Plasminogen Activator Inhibitor-2 - En Spontant Polymeriserende Serpin i et Præeklampsi Perspektiv

Abstract

Omtrent 2-5% af alle graviditeter bliver ramt af præeklampsia hvortil årsagen ikke er kendt. Patienter med etableret præeklampsi har et nedsat koncentrationsniveau af Plasminogen Activator Inhibitor-2 (PAI-2) i plasma, hvilket indikerer at PAI-2 kan spille en rolle i udviklingen af præeklampsi, eller faldet i PAI-2 kan være et resultat af præeklampsi. PAI-2 har evnen til spontant at danne polymerer, hvilket kan være relevant i et præeklampsi perspektiv. Dermed må rollen af PAI-2 i ætiologien af præeklampsi undersøges fuldt ud. Derfor var formålet med dette studie at udvikle metoder, modeller og værktøjer til at hjælpe undersøgelsen af PAI-2 og dens rolle i præeklampsi. Disse metoder stræbte efter at udvikle på metoder og værktøjer til at inducere polymeriseringen af PAI-2, og modeller til at visualisere graden af polymerdannelse i plasmaprøver fra gravide. Yderligere var formålet her at generere og karakterisere Monoklonale Antistoffer (mAbs), med hensigt på at etablere et ELISA med formål i at kunne kvantificere koncentrationsniveauet af PAI-2 i plasmaprøver fra gravide. In vitro modeller til polymerisering af PAI-2 blev etableret. Polymerisering blev induceret henholdsvist kemisk og termisk, ved eksposering af PAI-2 prøver til Guanidin Hydrochlorid (GuHCl) eller varme. Disse prøver blev visualiseret ved hjælp af SDS-PAGE, nativ PAGE og western blot teknikker. Muse anti-PAI-2 mAbs blev karakteriseret, og en ny generation rotte anti-PAI-2 mAbs blev udviklet og delvist karakteriseret. Et ELISA baseret assay blev etableret, med et muse mAb som det primære antistof, og et Polyklonalt Antistof (pAb) til detektion af det bundne PAI-2. Dette assay blev brugt til at måle PAI-2 koncentrationsniveau i plasmaprøver fra patienter med præeklampsi og kontroller. Endvidere blev PAI-2 i plasmaprøver fra gravide undersøgt, ved brug af magnetiske DynaBeadsTM med et muse mAb til at binde PAI-2 i prøven. Resultat af dette blev illustreret med western blotting teknikker. Endelig blev anti-PAI-2 mAbs produceret i rotter, i et forsøg på at danne antistoffer med høj affinitet og forskellige specificiteter, der formentlig kan være brugbare i yderligere optimering af ELISA og for detektion af polymeriseret PAI-2. Resultaterne af in vitro polymerisering af PAI-2, induceret kemisk eller termisk, gav polymerisering i prøverne. Ydermere, viste PAI-2 fra plasmaprøver både intakt, ikke polymeriseret PAI-2, samt PAI-2-polymerer. Det etablerede ELISA til kvantificering af PAI-2 niveauer i plasmaprøver fra præeklampsi patienter og kontroller, viste målbare koncentrationsniveauer PAI-2. Rotte mAbs kunne detektere PAI-2 ved brug i western blotting. Endvidere kunne de detektere både intakt og polymeriseret PAI-2, når PAI-2-prøver var bundet til ELISA plader. Imidlertid fremstod ingen af antistofferne til at have specificitet alene mod polymeriseret PAI-2 Det ELISA baseret assay etableret i dette studie kræver validering. Ydermere bør rotte mAbs videre karakteriseres i deres evne til at genkende nativt, humant PAI-2 fra plasmaprøver. Fremtidige studier bør designes til at undersøge om der findes en forskel i mængden af PAI-2 polymerer i plasma fra patienter påvirket af præeklampsi.

Approximately 2-5% of all pregnancies are affected by preeclampsia, the cause of which remains unknown. Patients with established preeclampsia have decreased Plasminogen Activator Inhibitor-2 (PAI-2) plasma levels, indicating that PAI-2 might have a role in the development of preeclampsia, or that the decrease in PAI-2 levels might be a result of preeclampsia. PAI-2 has the ability to spontaneously form polymers, which could be relevant in a preeclampsia perspective. Thus, the possible role of PAI-2 in the etiology of preeclampsia needs to be investigated in full. Therefore, the aim of this study was to develop experimental methods, models and tools to aid in the investigation of PAI-2 and its role in preeclampsia. These methods aimed to evolve on existing methods to induce polymerisation of PAI-2 and models to visualise the degree of polymer formation in samples. In addition, the aim herein was to generate and characterise Monoclonal Antibodies (mAbs), with the intent to establish an ELISA with the purpose of quantifying of the levels of PAI-2 in plasma samples from pregnant individuals. In vitro models for polymerisation of PAI-2 were established. The polymerisation was induced chemically and thermally, using exposure to Guanidine Hydrochloride (GuHCl) or heat, respectively. These samples were then visualised using SDS-PAGE, native PAGE and western blotting techniques. Mouse mAbs towards PAI-2 were characterised and a new generation of rat mAbs against PAI-2 was generated and partially characterised. An ELISA based assay was established, with a mouse mAb as the capture antibody, and a pAb to detect captured PAI-2 from samples. This assay was used on plasma samples from preeclamptic patients and controls. Moreover, PAI-2 in pregnancy plasma samples was investigated, utilising magnetic DynaBeadsTM with a mouse mAb, to capture PAI-2 from the sample. The results of this were illustrated with western blotting techniques. Lastly, anti-PAI-2 mAbs were raised in rats, in an attempt to create high-affinity antibodies with different specificities, which might be applicable in further optimisation of ELISA and for the detection of PAI-2 polymers. The results from in vitro polymerisation of PAI-2, induced chemically and and heat, showed polymerisation in the samples. Furthermore, PAI-2 in plasma samples showed intact, non-polymerised PAI-2 and polymerised PAI-2. The established ELISA for quantification of PAI-2 levels in plasma samples from preeclampsia patients compared with controls, showed detectable amounts of PAI-2. The rat mAbs were able to detect PAI-2, when used for western blotting applications. Furthermore, they could bind both intact and polymerised PAI-2, when PAI-2 samples were bound on an ELISA plate. However, none of these antibodies seemed to have specificity towards only polymerised PAI-2. The ELISA based assay established in this study remains to be validated. Additionally, rat mAbs must be characterised in their ability to recognise native, human PAI-2 from plasma samples. Future studies must be designed to investigate if there is a difference in the amount of PAI-2 polymers present in plasma from patients affected by preeclampsia.

A master thesis from Aalborg University

Plasminogen Activator Inhibitor-2 - En Spontant Polymeriserende Serpin i et Præeklampsi Perspektiv

[Plasminogen Activator Inhibitor-2 - A Spontaneously Polymerising Serpin in a Preeclampsia Perspective]