12-HETrE, An Endogenous Inhibitor of Platelet Activation,

Abstract 3254

Platelet activation plays a pivotal role in thrombosis and hemostasis. Understanding the signaling events mediating this process is essential in preventing unwanted clot formation, which can lead to the development of heart attack, stroke, and venous thromboembolism. Fatty acids may play an important role in determining the level of platelet reactivity, however the mechanism(s) by which this occurs are not entirely clear. 12-lipoxygenase (12-LOX) has been shown to oxidize the fatty acid, arachidonic acid (AA), in order to produce the bioactive eicosanoid 12-HETE, which has recently been shown to play a role in tissue factor activation and subsequent thrombin generation in the platelet (Thomas et al., 2010, J Biol Chem; 285:6891–903). 12-HETE has also been shown to signal to cells, in part, through the G protein-coupled receptor GPR31 (Guo et al, 2011, J Biol Chem; epub). While research on 12-LOX-mediated eicosanoid regulation of platelets has primarily focused on 12-HETE, the potential for regulation of platelets by eicosanoids derived from other fatty acids has been overlooked. As the fatty acid content on the platelet membrane is extremely dynamic and fatty acid supplementation is correlated with a reduced risk for cardiovascular disease, we hypothesized that 12-LOX oxidation of another fatty acid may play a direct role in regulating platelet function. To test this hypothesis, the Ω-6 fatty acid dihomo-γ-linolenic acid (DGLA) as well as its eicosanoid derived from 12-LOX oxidation, 12-hydroxyeicosatrienoic acid (12-HETrE), were applied to washed platelets followed by stimulation with thrombin, PAR1-AP, or PAR4-AP. Platelets treated with either DGLA or 12-HETrE showed significant attenuation in platelet aggregation following stimulation with thrombin or PAR-AP. A number of biochemical intermediates were also tested in the presence of 12-HETrE or DGLA including Rap1 activation, αIIbβ3 integrin activation, α-granule secretion, and dense granule secretion. All endpoints tested were attenuated in the presence of DGLA or 12-HETrE relative to control. To confirm the regulation was unique to DGLA and its metabolite, the same endpoints were measured in the presence of AA or 12-HETE. Pre-treatment with either AA or 12-HETE did not attenuate any of the agonist-mediated platelet activation endpoints. Thus, our data supports a unique role for 12-HETE and 12-HETrE and that fatty acid regulation of platelet function may be highly dependent on the lipid content of the platelet. Shifting the ratios of fatty acids in the platelet through dietary supplementation or pharmacological intervention may be sufficient to induce a cardio-protective state, in part, through increasing 12-HETrE formation and subsequently inhibiting platelet activation.