Potent Anti-Platelet Metabolite, 12-HETrE, Inhibits Platelet Activation and Thrombosis In Vivo Via Activation of the IP Receptor

Platelet activation is a critical component of clot formation. Under pathophysiological conditions platelet-mediated clot formation can lead to an occlusive thrombotic event resulting in myocardial infarction and stroke. Regulation of platelet reactivity relies on an understanding of the complex intra-platelet signaling that takes place following activation of various receptors on the platelet surface. Our lab recently identified a novel oxidized metabolite known as 12-HETrE that is formed from 12-lipoxygenase (12-LOX) oxidation of the ω-6 fatty acid DGLA in the platelet. Since DGLA constitutes one of the predominant fatty acids contained in the phospholipids on the platelet membrane and is also a rich source of dietary fatty acid intake through a variety of plant oils, we had originally sought to determine if this metabolite contained inherent regulatory properties on the human platelet. We successfully showed not only that 12-HETrE potently regulates hemostasis and thrombosis in vivo, but additionally identified that the metabolite signals inhibition of platelet activity through a Gα_S-linked GPCR resulting in formation of cAMP and activation of PKA and VASP. In the current study, we identified for the first time the receptor activated by 12-HETrE in the human platelet. To identify which GPCR is activated by 12-HETrE, we utilized both pharmacologic as well as genetic approaches. Our ex vivo pharmacological data suggested 12-HETrE predominantly signals through the IP receptor in the human platelet. This finding was confirmed using an IP receptor knockout mouse as well as human cell lines expressing or absent IP receptor on their surface. Finally, these findings were confirmed in vivo with wild type or IP knockout mice and assessing the ability of 12-HETrE to inhibit laser-induced thrombosis. Hence, these findings show for the first time that 12-HETrE does in fact regulate inhibition of platelets through the IP receptor. Since 12-HETrE has already been shown to inhibit thrombosis without a significant increase in bleeding diathesis, the current study has elucidated a new target for IP receptor activation and an inhibition of platelet activity and occlusive thrombus formation and should be evaluated for its potential as a new class of anti-thrombotic therapeutics.