11/14‐oxy‐DPA, the 12‐LOX derived metabolites of DPA, inhibit platelet activation through the cGMP‐PKG signaling pathway

Platelet‐mediated thrombosis is the primary underlying mechanism leading to cardiovascular life‐threatening clinical events. Regulating excessive platelet reactivity is an essential aspect of antithrombotic therapy. Previously, we had shown that lipid metabolites of 12‐lipoxygenase (12‐LOX) such as 12‐HETrE, a 12‐LOX oxylipin of the ω‐6 polyunsaturated fatty acid dihomo‐gamma‐linolenic acid, could function as an inhibitor to platelet activation. Here, we demonstrate for the first time that another ω‐6 PUFA, docosapentaenoic acid (DPA; 22:5n‐6), can be endogenously oxidized by 12‐LOX to generate two distinct oxylipins, 11‐oxy‐DPA and 14‐oxy‐DPA to regulate platelet reactivity. Although both 11‐oxy‐DPA and 14‐oxy‐DPA were able to significantly inhibit platelet activation, we observed differences in the potency of the 12‐LOX‐derived metabolites of DPA,n‐6. 11‐oxy‐DPA was able to significantly inhibit platelet aggregation at a lower concentration than 14‐oxy‐DPA. We also observed calcium mobilization was significantly attenuated in the presence of 11‐oxy‐DPA or 14‐oxy‐DPA. Furthermore, to determine whether 11‐oxy‐DPA and 14‐oxy‐DPA could be regulating platelet function through a Gα s signaling pathway by which it could be behaving similarly to prostacyclin or 12‐HETrE, we measured a downstream effector of cAMP, the phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) at serine 157 by PKA. We observed no differences in VASP phosphorylation at serine 157 in platelets following 11‐oxy‐DPA or 14‐oxy‐DPA treatment compared to vehicle control. However, VASP phosphorylation at serine 239, a substrate of PKG, was enhanced in platelets treated with 11‐oxy‐DPA or 14‐oxy‐DPA treatment. Thus, this suggest that 11‐oxy‐DPA and 14‐oxy‐DPA inhibit platelet activation through a novel cGMP‐PKG‐dependent pathway. This is the first study to demonstrate the novel metabolites of ω‐6 DPA generated by 12‐LOX have role in regulating platelet activation through the cGMP‐PKG signaling pathway and expands potential targets for future therapeutic intervention for the prevention of thrombotic events leading to myocardial infarction and stroke.