Binding and uptake of FVIII by conditioned DMVECs. (A) Confluent hDMVECs were pretreated with 100 nM IFN-γ (48 hours) and 100 nM TNF-α (24 hours) prior to incubation for 12 hours rhfF8–Alexa 488 (10 U/mL; in the presence of 1 U/mL VWF and 150 µg/mL FIB) together with Lysotracker Red (late endosome/lysosome marker; 50 nM). Cells were then rinsed and imaged live. Fluorescence microscopy showed significant rhfF8–Alexa 488 (green) accumulated into perinuclear punctae, which partially colocalized with late endosomes/lysosomes (red) as seen by the yellow signal in the merged image. This was validated by computational image analysis, which also identified substantial areas of rhfF8-488 and Lysotracker Red coincidence, as depicted by white pixels in the Costes mask (right panel) and reflected in an average Pearson correlation coefficient of 0.680 ± 0.033 (r² > 0.98; Costes P = 100%). Scale bar, 25 μm. (B) As a complementary and more sensitive approach to detect FVIII binding and uptake, rhfF8 was coated onto GMA-8021 (anti-FVIII MAb)-conjugated carboxylate-modified red fluorescent nanoparticles (rhfF8 fluorospheres). The same fluorescent particles were subjected to blocking of the carboxylate modification with 2% BSA to demonstrate the level of nonspecific binding and cellular uptake (Control fluorospheres). Fluorospheres were then incubated together with 150 µg/mL fibrinogen in the presence (not shown) or absence of 1 U/mL VWF for 1.5 hours on IFN-γ– and TNF-α–conditioned mDMVECs. Differential interference contrast (DIC) and confocal fluorescence images were superimposed. Arrowheads indicate patches of putative membrane-bound rhfF8. Arrows indicate putatively internalized perinuclear clusters of rhfF8 that presumably represent endosomes and lysosomes (left panel). The same pattern was observed in the presence of VWF. In the absence of rhfF8 coating, control fluorospheres showed very little EC binding or uptake (right panel, arrows). Scale bar, 20 μm.