VWF-ADAMTS13 axis dysfunction in children with sickle cell disease treated with hydroxycarbamide vs blood transfusion

Abstract

Previous studies have reported elevated von Willebrand factor (VWF) levels in patients with sickle cell disease (SCD) and demonstrated a key role for the VWF-ADAMTS13 axis in the pathobiology of SCD vaso-occlusion. Although blood transfusion is the gold standard for stroke prevention in SCD, the biological mechanisms underpinning its improved efficacy compared with hydroxycarbamide are not fully understood. We hypothesized that the improved efficacy of blood transfusion might relate to differences in VWF-ADAMTS13 axis dysfunction. In total, 180 children with a confirmed diagnosis of SCD (hemoglobin SS) on hydroxycarbamide (n = 96) or blood transfusion (n = 84) were included. Despite disease-modifying treatment, plasma VWF and VWF propeptide were elevated in a significant proportion of children with SCD (33% and 47%, respectively). Crucially, all VWF parameters were significantly higher in the hydroxycarbamide compared with the blood transfusion cohort (P < .05). Additionally, increased levels of other Weibel-Palade body-stored proteins, including factor VIII (FVIII), angiopoietin-2, and osteoprotegerin were observed, indicated ongoing endothelial cell activation. Children treated with hydroxycarbamide also had higher FVIII activity and enhanced thrombin generation compared with those in the blood transfusion cohort (P < .001). Finally, hemolysis markers strongly correlated with VWF levels (P < .001) and were significantly reduced in the blood transfusion cohort (P < .001). Cumulatively, to our knowledge, our findings demonstrate for the first time that despite treatment, ongoing dysfunction of the VWF-ADAMTS13 axis is present in a significant subgroup of pediatric patients with SCD, especially those treated with hydroxycarbamide.

Graphical abstract

Figure 1.

Plasma VWF levels in children with SCD treated with hydroxycarbamide compared to blood transfusion. Comparisons between children with SCD treated with HC or BT are shown for plasma levels of (A) VWF:Ag, (B) VWF:CB, and (C) VWFpp. (D) VWF:Ag levels are shown comparing group O and non-O blood groups. Dotted red lines denote the upper and lower limit of the local reference range, with green shaded area falling within normal limits. Data are presented as median and the IQR. Comparisons between groups were assessed by the Mann-Whitney U test. Correlations between plasma VWF:Ag levels and (E) Hb and (F) LDH are shown. Correlations were evaluated using the Spearman rank correlation test; ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.

Figure 2.

VWF multimer distribution in children with SCD treated with hydroxycarbamide compared to blood transfusion. (A) Representative images of VWF multimer distribution in HC and BT cohorts. Percentage of (B) high-, (C) intermediate-, and (D) low-molecular-weight VWF multimers. Comparisons between groups were assessed by the Mann-Whitney U test. Dotted red lines represent the percentage of high-, intermediate-, and low-molecular-weight multimers in normal pooled plasma (NPP); ∗P < .05, ∗∗∗P < .001, and ∗∗∗∗P < .0001.

Figure 3.

ADAMTS13 activity in SCD children treated with hydroxycarbamide compared to blood transfusion. (A)ADAMTS13 activity and (B) VWF:Ag/ADAMTS13 ratio are compared between the HC and BT cohorts. Dotted green line represents the local normal VWF:Ag/ADAMTS13 ratio (1.0). Plasma levels of PF4 (C) and IL-6 (D) are compared between the HC and BT cohorts. Comparisons between groups were assessed by the Mann-Whitney U test; ns, not significant; ∗∗P < .01 and ∗∗∗P < .001.

Figure 4.

VWF-ADAMTS13 axis and Weibel Palade body exocytosis in SCD children treated with hydroxycarbamide compared to blood transfusion. Comparisons between HC and BT cohorts are shown including (A) VWFpp/VWF:Ag ratio and (B) FVIIl:C levels. (C) Correlation between FVIII:C and VWF:Ag. (D) Comparisons between HC and BT cohorts are shown for FVIII:C/VWF:Ag ratio, (E) angiopoietin-2, and (F) osteoprotegerin. Comparisons between groups were assessed by the Mann-Whitney U test. Correlations were evaluated using the Spearman rank correlation test; ns, not significant; ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.

Figure 5.

Thrombin generation in children with SCD treated with hydroxycarbamide compared to blood transfusion. Comparisons between the HC and BT cohorts are shown including (A) thrombin generation curves from representative individuals within each cohort; (B) the quantitative parameters of endogenous thrombin potential, (C) peak thrombin, and (D) velocity index. Comparisons between groups were assessed by the Mann-Whitney U test. (E) Correlation between plasma FVIII:C levels and peak thrombin generated. Correlations were evaluated using the Spearman rank correlation test; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, and ∗∗∗∗P < .0001.

Figure 6.

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.

Figure 7.

Representation of proposed multifactorial mechanisms through which HC or BT disease-modifying therapies affect endothelial cell activation VWF–ADAMTS13 axis dysfunction in children with SCD. Key steps highlighted in red. (1) Significantly higher free heme levels were seen in children treated with HC compared with those treated with BT. (2) Plasma concentrations of free heme present in children treated with HC were sufficient to drive Weibel-Palade body exocytosis from human umbilical vein endothelial cells in vitro. (3) LDH and free heme were both independently associated with plasma VWF levels, suggesting that rate of hemolysis and amount of free heme can affect endothelial cell activation. (4) High-molecular-weight VWF multimers were significantly reduced in children treated with HC, consistent with ongoing high-molecular-weight VWF multimers consumption. (5) Although modified by ABO blood group, increased VWF levels were predominantly attributable to increased endothelial cell secretion. (6) FVIII:C levels were significantly higher in children treated with HC, driving enhanced thrombin generation. (7) Plasma levels of VWFpp, angiopoetin-2, and osteoprotegerin were all significantly elevated in children treated with HC. Downstream consequences are currently unknown. (8) Mediation analysis highlighted feedback mechanism through which increased plasma VWF levels may directly promote hemolysis.