Heme-mediated stasis and P-selectin and VWF expression require NOX, PKC, and oxidants. (A) Thirty minutes prior to infusion of heme (3.2 µmol/kg), NY1DD mice (n = 3 per treatment) were injected with NOX inhibitors apocynin (10 mg/kg, IP) or DPI (30 mg/kg, IP), the PKC inhibitor calphostin C (100µg/kg, IP), the antioxidant NAC (2.4 mg/kg, IP), or the iron chelator S-DFO (6 mL/kg, IV, every third day × 4). Controls include vehicle (0.012 mL/g dimethylsulfoxide [DMSO], IP) or hetastarch (6 mL/kg, IV, every third day × 4). Percent stasis was measured using intravital microscopy 1 hour after heme infusion. Bars are mean percent stasis + SD with mean stasis values written above the bars. *P < .05 vs control. (B) HUVECs were pretreated 30 minutes with apocynin (1 mM) or calphostin C (300 nm) followed by the addition of heme (10 µM) for 15 minutes. Control cells were untreated. Cells were fixed and immunostained for VWF (red) on the cell surface. Nuclei were counterstained with DAPI (blue). (C) Heme-induced oxidative stress was measured in HUVECs. Confluent HUVECs in 1% FBS were pretreated for 1 hour with vehicle (DMSO), apocynin (10 mM), DPI (20 µM), calphostin C (200 nM), NAC (10 mM), quercetin (50 µM), or DFO (100 µM). After 1 hour, cells were washed and loaded with the cell-permeable ROS probe DCFH-DA (100 µM) for 30 minutes. After 30 minutes, cells were washed and incubated with heme (20 µM) or LPS (20 ng/mL) in 0.1% FBS media for 4 hours. After 4 hours, cells were washed and ROS production was measured as accumulated cell fluorescence from oxidized DCFH-DA. Values are means + SD (n = 4 or 5 wells per treatment).