177P Queen Elizabeth II Conference Centre London
Pharmacology 2013

 

 

Identification of 11-HETE and 15-HETE as major products of the platelet COX-1 enzyme

F Rauzi1, NS Kirkby1,2, M Eldin3, JA Mitchell2, D Zeldin3, TD Warner1. 1William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK, 2National Heart & Lung Institute, Imperial College, London, UK, 3Laboratory of Respiratory Biology, Respiratory & Cardiovascular Diseases, National Institute of Environmental Health Sciences, Research Triangle Park, NC2770, USA

Background: Prostaglandins (PGs) and hydroxyeicosatetraeinoic acids (HETEs) are synthesized from arachidonic acid (AA), which is released from the membrane phospholipids of cells through the activity of cytosolic phospholipase A2 α (cPLA2 α). In platelets AA is metabolised through the enzymes cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) leading to the production of a range of eicosanoids. We have determined the enzyme sources of the major prostanoid and HETE products released by platelets following activation by selective platelet agonists and investigated whether anti-platelet therapy might affect the production of these eicosanoids.

Methods: Blood was collected into lepirudin (250µg/ml) by venepuncture from healthy volunteers (n=4). Intact blood and platelet rich plasma (PRP), obtained by centrifugation of blood (175 x g for 15 min), was incubated with collagen (30µg/ml), TRAP-6 (30µM) or vehicle, in a platelet aggregometer (1,2,3) with stirring (1200rpm; 37°C). Both blood and PRP were pre-treated with aspirin (100µM), the P2Y12 receptor blocker, prasugrel (0.03%DMSO; 3µM), aspirin+prasugrel or vehicle and incubated for 30min at 37°C. Plasmas were then separated by centrifugation (12000rpm) for 1 min at 4ºC and stored at -80ºC. The levels of prostanoids and HETEs were determined by LC/MS/MS analysis.

Results: Stimulation of blood or PRP with collagen or TRAP-6 (Table 1) caused large, >100 times, increases in the levels of thromboxane (TX) A2, prostaglandin (PG) E2, and PGD2, and similar increases in 11-, 12-, and 15-HETE. Aspirin inhibited the production of TXA2, PGE2 and PGD2, as well as inhibiting the productions of 11-HETE and 15-HETE, but not 12-HETE, in both PRP and blood. The levels of 12-HETE were reduced by aspirin and prasugrel used in combination (*p<0.05), consistent with these drugs inhibiting the processes of platelet activation but not directly inhibiting platelet 12-LOX. (Data shown as mean±SEM; ANOVA with Dunnett test vs vehicle). Prasugrel alone did not alter the levels of eicosanoids compared to vehicle treatment (data not shown).

Table 1. Eicosanoid production (ng/ml) in PRP and whole blood stimulated with collagen or TRAP6 (*P<0.05 vs vehicle)

agonist Drug TXB2 11-HETE 15-HETE 12-HETE
PRP WB PRP WB PRP WB PRP WB
PBS vehicle 0.4±0.2 0.6±0.3 0.06±0.04 0.08±0.02 0.3±0.2 0.4±0.1 7.8±5.8 14.9±10.8
collagen vehicle 66.±10.8 54.3±9.6 23.5±3.3 2.4±1.9 23.8±4.1 1.7±1.5 486.9±57.3 44.2±41.2
aspirin 0.4±0.2* 0.2±0.2* 0.7±0.08* 0.3±0.05* 3.0±0.6* 1.3±0.1* 529.3±52.8 188.8±72.3
TRAP6 vehicle 37.±4.4 28.2±3.8 31.8±8.6 16.5±2.6 30.6±7.7 17.7±3.2 275.7±138.5 114.4±53.6
aspirin 0.2±0.6* 0.2±0.1* 0.3±0.1* 0.3±0.1* 1.2±0.5* 1.2±0.5* 108.7±66.2 118.7±66.4

Conclusions: In blood activated by exposure to collagen or TRAP-6 platelets are the main source of both prostanoids and HETEs. Aspirin abolished the production of not only prostanoids, but also 11-HETE and 15-HETE. This identifies 11-HETE and 15-HETE as major COX-1 products in platelets.

(1) Born G. V. R., Nature, 194: 927, 1962. (2) O’Brien J. R, J Clin Path, 15:452, 1962. (3) Jennings L.K., McCabe M., Platelets (ed. Alan D. Michelson) Ch. 26:495, 2007.