Stabilin-1 and stabilin-2 in HSECs mediate tethering of damaged RBCs in a PS-dependent manner. (A) Representative images of binding of damaged RBCs (DR) or normal RBCs (NR) in HSECs. Scale bar, 10 μm. (B) Microscopic quantification of binding or engulfment of normal or damaged RBCs in HSECs. Binding or phagocytosis index was determined based on the number of bound or engulfed RBCs per HSEC. Results represent mean ± SD from at least 3 experiments; t test: *P < .01 between DR and NR. (C) Binding of damaged RBCs by HSECs in the presence of various phospholipid liposomes (10μM). PC indicates phosphatidylcholine; PS, phosphatidylserine; PE, phosphatidylethanolamine; and PI, phosphatidylinositol; t test: *P < .01 versus untreated control. (D) HSECs were incubated with normal of damaged RBCs in serum-free medium containing 5mM CaCl₂, and binding index was then determined; t test: *P < .01 between DR and NR. (E) HSECs were incubated with damaged RBCs in the presence of 3 different concentrations (0.1, 1, or 10μM) of PS liposomes. Binding assays were preformed under serum-free conditions, and the binding index of damaged RBCs was determined. (F) HSECs were preincubated with PS liposomes (10μM) during the indicated times and then incubated with damaged RBCs for 1 hour. Binding assays were preformed under serum-free conditions, and binding index of damaged RBCs was determined. (G) Expression levels of PS recognition receptors in HSECs were analyzed by quantitative real-time PCR. (H) HSECs were incubated with NBD-PC–labeled PS or PC beads (green) for 1 hour at 37°C. The cells were fixed and stained with antibodies directed against stabilin-1 (yellow) and stabilin-2 (red). The white arrowhead indicates the bound PC bead. (I) Down-regulation of stabilin-1 and stabilin-2 expression decreased damaged RBC binding in HSECs. Results represent mean ± SD from at least 3 experiments; t test: *P < .01 versus untreated control.