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

Yeung, Jennifer; Tourdot, Benjamin E.; Fernandez-Perez, Pilar; Vesci, Joanne; Ren, Jin; Smyrniotis, Christopher J.; Luci, Diane K.; Jadhav, Ajit; Simeonov, Anton; Maloney, David J.; Holman, Theodore R.; McKenzie, Steven E.; Holinstat, Michael

doi:10.1182/blood-2014-05-575878

Cited by 76 publications

(77 citation statements)

References 52 publications

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“…The proposed inhibitory effect mediated through 12-LOX appears paradoxical based on previous work in our lab and others showing that 12-LOX is a positive mediator of platelet function 17, 21, 42, 43 . However, due to the fact that 12-LOX is an enzyme whose function is to add an oxygen to a free fatty acid in order to produce a bioactive oxylipin, it is reasonable to conclude from the data presented here and elsewhere 5, 8 that the substrate for 12-LOX is the determining factor in its effect on platelets and ultimately thrombosis.…”

Section: Discussionmentioning

confidence: 78%

“…To determine if DGLA inhibited platelet aggregation by impinging on intracellular signaling, the activation of Rap1, a common signaling effector required for integrin α IIb β 3 activation (Shattil et al, 2010; Shattil and Newman, 2004) was assessed in DGLA treated platelets stimulated with PAR4-AP 19–21 . In platelets isolated from WT mice, DGLA inhibited Rap1 activation at all concentrations of PAR4-AP tested (Figure 1C).…”

Section: Resultsmentioning

confidence: 99%

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12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gα _s Signaling in Platelets

Yeung

Tourdot

Adili

et al. 2016

ATVB

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Objective Dietary supplementation with polyunsaturated fatty acids (PUFAs) has been widely used for primary and secondary prevention of CVD in individuals at risk; however, the cardioprotective benefits of PUFAs remain controversial due to lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 PUFA, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-HETrE, inhibiting platelet activation and thrombosis. Approach and Results DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX−/−). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) following laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX−/− mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, two hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis following 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked GPCR pathway in human platelets. Conclusions This study provides the first direct evidence that an omega-6 PUFA, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gαs-linked GPCR-dependent manner.

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Section: Discussionmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 99%

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gα _s Signaling in Platelets

Yeung

Tourdot

Adili

et al. 2016

ATVB

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“…11 Further, pharmacologic inhibition of 12-LOX by ML355 was shown to attenuate FcγRIIa-mediated human platelet aggregation via modulation of the proximal signaling components of the pathway. 12 To identify if 12-LOX is a key regulator of platelet activation following activation of the coagulation pathway, the effect of ML355 on platelet aggregation induced by thrombin was assessed. Platelet aggregation in washed human platelets was measured following stimulation with multiple concentrations of thrombin (Figure 1A).…”

Section: Resultsmentioning

confidence: 99%

First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis

Adili

Tourdot

Mast

et al. 2017

ATVB

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Objective Adequate platelet reactivity is required for maintain hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. 12-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated to regulate platelet function and thrombosis ex vivo, supporting a key role for 12-LOX in the regulation of in vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Here, we studied the effect of the first highly selective 12-LOX inhibitor, ML355, on in vivo thrombosis and hemostasis. Approach and Results ML355 dose-dependently inhibited human platelet aggregation and 12-LOX oxylipin production, as confirmed by mass spectrometry. Interestingly, the antiplatelet effects of ML355 were reversed following exposure to high concentrations of thrombin in vitro. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen were attenuated in whole blood treated with ML355 comparable to aspirin. Oral administration of ML355 in mice showed reasonable plasma drug levels by pharmacokinetic assessment. ML355 treatment impaired thrombus growth and vessel occlusion in FeCl3-induced mesenteric and laser-induced cremaster arteriole thrombosis models in mice. Importantly, hemostatic plug formation and bleeding following treatment with ML355 was minimal in mice in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model. Conclusions Our data strongly supports 12-LOX as a key determinant of platelet reactivity in vivo and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy.

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“…12 Aiming to identify genetic variations that affect FcgRIIA and HIT, our Platelet RNA and eXpression-1 (PRAX-1) study 13 was designed to find differentially expressed genes among hypo-and hyperresponders to FcgRIIA activation. Although many recent studies 11,14,15 have focused on the molecular mechanism by which FcgRIIA promotes platelet activation, less is known about negative regulators of the signaling pathway. T-cell ubiquitin ligand-2 (TULA-2), a protein tyrosine phosphatase identified as a negative effector of FcgRIIA in this study, is encoded by the UBASH3B (ubiquitin associated and SH3 domain-containing protein B) gene.…”

Section: Introductionmentioning

confidence: 99%

Anti–miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice

Zhou

Abraham

André

et al. 2015

Blood

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

Abstract: Key Points Platelet 12-LOX modulates FcγRIIa signaling and presents a viable therapeutic target in the prevention of immune-mediated thrombosis. This novel therapeutic approach is supported by pharmacologic inhibition and genetic ablation of 12-LOX in human and mouse platelets.

Cited by 76 publications

References 52 publications

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gα _s Signaling in Platelets

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gα _s Signaling in Platelets

First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis

Anti–miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice

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

Abstract: Key Points Platelet 12-LOX modulates FcγRIIa signaling and presents a viable therapeutic target in the prevention of immune-mediated thrombosis. This novel therapeutic approach is supported by pharmacologic inhibition and genetic ablation of 12-LOX in human and mouse platelets.

Cited by 76 publications

References 52 publications

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gα s Signaling in Platelets

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gα s Signaling in Platelets

First Selective 12-LOX Inhibitor, ML355, Impairs Thrombus Formation and Vessel Occlusion In Vivo With Minimal Effects on Hemostasis

Anti–miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice

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12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gα _s Signaling in Platelets

12(S)-HETrE, a 12-Lipoxygenase Oxylipin of Dihomo-γ-Linolenic Acid, Inhibits Thrombosis via Gα _s Signaling in Platelets