Protease-Activated Receptor Signaling in Platelets Activates Cytosolic Phospholipase A _2α Differently for Cyclooxygenase-1 and 12-Lipoxygenase Catalysis

Abstract

Objective The rate-limiting step in the biosynthesis of thromboxane A2 (TxA2) and 12-hydroxyeicosatetraenoic acid (12-HETE) by platelets is activation of cytosolic phospholipase A2α (cPLA2α), which releases arachidonic acid, which is the substrate for cyclooxygenase-1 (COX-1) and 12-lipoxygenase. We evaluated signaling via the human platelet thrombin receptors, protease-activated receptor (PAR) 1 and PAR4, to the activation of cPLA2α, which provides a substrate for the biosynthesis of TxA2 and 12-HETE. Methods and Results Stimulating washed human platelets resulted in delayed biosynthesis of 12-HETE, which continues after maximal formation of TxA2 is completed, suggesting that 12-HETE is not formed by the same pool of arachidonic acid that provides a substrate to COX-1. PAR1-induced formation of TxA2 was inhibited by the phosphatidylinositol kinase inhibitor LY294002, whereas this inhibitor did not block 12-HETE biosynthesis. Both 1-butanol and propranolol also blocked TxA2 biosynthesis but did not inhibit 12-HETE formation. Conclusion The concerted evidence indicates that the platelet thrombin receptors signal activation of cPLA2α coupled to COX-1 by a pathway different from that signaling activation of the cPLA2α coupled to 12-lipoxygenase.

Investigations of human platelet-type 12-lipoxygenase: role of lipoxygenase products in platelet activation

“…However, due to the high level of peroxidases present in the platelet, the biological effect of 12(S)-HPETrE on the platelet clot may be limited to localized regions of metabolite production. Taken together, these observations further support a potential role for unique pools of AA and DGLA in the platelet that are independently regulated to mediate either a prothrombotic or antithrombotic environment depending on a number of factors, including 12-LOX localization, fatty acid compartmentalization, and specifi c agonist used ( 21 ).…”

“…Although AA makes up a large proportion of the phospholipid content, other fatty acids are also available from the platelet membrane, and their content, relative to AA, has been shown to fl uctuate depending on diet ( 27,29,30 ). Whereas the catalysis of 12-LOX with AA has been studied previously ( 19,21,31,32 ), little is known about the relative 12-LOX kinetic rates of the other fatty acid substrates and their physiological effects on platelet activation. LOX are known to have promiscuous substrate selectivity and can react with a variety of fatty acids, ranging from 18 to 22 carbons long and having 2 to 6 sites of unsaturation.…”

“…These studies were approved by the Thomas Jefferson University Institutional Review Board, and informed consent was obtained from all donors before blood draw. Washed platelets were prepared as previously described ( 21,38 ). The fi nal platelet concentration of washed platelets in Tyrodes buffer was adjusted to a physiological concentration of 3 × 10 8 platelets/ml.…”

12-hydroxyeicosatetraenoic acid is associated with variability in aspirin-induced platelet inhibition

“…Collagen is known to stimulate platelet 12-LOX activity via the glycoprotein VI receptor, in a PECAM-1 and PKCdependent pathway [33]. Release of AA from membrane phospholipids by PLA 2 activation is a critical step in eicosanoid biosynthesis and a recent study has shown that COX-1 and 12-LOX are linked to different PLA 2 subsets, suggesting that TxA 2 and 12-HETE are generated from different intracellular pools of AA [34]. 12-HpETE is known to modulate platelet COX activity, suggesting a degree of 'cross-talk' between the LOX/COX pathways although the exact mechanism is unclear.…”

Platelet 12-LOX is essential for FcγRIIa-mediated platelet activation

“…The kinetics of 12-HETE production in PAR-stimulated platelets was previously determined 31 ; however, the kinetics of 12-HETE production in FcgRIIa-stimulated platelets remains unknown. Therefore, we sought to determine the kinetics of 12-HETE production in FcgRIIa-stimulated platelets.…”