12-HETrE, a Novel 12-LOX Oxylipin, Prevents Platelet Activation in a Gα_s-like Manner

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.