12-lipoxygenase activity plays an important role in PAR4 and GPVI-mediated platelet reactivity

Abstract

Summary Following initial platelet activation, arachidonic acid is metabolised by cyclooxygenase-1 and 12-lipoxygenase (12-LOX). While the role of 12-LOX in the platelet is not well defined, recent evidence suggests that it may be important for regulation of platelet activity and is agonist-specific in the manner in which it regulates platelet function. Using small molecule inhibitors selective for 12-LOX and 12-LOX-deficient mice, the role of 12-LOX in regulation of human platelet activation and thrombosis was investigated. Pharmacologically inhibiting 12-LOX resulted in attenuation of platelet aggregation, selective in hibition of dense versus alpha granule secretion, and inhibition of platelet adhesion under flow for PAR4 and collagen. Additionally, 12-LOX-deficient mice showed attenuated integrin activity to PAR4-AP and convulxin compared to wild-type mice. Finally, platelet activation by PARs was shown to be differentially dependent on COX-1 and 12-LOX with PAR1 relying on COX-1 oxidation of arachi donic acid while PAR4 being more dependent on 12-LOX for normal platelet function. These studies demonstrate an important role for 12-LOX in regulating platelet activation and thrombosis. Furthermore, the data presented here provide a basis for potentially targeting 12-LOX as a means to attenuate unwanted platelet activation and clot formation.

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

“…Washed platelets were resuspended in Tyrode's buffer as previously described 21 at a concentration of 3 3 10 8 platelets/mL unless otherwise indicated. ).…”

“…Mouse platelet preparation was prepared as previously described. 21 Murine platelets of 2.5 3 10 8 platelets/mL were resuspended in Tyrode's buffer containing human fibrinogen (75 mg/mL) and CaCl 2 (1 mM).…”

“…20 Platelet 12(S)-lipoxygenase (12-LOX), an oxygenase highly expressed in platelets, has been shown to potentiate the activation of select signaling pathways, including protease-activated receptor 4 (PAR4) and an ITAM-containing receptor complex (GPVI-FcRg). [21][22][23][24] The most well understood function of 12-LOX is the production of oxylipins, most notably the conversion of arachidonic acid (AA) to 12-hydroxyeicosatretraenoic acid (12-HETE) upon agonist stimulation of platelets through both G protein-coupled receptor-and non-G protein-coupled receptor-mediated pathways. 23 The oxylipin 12-HETE has been shown to be prothrombotic in platelets.…”

“…23 The oxylipin 12-HETE has been shown to be prothrombotic in platelets. 25,26 Although the mechanism by which 12-LOX regulates platelet activity is not fully understood, previous publications have demonstrated the ability of 12-LOX activity to augment key signaling components of platelet activation, including Rap1, Ca 21 mobilization, aIIbb3 activation, and dense granule secretion. 21,22,24,26 As 12-LOX activity was recently shown to be required for normal GPVI-mediated platelet activation, 21,22 we sought to determine if 12-LOX activity is an essential component of FcgRIIa signaling in platelets.…”

“…25,26 Although the mechanism by which 12-LOX regulates platelet activity is not fully understood, previous publications have demonstrated the ability of 12-LOX activity to augment key signaling components of platelet activation, including Rap1, Ca 21 mobilization, aIIbb3 activation, and dense granule secretion. 21,22,24,26 As 12-LOX activity was recently shown to be required for normal GPVI-mediated platelet activation, 21,22 we sought to determine if 12-LOX activity is an essential component of FcgRIIa signaling in platelets. 10 In this study, human platelets were treated with the selective 12-LOX inhibitor, ML-355, 27 or vehicle control prior to FcgRIIa stimulation to determine if 12-LOX plays a role in the FcgRIIa signaling pathway.…”