Solid-supported lipid bilayer in combination with atomic force microscope enables in vitro studies of (patho)physiological processes of the cell membrane under physiological conditions. Antiphospholipid syndrome is a model disease, in which the presence of autoantibodies against various proteins of haemostasis system disrupt processes of activated cell membrane, leading to recurring thrombosis and obstetric complications.
In our work we have studied the effect of isolated antiphospholipid antibodies and β2-glycoprotein I on the integrity of anticoagulant crystalline lattice of annexin A5 formed on phospholipid bilayer using fluid cell atomic force microscope. Specific antibodies against prothrombin and β2-glycoprotein I were isolated from a patient diagnosed with primary antiphospholipid syndrome using affinity chromatography. Purity and specificity were confirmed with enzyme-linked immunosorbent assay. Phospholipid vesicles were prepared by evaporation-based film formation, which was hydrated and sonicated. Vesicles were incubated on mica to form a bilayer. Atomic force microscope measurements were performed in contact mode by scanning the surface of the bilayer and observing the changes after injecting annexin A5, β2-glycoprotein I and antibodies against β2-glycoprotein I.
The analysis of isolated specific antibodies rises the question of existence of subpopulation of antibodies which recognize both prothrombin and β2-glycoprotein I or their possible shared epitopes. The size of vesicles depended mostly on the power and less on the time of sonication. The highest bilayer coverage of mica was achieved using vesicles sized 100–200 nm. We showed that the crystallization rate and crystal form of annexin A5 depends on the concentration of annexin A5 and mass fraction of phosphatidylserine. Agglomeration of β2-glycoprotein I on the bilayer was not noticed, even in the presence of antibodies. The presence of immune complex did not disrupt annexin A5 crystallization. The lack of bilayer binding of β2-glycoprotein I may occur due to experimental conditions or protein source.
The model used in our work represents an important role in diagnostics of antiphospholipid syndrome by assessing different antiphospholipid antibodies. It can also function as a searching tool for new therapeutic substances in antiphosholipid syndrome therapy.