Identification of HETE isoforms produced by activated platelets and study of their roles within the vasculature.

PhDBackground: Prostaglandins (PGs) and hydroxyeicosatetraeinoic acids (HETEs) are synthesized from arachidonic acid (AA) released from the membrane phospholipids of cells through the activity of cytosolic phospholipase A2α (cPLA2α). In platelets AA is metabolised through cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) leading to the production of a range of eicosanoids. This project determined the enzymes responsible for the production of the major prostanoid and HETE metabolites released by activated platelets and investigated the roles of these within the vasculature. Methods: The responses of whole blood and platelets collected from healthy volunteers or patients lacking cPLA2α were examined in the absence and presence of anti-platelet drugs (aspirin and/or prasugrel). The effects of selective platelet agonists and global stimulants of blood cells were tested to provide multiple functional responses against which to compare the ability of platelets and blood to produce eicosanoid molecules, as measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Stimulation of blood and PRP produced large increases in the levels of TXA2, PGE2, PGD2, 11- and 15-HETE which were found to be produced by platelets via the activity of cPLA2α and COX-1 enzymes. 12-HETE was dependent upon cPLA2α but not COX-1. 11- and 15-HETE have not previously been reported as major platelet products. A series of experiments indicated that 11-, 12- and 15-HETE had very weak influences on platelet aggregation/adhesion responses. Notably, 12(S)-HETE, which represents the enantiomer released by platelets, strongly promoted neutrophil chemotactic responses. Furthermore, 15(S)-HETE was identified as the bioactive enantiomer produced by activated platelets and was able to induce tube formation and cell migration in cultures of human microvascular endothelial cells (HMEC-1) and promote the formation of sprouts from rat aortic rings. Conclusions: Platelets are major producers of prostanoids and HETEs in activated blood. Notably, aspirin abolished the production of not only prostanoids, but also 11-HETE and 15-HETE, identifying these as major COX-1 products. Moreover, 12(S)- Abstract II HETE robustly induced neutrophil chemotaxis, while 15(S)-HETE promoted angiogenic responses both in vitro and ex vivo. These data identified 15(S)-HETE as a potential target for the development of therapies able to limit angiogenic processes in conditions such as tumour progression and metastasis