Figure 6.
Binding to ICAM-1 and EPCR Increases Adhesion of Infected Erythrocytes
(A–F) Erythrocytes infected with (A and D) HB3VAR03 (predicted ICAM-1 and EPCR binder), (B and E) 3D7PFD1235w (predicted ICAM-1 and EPCR binder), and (C and F) IT4VAR13 (ICAM-1 and CD36 binder) binding to (A–C) receptor-coated microslides under flow conditions using recombinant ICAM-1, rEPCR, rICAM-1, and rEPCR; rCD36; or rICAM-1 and rCD36, and to (D–F) resting human brain microvascular endothelial cells (HBMEC). To demonstrate specific adhesion, channels coated with HBMEC were pre-incubated with anti-ICAM-1 (40 μg/mL), anti-EPCR (10 μg/mL), or anti-ICAM1 and EPCR combined (40 and 10 μg/mL, respectively). Flow experiments were done in parallel with the same conditions for immobilizing proteins or cell-coated chips. Values are bound infected erythrocyte per mm2 ± SEM. Data were also analyzed by two-way ANOVA with Tukey’s multiple comparison (Tables S7A and S7B).
(G–I) Immunofluorescence images of erythrocytes infected with parasite strains (G) HB3VAR03, (H) 3D7PFD1235w, and (I) IT4VAR13. Complexes of ICAM-1 or EPCR were added to measure binding by immunofluorescence. Main panels show overlays of ICAM-1 (green), EPCR (red), and nuclear (blue) staining. Inserts show single channels. Scale bar, 2 μm.