Hemolysate induced HUVEC dysfunction. HUVECs cultured in a flow system were exposed to either AA hemolysate, TNFα, or serum for 4 hours. Functional assays were performed by infusing heparinized WB on hemolysate-preconditioned HUVECs for 10 minutes. The infusion flow rate in the fluidic model was adjusted to reach a shear stress of 1 dyne·cm⁻². (A) RBC adhesion after infusion of heparinized WB from AA donors on either AA hemolysate, TNFα, or serum-preconditioned HUVECs for 10 minutes at shear stress 1 dyne·cm⁻². Data represent mean ± SEM (n = 8), the Kruskal-Wallis test with ∗P < .05 and ∗∗∗P < .001. (B-C) Platelet aggregation and activation at endothelial injury sites after infusion of heparinized WB from AA donors on either AA hemolysate, TNFα or serum-preconditioned HUVECs for 10 minutes at shear stress 1 dyne·cm⁻². (C) Fixed cells were stained with PECAM-1 FITC (green), CD41a PE (orange), and CD62P (mouse antihuman primary antibody and Alexa Fluor 647 goat antimouse secondary antibody, red). Data represent mean ± SEM (n = 5), the Kruskal-Wallis test with ∗P < .05 and ∗∗∗P < .001. (D) Inhibitory effect of integrilin on platelets aggregation. HUVECs cultured in a flow system were exposed to AA hemolysate for 4 hours. AA heparinized WB was pretreated or not with integrilin, a GPIIbIIIa antagonist, at 10 μg/mL for 30 minutes before infusion on hemolysate-preconditioned HUVECs for 10 minutes. The infusion flow rate in the fluidic model was adjusted to reach a shear stress of 1 dyne·cm⁻². After the washing step, fixed cells were stained with PECAM-1 FITC (green) and CD41a PE (orange). Images representative of 5 different experiments, data represent mean ± SEM (n = 5) (∗∗) paired t test with P < .01. FITC, fluorescein isothiocyanate.