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By S.E. van Tol


LRP8 is a transmembrane receptor present on multiple celltypes. It is known to be involved in the Reelin pathway in neuronal cells, leading to formation of layers in the neocortex during embryogenesis. LRP8 is also known to be present on platelets, endothelial cells and monocytes. Different ligands present in the circulation bind LRP8 and induce several effects. Dimeric β2-GPI, apoB100, protein C and factor XI enhance platelet aggregation when binding to LRP8 on platelets. Binding of dimeric β2-GPI on endothelial cells enhances leukocyte adhesion and thrombus formation. On the contrary, apoE interacting with LRP8 on platelets and endothelial cells inhibits platelet aggregation via activation of nitric oxide synthase. In macrophages apoE can induce a switch from M1 macrophages to M2 macrophages, an anti-inflammatory and anti-atherogenic phenotype of macrophages. APC on monocytes can activate nitric oxide synthase and thereby its cytoprotective effects are established. The majority of the ligands induce platelet aggregation and thrombosis. Indeed in LRP8 deficient mice a reduced thrombus formation was found, indicating LRP8 contributes to thrombosis. The aim of this study was to evaluate the binding epitopes of different anti-LRP8 VHHs on LRP8. Therefore, first anti-LRP8 VHHs were produced and best binding VHHs were selected. These VHHs, anti-LRP8 VHH4, 7 and 8 were produced on a large scale. Their purity and concentration was tested and compared with an old production of the same VHHs. To enable evaluating binding epitopes, biotinylation of VHH7 and 8 was performed. With an ELISA it was confirmed biotinylation succeeded. Then with a competitive ELISA the binding epitopes of the anti-LRP8 VHHs was defined. Anti-LRP8 VHH4 and 7 bind the same epitope on LRP8, VHH8 binds another epitope. This knowledge can be used for further research on LRP8, for example performing a sandwich ELISA or making a bihead of two different anti-LRP8 VHHs

Topics: LRP8, apoER2, platelets, endothelial cells, monocytes, neuronal cells, thrombosis, beta2-glycoprotein I, apolipoprotein E, apolipoprotein B100, Factor XI, protein C
Year: 2012
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