Topology-related assembly of prothrombinase complexes on TNF-α–activated HUVECs. Subconfluent HUVECs were treated with TNF-α (50 ng/mL, 4 hours) and then incubated with factor Va-Alexa 488, 10 nM (green) and 10 nM factor Xa-647 (red). They were then imaged, live, with differential interference contrast and confocal fluorescence. (A-B) Representative wide and enlarged views of a field of HUVECs with low levels of factor Va and Xa binding. Thin cellular projections did not have visible staining (arrow). (C-D) Representative wide and enlarged views of a field of HUVECs containing colocalized factors Va and Xa on filaments and filopodialike structures at the cell periphery. (E-F). Representative wide and enlarged views of a field of HUVECs showing colocalized binding of factor Va and Xa on punctate structures of the cell body. (G) HUVECs were cultured in 96-well plates in the presence of TNF-α. Prothrombinase activity was measured in a discontinuous assay with a chromogenic thrombin substrate. Prothrombinase activity was inhibited to a greater degree by lactadherin compared with annexin A5. Data are mean ± standard deviation for 2 experiments, each performed in triplicate.