Physical and biological characterization of hr-anxA1.

(A) Hr-anxA1 induced concentration dependent calcium flux in FPR2 transfected HEK-293 cells, 1 μM ionomycin is taken as reference value (100%). (B) Ellipsometry analysis shows calcium dependent binding of 1 µg/ml purified annexin to a 20/80 mol% PS/PC bilayer. (C) Internalization of fluorescent annexin by apoptotic Jurkat cells in presence and absence of inhibitors of FPR1 (cyclosporin H, 1 μM) and FPR2/3 (WRW, 10 μM) as analyzed by flow cytometry. Mean fluorescence intensity (MFI) is normalized to MFI of annexin internalization on ice. (D) Hr-anxA1 internalization (green) as visualized by fluorescent confocal laser scanning microscopy (CLSM). Nuclei are stained with DAPI (blue) and PS-expression is stained with anxA5-AF568 (red). PS negative cells (indicated with white arrow) did not internalize hr-anxA1. (E) Pretreatment of PMN with 10 nM hr-anxA1 inhibited both rolling and adhesion of PMN on a TNF-α activated HUVEC monolayer. (F) One frame out of 32 is shown of a flow chamber model with rolling and adhering fluorescent THP-1 monocytes, flowing over TNF-α activated HUVEC monolayer. Rolling and adhering THP-1 monocytes are indicated by white and red arrows respectively. (G) Pretreatment of THP-1 cells with 10 nM hr-anxA1 has effect on neither rolling nor adhesion of THP-1 cells. All values are represented as mean ± SEM of 3 independent experiments.