Blood Transfusion Ameliorates Free Haem-Induced Loss of Thrombomodulin and Endothelial Cell Activation in Paediatric Sickle Cell Anaemia

Background Sickle cell anaemia (SCA) is responsible for major paediatric morbidity including risk of stroke in up to 10% of children. Mainstays of therapy include hydroxyurea (HU) and chronic blood transfusion (BT), with BT considered gold standard for stroke prevention. However, the biological mechanisms through which BT is significantly superior to HU in reducing stroke risk have not been clearly defined. SCA complications are due in part to free haem-mediated damage, leading to endothelial cell (EC) activation, tissue factor (TF) expression and release of von Willebrand Factor (VWF), which binds platelets and sickle RBCs. Recently loss of EC Thrombomodulin (TM) has been shown to promote EC activation and VWF release and soluble TM (sTM) plays a role in disseminated intravascular coagulation and COVID-19.

Hypothesis We hypothesize free haem alters EC TM expression, promoting EC activation and VWF release. Intravascular hemolysis and free haem may be reduced in children on chronic BT, maintaining EC quiescence and enhancing protection against stroke compared with HU.

Methods Following ethical approval and informed consent 180 SCA children (median age 12 years, 49% male) were recruited. Steady-state plasma samples were collected prior to BT (n=84) or at outpatient clinic for HU (n=96). Paired crisis samples were obtained in a subset of HU (n=30). EC activation markers and Weibel Palade Body (WPB) proteins were evaluated by ELISA, VWF multimers were assessed by 1.8% agarose gels and ADAMTS13 activity assessed by FRET. Free haem levels were quantified and thrombin generation (TG) assays performed. Factor VIII:C was measured in the National Coagulation Centre at St James's Hospital. Human umbilical vein endothelial cells (HUVEC) were exposed to cell-free haem (Hemin), hydroxyurea, cytokines (IL-1β and TNFα) or patient plasma; VWF was measured in the supernatant and HUVEC TM expression assessed by flow cytometry.

Results VWF:Ag and VWF propeptide (VWFpp) levels were significantly increased in HU compared to BT children, suggesting acute EC activation (median plasma VWF:Ag levels 1.29 v 1.03 IU/ml; p=0.0005, median VWFpp levels 1.63 v 1.17 IU/ml p=0.006). Furthermore, plasma levels of other WPB stored proteins were also significantly elevated in the HU cohort, including Angiopoietin-2 (Ang-2) (4214 v 2337 pg/ml, p<0.0001 and Osteoprotegerin (OPG) (1825 v 1507 pg/ml p=0.006). Abnormalities of high-, intermediate- and low molecular weight VWF multimers were noted in HU but not BT. While putative inhibitors of ADAMTS13 were increased in HU, including Platelet Factor 4 and Interleukin-6, ADAMTS13 activity did not differ between groups. FVIII:C levels were significantly elevated in 65% of the total cohort and were higher in HU than BT (1.62 IU/ml v 1.30 IU/ml p<0.0001). Free haem levels were increased in HU versus BT (12.5 v 9.5μM p=0.0003), as well as levels of LDH and reticulocyte %, pointing to increased intravascular hemolysis. EC markers and FVIII:C correlated strongly with hemolytic indices. In addition to increased EC activation, increased endogenous thrombin potential (ETP), peak thrombin and velocity index were observed in HU versus BT, pointing to enhanced TG, possibly driven by elevated circulating TF. TG parameters correlated with plasma FVIII:C levels. Exposure of HUVEC to free haem in vitro resulted in a significant decrease in surface TM expression and increased VWF secretion (p<0.05). Conversely exposure to hydroxyurea or cytokines did not alter TM expression. Exposure of HUVEC to plasma from crisis but not paired steady-state plasma induced EC activation and VWF release compared with control plasma. Importantly, free haem levels ≥20 μM were observed in 25% HU children in steady state and 40% in crisis, compared with 0% of BT. Furthermore, significantly elevated sTM levels in SCA children with a history of cerebrovascular disease compared to those without (5.7 v 4.6 ng/ml p=0.02), suggesting that sTM may predict end-organ dysfunction.

Conclusions We have shown in a large cohort of SCA children that EC activation, TG and intravascular haemolysis are increased in HU-treated children compared with BT. Free haem induces loss of EC TM and VWF release in vitro, leading to EC activation. Elevated sTM levels were a biomarker of end-organ damage. These data suggest that reduction of free haem in children undergoing regular BT is a key mechanism through which EC quiescence is maintained in paediatric SCA.

Karampini:CSL Behring: Research Funding. O'Donnell:J.S.O'D has served on the speaker's bureau for Baxter, Bayer, Novo Nordisk, Sobi, Boehringer Ingelheim, Leo Pharma, Takeda and Octapharma. He has also served on the advisory boards of Baxter, Sobi, Bayer, Octapharma CSL Behring, Daiichi Sankyo, Boehringer: Research Funding, Speakers Bureau.