Circulating Microparticles, Plasma Protein C and Free Protein S Levels in Children with Sickle Cell Anemia.

Abstract

Microparticles (MP) originate from blebbing and shedding from cell membrane surfaces in physiological and pathological conditions. Increased levels are generated by a number of mechanisms including platelet activation, vascular endothelial damage, thrombin activity, C5b-9 activation, and PF4-heparin-antibody interaction. Increased circulating MP have been described in patients with sickle cell anemia (SCA). Elevated monocyte-derived MP expressing tissue factor have been reported in patients in crisis. The lack of a standardised method for MP quantification remains problematic. We measured MP (numbers and functional markers), protein C and free protein S, in a large cohort of pediatric patients to investigate the role of MP in SCA and their relation to protein C and free protein S plasma levels. One hundred and eleven children of sub-Saharan African ethnicity with SCA (hemoglobin (Hb) SS) were studied: 51 without previous history of crisis in steady state (mean age 5.3 years); 15 in crisis (9 chest crises, 6 other, mean age 5.4 years); 30 on hydroxyurea (mean age 7.2 years); 15 on transfusion therapy (mean age 6.5 years); 17 children of sub-Saharan African ethnicity of similar age (mean age 4.6 years) were used as control group (Hb AA). MP were analyzed by flow cytometry, according to Biró et al (J Thromb Haemost.2004; 2(10):1842–51), using Annexin V and antibodies against, CD61, CD42a, CD62P (P-selectin), CD235a, CD14, CD142 (tissue factor), CD201 (endothelial protein C receptor or EPCR), CD62E (E-selectin), CD36 (thrombospondin or TSP-1), CD47 (TSP-1 receptor), CD31 PECAM (platelet-endothelial cellular adhesion marker), CD144 (VE-cadherin). Protein C (chromogenic) and free protein S (latex based assay) were measured in all subjects. Correlation was measured by Pearson Rank test, and comparisons between groups were analyzed by Mann-Whitney test. Total MP AV were lower in crisis (1.26 × 106/ml; 0.56–2.44 × 106) and steady state (1.35 × 106/ml; 0.71– 3.0 × 106) compared to transfusion (4.33 × 106/ml; 1.6–9.2 × 106p<0.01). Protein C levels were significantly lower in crisis (median 0.52 IU/ml; interquartile range 0.43–0.62) compared to all other groups: HbAA (0.72 IU/ml; 0.66–0.82, p<0.001); hydroxyurea (0.67 IU/ml; 0.58–0.77, p<0.001); steady state (0.63 IU/ml; 0.54–0.70, p<0.05) and transfusion (0.60 IU/ml; 0.54–0.70, p<0.05). In addition levels were significantly reduced in steady state (0.63 IU/ml; 0.54–0.70) compared to HbAA (0.72 IU/ml; 0.66–0.80, p<0.01). Protein S levels were significantly higher in HbAA (0.85 IU/ml; 0.72–0.97) compared with crisis (0.49 IU/ml; 0.42–0.64, p<0.001), hydroxyurea (0.65 IU/ml; 0.56–0.74, p<0.01), and transfusion (0.59 IU/ml; 0.47–0.71, p<0.01). There was also a significant difference in crisis patients compared to steady state (0.49 IU/ml; 0.42–0.64 v 0.68 IU/ml; 0.58–0.79, p<0.05). There was high correlation (R>0.9, p<0.05) between total numbers of Annexin V 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 to HbAA (3.2%, p<0.05); %MP CD144 was higher in crisis (7.6%) compared to transfusion (2.1%, p<0.05); %CD14 (0.77%) was higher in crisis compared to 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 to the rest (40%); %MP CD235a was higher in crisis (17.9%) compared to transfusion (8.9%), hydroxurea (8.7%) and steady state (9.9%, p<0.05); %CD62E did not differ significantly across the groups and CD142 was undetectable. These studies indicate that there are significantly lower levels of protein C and free protein S in children with sickle cell crisis. In addition there are significantly lower numbers of circulating platelet MP in steady state and crisis patients; however in crisis a significantly higher percentage of MP express markers of endothelial and vascular damage, and of red cell origin. Among these are composite hybrid microparticles expressing markers of more than one cell type, probably brought about by severe vascular stress and close contact of various circulating cell types with vascular endothelium.

The APC-EPCR-PAR1 axis in sickle cell disease

“…Dysfunction or deficiencies in the protein C—protein S system are associated with venous thrombosis ( 80 – 82 ). It is well-documented that individuals with SCD have deficiencies in both the antigen and activity levels of protein C and protein S ( 29 , 83 – 89 ), and that they are further decreased during crisis ( 90 ). Protein C and protein S levels negatively correlate with markers of coagulation activation ( 88 ).…”

“…EPCR shedding is mediated by pro-inflammatory cytokines and proteases such as TNFα converting enzyme (TACE), A Disintegrin and Metalloproteinase-10 (ADAM-10) and ADAM-17. Interestingly, EPCR shedding has been observed in individuals and mice with SCD ( 100 , 101 ), and EPCR-positive microparticles are found in the circulation of individuals with SCD ( 90 ). A recent abstract described loss of EPCR expression in the kidney vasculature and presence of soluble EPCR in the urine of aged sickle mice, a phenomenon that could also be triggered in young sickle mice by infusion of a low dose of heme to mimic an acute sickling event ( 102 ).…”