Multiple EC adhesion molecules are required for stasis including mobilization of WPB constituents P-selectin and VWF to the EC surface. (A) Blocking antibodies to adhesion molecules P-selectin, E-selectin, VWF, VCAM-1, α4β1 integrin, ICAM-1, PECAM-1, αVβ3 integrin, thrombomodulin, or control IgG (30 µg per mouse, IV) were infused into NY1DD sickle mice with DSFCs 5 minutes prior to infusion of heme (3.2 µmol/kg). Percent stasis was measured at 1 hour after heme infusion as described in Figure 1. *P < .001 vs control IgG and #P < .001 antithrombomodulin vs anti-P-selectin, E-selectin, VWF, VCAM-1, α4β1 integrin, ICAM-1, PECAM-1, or αVβ3 integrin. (B) HUVECs were fixed in 4% paraformaldehyde and stained for surface P-selectin (green) or VWF (red) after the indicated incubations. Nuclei were counterstained with DAPI (blue). White bars represent 40 µm. Representative cells are shown. Top row: HUVECs in 0.1% FBS were incubated with heme (20 µM) for 15 minutes, HbA or HbS (20 µM heme) for 24 hours, or HbA for 24 hours followed by heme for 15 minutes. Middle row: HUVECs were incubated with heme + hemopexin (20 μM), HbA + haptoglobin (20 µM), or HbA + hemopexin for 24 hours. Bottom row: HUVECs were incubated for 24 hours in 0.1% FBS with or without HbA or HbS followed by H/R (5% CO₂/95% N₂ for 3 hours and 5% CO₂/95% air for 1 hour). Control cells were incubated in 0.1% FBS for 24 hours. Cells were not permeabilized; all pictures represent cell-surface expression. (C) Normal C57BL/6 and NY1DD sickle mice were infused with saline (12 mL/kg, negative control), histamine (1200 µmol/kg, positive control), or heme (3.2 µmol/kg). Fifteen minutes after infusion, mice were sacrificed in CO₂ and dorsal skin (n = 1/treatment) was removed and fixed in Zamboni’s for immunofluorescence staining of VWF (red) and counterstaining of nuclei with DAPI (blue). White bars represent 30 µm. Representative venules are shown.