12-Lipoxygenase Regulates Platelet Reactivity and Thrombosis Through Formation Of Unique Oxylipins

Platelet activation plays a central role in hemostasis and thrombosis and is regulated through a complex milieu of intracellular signaling events vital for prevention of excessive clot formation and vessel occlusion. There is strong evidence that oxylipins (oxygenated fatty acids) derived from omega-3 (ω-3) and w-6 fatty acids offer cardio-protection; however the underlying mechanism for this protection is not well understood. Previously, we showed that ex vivo fatty acid supplementation of human platelets with the ω-6 fatty acid (dihomo-γ-linolenic acid (DGLA)) significantly inhibited platelet-induced aggregation. The anti-platelet effect exerted by DGLA depended on oxidation by 12-lipoxygenase (12-LOX) to form unique oxylipins with inhibitory activity toward platelet function. Here, for the first time, we show the underlying signaling mechanisms by which 12-LOX functions as an essential regulator of endogenous fatty acid metabolism protecting against excessive platelet activation and thrombosis. To delineate the mechanisms by which 12-LOX regulates platelet reactivity, we investigated upstream signaling correlates leading to platelet aggregation, including calcium mobilization, Rap1 activation, and GP IIb/IIIa activation. Using mice deficient in platelet 12-LOX (12-LOX^-/-), we were able to show functional 12-LOX is required for the regulation of DGLA inhibition of PAR4- and collagen-mediated platelet aggregation. Platelets from wild-type mice treated with DGLA or 12-HETrE (oxidized form of DGLA) showed significant suppression of platelet-mediated aggregation, whereas DGLA was unable to suppress platelet aggregation in platelets from 12-LOX^-/- mice. Importantly, treatment with 12-HETrE significantly attenuated platelet aggregation in 12-LOX^-/- mice. Further, DGLA was unable inhibit calcium mobilization and Rap1 activation in mice deficient for 12-LOX, while calcium mobilization and Rap1 activation were significantly inhibited by 12-HETrE. Overall, our data support 12-LOX playing an important role in both hemostasis and thrombosis. The loss of 12-LOX activity results in an inability of fatty acids to be oxidized to their respective bioactive lipid products. The formation of these oxylipins (12-HETrE in particular) is essential for prevention of platelet activation leading to atherothrombus formation and stroke. Hence, this study is the first to show that the anti-platelet effect mediated by fatty acids is due in large part to their oxylipins production by 12-LOX and the formation of potently anti-platelet metabolites.