Effect of factor VIII–deficient plasma on VE-cadherin distribution on ECs.

HUVECs were grown on fibronectin-coated glass cover slides, stained for VE-cadherin and F-actin, and visualized by double immunofluorescence (original magnification × 40) as described in “Materials and methods.” Overlays show F-actin in red and VE-cadherin in green; colocalization appears in yellow. (A-C) Staining for VE-cadherin and F-actin in untreated cells. VE-cadherin and F-actin staining of ECs exposed to factor VIII–deficient plasma without TNF (D-F), TNF alone (G-I), factor VIII–deficient plasma on ECs pretreated with TNF (J-L), factor VIII–deficient plasma pretreated with TNF in the presence of AT III (M-O), and AT III alone (P-R). Slides from a 90-minute time point are shown. Note the gaps between the adjacent ECs (white arrows) and the lack of VE-cadherin (white arrowheads) only on ECs exposed to factor VIII–deficient plasma pretreated with TNF (J-L). Preabsorbtion of anti–VE-cadherin antibody with an appropriate peptide abolished the VE-cadherin staining (S).