Erythrocyte PS exposure and VWF-binding in SCD patients treated with hydroxycarbamide compared to blood transfusion. Comparisons between the HC and BT cohorts are shown for (A) LDH levels and (B) free heme levels. Comparisons between groups were assessed by the Mann-Whitney U test. (C) Washed RBCs were prepared from children with SCD on HC or BT therapy, and flow cytometry used to assess PS exposure, together with VWF-binding capacity. (D) Representative examples of annexin V binding for washed RBCs from a healthy (HbAA) control compared with children with SCD on either HC or BT treatment. (E) Data are presented as mean of the mean fluorescence intensity (MFI) of PS exposure on RBCs normalized to control for HC and BT subgroups. Statistical analyses were performed using the Mann-Whitney U test. (F) Representative examples of recombinant VWF (rVWF) binding for washed RBCs from a healthy (HbAA) control compared with children with SCD on either HC or BT treatment. (G) Data are presented as mean MFI of rVWF binding to washed RBCs normalized to control for HC and BT subgroups. Statistical analyses were performed using the Mann-Whitney U test. (H) To determine whether VWF interacts with sickle RBCs in vivo, unwashed RBCs from children on BT and HC treatment were isolated, and bound VWF assessed by flow cytometry. Statistical analyses were performed using the Mann-Whitney U test with a significant P value < .05; ∗∗∗P < .001 and ∗∗∗∗P < .0001.