Platelet, leukocyte and endothelial cell eicosanoid formation in two patients possessing a homozygous mutation in the cytosolic phospholipase A2 α gene

Nicholas Kirkby1,2, Matthew Edin3, Ginger Milne4, Daniel Reed2, Francesca Rauzi1, Hilary Longhurst1, Daryl Zeldin3, Jane Mitchell2, Timothy Warner1. 1Barts & the London School of Medicine, London, UK, 2Imperial College London, London, UK, 3National Institute of Environmental Health Sciences, North Carolina, USA, 4Vanderbilt University, Tennessee, USA

Activated platelets, leukocytes and endothelial cells synthesise eicosanoid mediators such as thromboxane (TX)A2, prostaglandin (PG)E2 and prostacyclin (PGI2) which are crucial for the regulation of thrombosis and inflammation. This requires arachidonic acid release from membrane phospholipids, a process catalysed by phospholipase (PL)A2 enzymes. Although 19 PLA2 isoforms have been identified, only cytosolic PLA2 α (cPLA2 α) has specificity for arachidonate-containing phospholipids. We have recently reported two patients bearing a homozygous mutation in the gene encoding cPLA2 α, which results in loss of enzyme activity1. Here we have characterised eicosanoid levels from stimulated blood, blood-outgrowth endothelial cells (BOECs) and urine from these patients to determine the requirement for cPLA2 α for eicosanoid synthesis.

Blood samples were collected from healthy volunteers (n=4) and cPLA2 α-deficient patients (n=2). Platelet and leuckocyte eicosanoid formation was stimulated by incubating whole blood with collagen (30μg/ml in isotonic glucose; 30 mins; 37°C) or LPS (10μg/ml in PBS; 24 hrs; 37°C), respectively. Endothelial cell eicosanoid formation (stimulated with A23187 calcium ionophore; 30μM in PBS; 30 mins; 37°C) was studied by using blood outgrowth endothelial cells (BOEC), isolated and cultured as previously described2. Levels of a range of eicosanoid mediators including prostanoids, isoprostanes, HETEs, EETs, and DHETs were measured in conditioned plasma and medium by LC/MS/MS3. Urine was collected from healthy volunteers (n=15) and cPLA2 α-deficient patients (n=2 in quadruplicate) and eicosanoid metabolites measured by GC/MS4.

Whole blood from healthy volunteers stimulated with collagen or LPS produced distinct patterns of eicosanoid release. Collagen produced large increases in levels of TXB2, PGD2, 11-, 12- and 15-HETE, whereas in LPS-stimulated blood, PGE2 and 11-HETE were the dominant products. Blood from cPLA2 α-deficient patients failed to synthesise eicosanoids in response to either collagen or LPS. For example the concentrations of TXB2 measured after incubation with collagen were 18.0±2.7ng/ml in healthy volunteer blood and <0.1ng/ml in cPLA2 α-deficient patient blood. Similarly, A23187-stimulated BOECs produced a distinct eicosanoid fingerprint, with the prostacyclin metabolite 6-keto-PGF1 α, PGE2 and PGD2 being the primary products. Eicosanoid release was almost abolished in BOECs derived from cPLA2 α-deficient patient blood. Urinary metabolites of PGI2, TXA2, PGE2, PGD2 and leukotrienes (LT) were measured in order to assess eicosanoid formation in vivo. Interestingly, whilst urinary levels of PGI2 and LT metabolites were reduced >95% in cPLA2 α-deficient patients, metabolites of TXA2, PGE2 and PGD2 were reduced only 30-50%.

Taken together, these data definitively demonstrate the requirement of the cPLA2 α for eicosanoid formation by platelets, leukocytes and differentiated endothelial cells (BOECs). The presence of relatively high levels of some prostanoid metabolites in cPLA2 α-deficient patient urine, however, suggests that substantial cPLA2 α-independent eicosanoid formation must occur at other sites in the body.

(1) Brooke MA et al. Gut. In press; (2) Starke RD et al. Blood 104:9, 2013; (3) Newman JW et al. J Lipid Res 43:9, 2002; (4) Daniel VC et al. J Chromatogr B Biomed Appl 653:117, 1994