Proučevanje medmolekulskih interakcij med fosfolipidnim dvoslojem na trdnem nosilcu, aneksinom A5, β2-glikoproteinom I in protitelesi proti β2-glikoproteinu I z mikroskopom na atomsko silo

Kobold, Andrej (author), Božič, Borut (mentor), Čučnik, Saša (comentor)

Lipidni dvosloj na trdnem nosilcu v kombinaciji z mikroskopom na atomsko silo omogoča in vitro študije (pato)fizioloških procesov na celični membrani pod fiziološkimi pogoji. Antifosfolipidni sindrom je primer stanja, kjer avtoprotitelesa proti različnim proteinom, udeleženih v hemostazi, spremenijo procese na aktivirani celični membrani, kar vodi v ponavljajoče tromboze in zaplete v nosečnosti. V našem delu smo v tekočinski celici mikroskopa na atomsko silo pri fizioloških pogojih proučevali vpliv izoliranih antifosfolipidnih protiteles in β2-glikoproteina I na celovitost antikoagulacijske kristalne mreže aneksina A5 na fosfolipidnem dvosloju. Specifična protitelesa proti protrombinu in β2-glikoproteinu smo izolirali iz pacienta s primarnim antifosfolipidnim sindromom z metodami afinitetne kromatografije, čistost in specifičnost pa potrdili z encimskoimunokemijskimi metodami. Fosfolipidne vezikle smo pripravili z odparevanjem topila in tvorbo filma, ki smo ga hidrirali in ultrazvočno obdelali. Vezikli so pri inkubaciji na sljudi tvorili fosfolipidni dvosloj. Z mikroskopom na atomsko silo smo v kontaktnem načinu slikali površino dvosloja in opazovali spremembe po injiciranju aneksina A5, β2-glikoproteina I in protiteles proti β2-glikoproteinu I. Po analizi izoliranih specifičnih protiteles se nam poraja vprašanje o obstoju podpopulacije protiteles, ki prepoznavajo tako protrombin kot β2-glikoprotein I oziroma morebitne skupne epitope. Ugotovili smo, da je velikost veziklov odvisna predvsem od moči ultrazvočne obdelave, manj pa od časa obdelave. Največji delež pokritosti sljude z dvoslojem smo dosegli z vezikli velikosti 100–200 nm. Pokazali smo, da je hitrost kristalizacije in kristalna oblika aneksina A5 odvisna od koncentracije aneksina A5 in deleža fosfatidilserina. Vezave β2-glikoproteina I na dvosloj nismo zaznali niti v prisotnosti protiteles. Prisotnost imunskega kompleksa ni vplivala na kristalizacijo aneksina A5. Nevezava β2-glikoproteina I je lahko posledica eksperimentalnih pogojev ali njegovega izvora. Uporabljeni model ima vlogo v diagnostiki antifosfolipidnega sindroma z vrednotenjem različnih antifosfolipidnih protiteles. Služi lahko tudi za iskanje novih učinkovin za zdravljenje antifosfolipidnega sindroma.

• Language: Slovenian
• Keywords: antifosfolipidna protitelesa, aneksin A5, β2-glikoprotein I, lipidni dvosloj na trdnem nosilcu, mikroskop na atomsko silo
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121912
• Publication date in RUL: 07.11.2020

Secondary language

• Language: English
• Title: A study of intermolecular interactions between solid-supported phospholipid bilayer, annexin A5, β2-glycoprotein I and anti-β2-glycoprotein I antibodies using atomic force microscope

Abstract

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.

• Keywords: antiphospholipid antibodies, annexin A5, β2-glycoprotein I, solid-supported lipid bilayer, atomic force microscopy