12-LOX-derived DPAn-6and oxylipins 11-HpDPAn-6and 14-HpDPAn-6inhibit platelet activation. (A) Oxylipins derived from platelets (n = 4) were assessed by incubating human platelets with 10 μM DPA in the presence of vehicle control (ctrl), 12-LOX inhibitor (ML355), or COX-1 inhibitor (indomethacin) prior to mass spectrometry. (B) The major metabolites produced from DPAn-6 in platelets are 12-LOX–derived oxylipins, 11-HpDPAn-6, and 14-HpDPAn-6. The effects of 11-HpDPAn-6 and 14-HpDPAn-6 on platelet activity were assessed by incubating human platelets with increasing concentrations of DPAn-6 metabolites for 5 minutes prior to stimulation with thrombin (n = 10) (C) or collagen (n = 10) (D). (E) Calcium mobilization induced by the glycoprotein VI agonist convulxin was assessed in the absence or presence of DPAn-6 (left panel), 11-HpDPAn-6 (middle panel), or 14-HpDPAn-6 (right panel) (n = 5). (F) PKC activity was assessed in the absence or presence of DPAn-6 (2.5 μΜ), 11-HpDPAn-6, (2.5 μΜ), or 14-HpDPAn-6 (2.5 μΜ) prior to stimulation with 0.2 nM thrombin (n = 4) or 10 ng/mL convulxin (n = 4). (G) Platelet ATP secretion was measured in the absence or presence of DPAn-6 (1 μM), 11-HpDPAn-6 (1 μM), or 14-HpDPAn-6 (1 μM) prior to stimulation with 0.5 nM thrombin (left panel; n = 6) or 2 μg/mL collagen (right panel; n = 4). (H) α-Granule secretion was assessed in the absence or presence of DPAn-6 (1 μM), 11-HpDPAn-6 (1 μM), or 14-HpDPAn-6 (1 μM) prior to stimulation with thrombin 0.5 nM (n = 5). Data are means ± standard deviation (SD) for PKC activation means ± SEM for others. *P < .05, **P < .01, ***P < .001, ****P < .0001, 1- or 2-way ANOVA. DMSO, dimethyl sulfoxide.