title: Circulating microparticles, protein C, free protein S and endothelial vascular markers in children with sickle cell anaemia.
abstract: BACKGROUND
  Circulating microparticles (MP) have been described in sickle cell anaemia (SCA); however, their interaction with endothelial markers remains unclear. We investigated the relationship between MP, protein C (PC), free protein S (PS), nitric oxide (NO), endothelin-1 (ET-1) and adrenomedullin (ADM) in a large cohort of paediatric patients.
  
  
  METHODS
  A total of 111 children of African ethnicity with SCA: 51 in steady state; 15 in crises; 30 on hydroxyurea (HU) therapy; 15 on transfusion; 17 controls (HbAA) of similar age/ethnicity. MP were analysed by flow cytometry using: Annexin V (AV), CD61, CD42a, CD62P, CD235a, CD14, CD142 (tissue factor), CD201 (endothelial PC receptor), CD62E, CD36 (TSP-1), CD47 (TSP-1 receptor), CD31 (PECAM), CD144 (VE-cadherin). Protein C, free PS, NO, pro-ADM and C-terminal ET-1 were also measured.
  
  
  RESULTS
  Total MP AV was lower in crisis (1.26×10(6) ml(-1); 0.56-2.44×10(6)) and steady state (1.35×10(6) ml(-1); 0.71-3.0×10(6)) compared to transfusion (4.33×10(6) ml(-1); 1.6-9.2×10(6), p<0.01). Protein C levels were significantly lower in crisis (median 0.52 IU ml(-1); interquartile range 0.43-0.62) compared with all other groups: HbAA (0.72 IU ml(-1); 0.66-0.82, p<0.001); HU (0.67 IU ml(-1); 0.58-0.77, p<0.001); steady state (0.63 IU ml(-1); 0.54-0.70, p<0.05) and transfusion (0.60 IU ml(-1); 0.54-0.70, p<0.05). In addition, levels were significantly reduced in steady state (0.63 IU ml(-1); 0.54-0.70) compared with HbAA (0.72 IU ml(-1); 0.66-0.80, p<0.01). PS levels were significantly higher in HbAA (0.85 IU ml(-1); 0.72-0.97) compared with crisis (0.49 IU ml(-1); 0.42-0.64, p<0.001), HU (0.65 IU ml(-1); 0.56-0.74, p<0.01) and transfusion (0.59 IU ml(-1); 0.47-0.71, p<0.01). There was also a significant difference in crisis patients compared with steady state (0.49 IU ml(-1); 0.42-0.64 vs. 0.68 IU ml(-1); 0.58-0.79, p<0.05). There was high correlation (R>0.9, p<0.05) between total numbers of AV-positive MP (MP AV) and platelet MP expressing non-activation platelet markers. There was a lower correlation between MP AV and MP CD62P (R=0.73, p<0.05) (platelet activation marker), and also a lower correlation between percentage of MP expressing CD201 (%MP CD201) and %MP CD14 (R=0.627, p<0.001). %MP CD201 was higher in crisis (11.6%) compared with HbAA (3.2%, p<0.05); %MP CD144 was higher in crisis (7.6%) compared with transfusion (2.1%, p<0.05); %CD14 (0.77%) was higher in crisis compared with transfusion (0.0%, p<0.05) and steady state (0.0%, p<0.01); MP CD14 was detectable in a higher number of samples (92%) in crisis compared with the rest (40%); %MP CD235a was higher in crisis (17.9%) compared with transfusion (8.9%), HU (8.7%) and steady state (9.9%, p<0.05); %CD62E did not differ significantly across the groups and CD142 was undetectable. Pro-ADM levels were raised in chest crisis: 0.38 nmol L(-1) (0.31-0.49) versus steady state: 0.27 nmol L(-1) (0.25-0.32; p<0.01) and control: 0.28 nmol L(-1) (0.27-0.31; p<0.01). CT-proET-1 levels were reduced in patients on HU therapy: 43.6 pmol L(-1) (12.6-49.6) versus control: 55.1 pmol L(-1) (45.2-63.9; p<0.05). NO levels were significantly lower in chest crisis (19.3 mmol L(-1) plasma; 10.7-19.9) compared with HU (22.2 mmol L(-1) plasma; 18.3-28.4; p<0.05), and HbSC (30.6 mmol L(-1) plasma; 20.8-39.5; p<0.05) and approach significance when compared with steady state (22.5mmol L(-1) plasma; 16.9-28.2; p=0.07).
  
  
  CONCLUSIONS
  Protein C and free PS are reduced in crisis with lower numbers of platelet MP and higher percentage of markers of endothelial damage and of red cell origin. During chest crisis, ADM and ET-1 were elevated suggesting a role for therapy inhibiting ET-1 in chest crisis.
authors.affiliation: Irish Blood Transfusion Service, Dublin, Ireland.|Our Lady's Children Hospital, Dublin, Ireland.|Trinity College Dublin, Dublin, Ireland.|Haematology Department, San Maurizio Regional Hospital, Bolzano, Italy; apiccin@gmail.com.
authors.forename: Andrea
authors.initials: A
authors.lastname: Piccin
authors.affiliation: Irish Blood Transfusion Service, Dublin, Ireland.
authors.forename: Ciaran
authors.initials: C
authors.lastname: Murphy
authors.affiliation: Irish Blood Transfusion Service, Dublin, Ireland.
authors.forename: Elva
authors.initials: E
authors.lastname: Eakins
authors.affiliation: B.R.A.H.M.S. AG, Biotechnology Centre, Berlin, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Kunde
authors.affiliation: Haematology Department, San Maurizio Regional Hospital, Bolzano, Italy.|Pathology Department, San Maurizio Regional Hospital, Bolzano, Italy.
authors.forename: Daisy
authors.initials: D
authors.lastname: Corvetta
authors.affiliation: Haematology Department, San Maurizio Regional Hospital, Bolzano, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Di Pierro
authors.affiliation: Pathology Department, San Maurizio Regional Hospital, Bolzano, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Negri
authors.affiliation: Pathology Department, San Maurizio Regional Hospital, Bolzano, Italy.
authors.forename: Mazzoleni
authors.initials: M
authors.lastname: Guido
authors.affiliation: Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera, University of Medicine of Padova, Padova, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Sainati
authors.affiliation: Our Lady's Children Hospital, Dublin, Ireland.
authors.forename: Corrina
authors.initials: C
authors.lastname: Mc Mahon
authors.affiliation: Our Lady's Children Hospital, Dublin, Ireland.|Trinity College Dublin, Dublin, Ireland.
authors.forename: Owen Patrick
authors.initials: OP
authors.lastname: Smith
authors.affiliation: Irish Blood Transfusion Service, Dublin, Ireland.|University College Dublin, Dublin, Ireland.
authors.forename: William
authors.initials: W
authors.lastname: Murphy
keywords: adrenomedullin; endothelin-1; microparticles; nitric oxide; protein C; protein S; sickle cell anaemia