Background: Secretory phospholipase A2 is an enzyme that is elevated in SCD patients with acute chest syndrome (ACS) and vaso-occlusive pain crisis (VOC) and inhibition of its enzymatic activity is in trials for ACS prevention (

Styles LA. Blood. 1996; 87:2573
). This enzyme cleaves arachidonic acid from the phospholipids of red blood cell (RBC) membranes; and arachidonic acid can be converted to other pro-inflammatory lipid compounds (
Murakami M. J Biochem. 2002; 131:285
). sPLA2 cleaves phosphatidylserine (PS) expressing lipid membranes. PS is usually in the inner leaflet (unexposed) in normal red blood cells (RBCs) but becomes externalized in sickle RBCs especially during VOC or ACS. There is modest exposure of PS during in the steady state among certain RBC populations (
de Jong K. Br J Haematol. 2006; 133:427
). PS exposure allows sPLA2 to cleave RBC membranes, producing lipids which prime neutrophils (PMNs) and cause PMN-mediated pulmonary endothelial cell injury as the second event in a two-event model (Ball JB, Blood submitted).

Objective: We hypothesize that sickle RBCs treated with hydroxyurea (HU) will be more resistant to digestion by sPLA2, thereby inhibiting the release of bioactive lipids.

Design/Methods: Whole blood was collected from children with SCD when healthy or daily during admissions for VOC or ACS. The plasma and RBCs were separated. Plasma and lipids extracted from the plasma were used as priming agents of quiescent PMNs isolated from healthy donors. Additionally, the separated RBCs were treated with exogenous sPLA2, creating sPLA2-liberated lipids, which were also used as priming agents. The plasma sPLA2 levels were measured.

Results: There was no difference in the sPLA2 levels of untreated and HU-treated SCD patients in the healthy condition (non-treated SCD: 12.8±3.1 ng/ml, HU: 12.2±4.2 ng/ml, p=0.95) and in VOC (non-treated SCD: 89.2 ± 79.3 ng/ml, HU: 86.5 ± 44.3 ng/ml, p=0.85). The PMN priming of the sPLA2-liberated lipids of untreated SCD patients, both healthy (*, Table 1) and those with VOC (#, Table 1), was elevated compared to the sPLA2-liberated lipids from HU-treated patients. We conclude that hydroxyurea treatment in vivo induces resistance to sPLA2 cleavage of RBC membrane lipids thereby inhibiting priming of PMNs, which has been implicated in PMN-mediated endothelial cell injury (Ball JB, Blood submitted).

Table 1: PMN priming activity of plasma lipids and sPLA2-liberated lipids from patients with SCD.

HealthyVOC pain
plasma lipidssPLA2-liberated lipidsplasma lipidssPLA2-liberated lipids
* - p<0.01 compared to untreated plasma lipids from healthy untreated SCD patients 
# - p<0.01 compared to untreated plasma lipids from untreated SCD patients with VOC 
Hgb AA 2.55 ± 0.62 nmol O2-/min 1.94 ± 0.20 nmol O2-/min   
untreated SCD 2.21 ± 0.17 nmol O2-/min 3.19 ± 0.25 * nmol O2-/min 2.73 ± 0.24 nmol O2-/min 3.99 ± 0.28 # nmol O2-/min 
HU-treated SCD 2.34 ± 0.47 nmol O2-/min 2.75 ± 0.49 nmol O2-/min 2.23 ± 0.4 nmol O2-/min 2.69 ± 0.52 nmol O2-/min 
HealthyVOC pain
plasma lipidssPLA2-liberated lipidsplasma lipidssPLA2-liberated lipids
* - p<0.01 compared to untreated plasma lipids from healthy untreated SCD patients 
# - p<0.01 compared to untreated plasma lipids from untreated SCD patients with VOC 
Hgb AA 2.55 ± 0.62 nmol O2-/min 1.94 ± 0.20 nmol O2-/min   
untreated SCD 2.21 ± 0.17 nmol O2-/min 3.19 ± 0.25 * nmol O2-/min 2.73 ± 0.24 nmol O2-/min 3.99 ± 0.28 # nmol O2-/min 
HU-treated SCD 2.34 ± 0.47 nmol O2-/min 2.75 ± 0.49 nmol O2-/min 2.23 ± 0.4 nmol O2-/min 2.69 ± 0.52 nmol O2-/min 
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Topics:
child, hydroxyurea, phospholipases a, sickle cell anemia, tissue membrane, catabolism, lipids, arachidonic acid, enzymes, acute chest syndrome

Disclosures: No relevant conflicts of interest to declare.

Author notes

Corresponding author

2008, The American Society of Hematology
2008
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