were observed. The binding affinity of annexin A5 to phosphatylidylserine and, consequently, the formation of the crystalline layer, is greater than the binding affinity of the antigen-antibody complex. The antigen-antibody complex, under experimental conditions, preferentially binds to the mica, which is more negatively charged than the phospholipid bilayer. In the presence of fully crystallized annexin A5 the complex is unable to expand along the phospholipid bilayer. The annexin crystal mantle thus offers protection against antibody binding, which can also be understood as inhibiting disease progression. We confirmed the assumption that the mere presence of antibodies against β2-glycoprotein I at the annexin mantle on the phospholipid membrane could not explain the development and / or progression of the disease.