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.

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.

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-mediated thrombosis is the primary underlying mechanism leading to cardiovascular life-threatening clinical events. Control of excessive platelet responses is an essential aspect of antithrombotic therapy. A number of anti-platelet drugs have been developed to target specific signaling pathways or endpoints involved in platelet activation. Despite the effectiveness of current anti-platelet therapies, uncontrolled thrombosis or bleeding complications still persist. We had proposed a potential novel therapeutic approach by which oxylipins generated by 12-lipoxygenase (12-LOX) oxidation of ω-6 could modulate platelet reactivity. We observed 12-hydroxyeicosatrienoic acid (12-HETrE), a 12-LOX oxidized oxylipin of ω-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), significantly attenuated human platelet activation. We then verified that DGLA oxidation to 12-HETrE depended on functional platelet 12-lipoxygenase (12-LOX) in our transgenic mouse model deficient in 12-LOX enzyme in the platelets (12-LOX-/-). To determine whether 12-HETrE could be inducing its inhibitory regulation in a GPCR-like manner by which it could potentially be behaving similarly to prostacyclin to activate adenylyl cyclase and increase cAMP through the Gs pathway, we measured cAMP level in the presence of 12-HETrE. We observed 12-HETrE significantly increased cAMP levels. We investigated a downstream effector of cAMP, such as VASP 157 phopshorylation, which is a PKA substrate. Also observed both Rap1 and GPIIbIIa activation to be attenuated in the presence 12-HETrE, confirming our aggregation result. This the first study to show the signaling mechanism of 12-HETrE which is dependent on active 12-LOX oxidation of DGLA to regulate platelet reactivity in a Gs-like manner. Disclosures No relevant conflicts of interest to declare.

FcγRIIa is central to the pathophysiology of a number of immune‐mediated thrombocytopenia and thrombosis (ITT) syndromes. ITT is associated with significant morbidity and mortality due to unwanted platelet‐mediated vessel occlusion. Therapeutic approaches for prevention of ITT remain a challenge since the currently approved treatment reduces, but does not eliminate incidence of thrombosis and is complicated by prolonged bleeding risk. Hence novel therapeutic strategies for prevention of ITT are warranted. Our lab has demonstrated that platelet 12‐lipoxygenase (12‐LOX), an enzyme that oxidizes free fatty acid, is an important regulator of FcγRIIa–mediated platelet activation in humans and mice. The pharmacological inhibition of 12‐LOX in human and genetic ablation of 12‐LOX in transgenic mouse (FcR/ALOX12‐/‐) platelets resulted in significant decrease in activation of FcγRIIa‐mediated effectors (PLCγ2, calcium mobilization, Rap1, and PKC) as well as platelet aggregation. To further evaluate the role of 12‐LOX regulation in this pathway, a pull‐down of 12‐LOX was performed to assess protein interactions that are required for FcγRIIa‐mediated platelet activation. A number of novel proteins were found to interact with 12‐LOX in an activation‐dependent manner. Our data supports an essential role for 12‐LOX in regulating FcγRIIa‐mediated platelet function through its complex formation, and demonstrates 12‐LOX as a viable novel therapeutic target to prevent immune‐mediated thromboses such as heparin‐induced thrombocytopenia and thrombosis (HITT) and sepsis.