Adhesion of senescent erythrocytes to laminin-α5 in the human spleen drives ghost formation. (A) Laminin-α5 flow assay using control, old, and spleen-derived erythrocytes. Anti-Lu/BCAM blocking antibody was used to assess whether adhesion to laminin-α5 was Lu/BCAM dependent (n = 4-7, mean ± SEM). (B) Representative flow assay confocal micrograph depicting erythrocytes that interact with laminin-α5 for extended durations at low shear stresses of 0.5 dyn/cm². Erythrocyte shape abnormalities are observed after roughly 1 hour (T = 60 minutes, arrows), and ghost formation is observed after roughly 2 hours. The lower panel depicts a flow experiment in which phalloidin has been added to the flow medium. Upon ghost formation through hemolysis, the erythrocyte membrane becomes permeable, leading to intracellular F-actin staining by phalloidin. (C) Correlation between adhesion of fresh, control, and neuraminidase-treated erythrocytes to laminin-α5 and the rate at which ghost formation is observed. (D-G). Laminin-α5 localization with respect to the white pulp (WP), red pulp (RP), the RP sinus (S) CD163⁺ cells and trabeculae (T). Continuous VE-cadherin distribution (arrowhead) was observed on protruding veins but not within the sinuses. (H) A 2-photon micrograph showing laminin-α5 distribution in the splenic sinusoids of the RP and their interaction (white arrows) with erythrocytes (glycophorin-A, CD235a) up to 100 µm deep within an intact piece of human spleen. (I) Flow cytometric gating strategy and quantification of nucleated cells that interact with laminin-α5 (Hoechst⁺), erythrocytes (Hoechst⁻CD235a⁺), and other cells (Hoechst⁻ CD235a⁻) from human spleen. (J) Hemoglobin content of laminin-α5 MACS-isolated erythrocytes was assessed by using benzidine staining. Hemoglobin leakage was observed as indicated by the arrows. (K) Quantification of laminin-α5 positive and negative erythrocyte ghosts within human spleen tissue. *P < .05; **P < .01 (mean ± SD).