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ObjectiveAdequate platelet reactivity is required for maintaining hemostasis. However, excessive platelet reactivity can also lead to the formation of occlusive thrombi. Platelet 12(S)-lipoxygenase (12-LOX), an oxygenase highly expressed in the platelet, has been demonstrated to regulate platelet function and thrombosis ex vivo, supporting a key role for 12-LOX in the regulation of in vivo thrombosis. However, the ability to pharmacologically target 12-LOX in vivo has not been established to date. Here, we studied the effect of the first highly selective 12-LOX inhibitor, ML355, on in vivo thrombosis and hemostasis.Approach and resultsML355 dose-dependently inhibited human platelet aggregation and 12-LOX oxylipin production, as confirmed by mass spectrometry. Interestingly, the antiplatelet effects of ML355 were reversed after exposure to high concentrations of thrombin in vitro. Ex vivo flow chamber assays confirmed that human platelet adhesion and thrombus formation at arterial shear over collagen were attenuated in whole blood treated with ML355 comparable to aspirin. Oral administration of ML355 in mice showed reasonable plasma drug levels by pharmacokinetic assessment. ML355 treatment impaired thrombus growth and vessel occlusion in FeCl3-induced mesenteric and laser-induced cremaster arteriole thrombosis models in mice. Importantly, hemostatic plug formation and bleeding after treatment with ML355 was minimal in mice in response to laser ablation on the saphenous vein or in a cremaster microvasculature laser-induced rupture model.ConclusionsOur data strongly support 12-LOX as a key determinant of platelet reactivity in vivo, and inhibition of platelet 12-LOX with ML355 may represent a new class of antiplatelet therapy.

ObjectiveDietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis.Approach and resultsDGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets.ConclusionsThis study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner.

Objective: Platelet activation after stimulation of PAR (protease-activated receptor) 4 is heightened in platelets from blacks compared with those from whites. The difference in PAR4 signaling by race is partially explained by a single-nucleotide variant in PAR4 encoding for either an alanine or threonine at amino acid 120 in the second transmembrane domain. The current study sought to determine whether the difference in PAR4 signaling by this PAR4 variant is because of biased G q signaling and whether the difference in PAR4 activity results in resistance to traditional antiplatelet intervention., Approach and Results: Membranes expressing human PAR4-120 variants were reconstituted with either G q or G 13 to determine the kinetics of G protein activation. The kinetics of G q and G 13 activation were both increased in membranes expressing PAR4-Thr120 compared with those expressing PAR4-Ala120. Further, inhibiting PAR4-mediated platelet activation by targeting COX (cyclooxygenase) and P2Y 12 receptor was less effective in platelets from subjects expressing PAR4-Thr120 compared with PAR4-Ala120. Additionally, ex vivo thrombus formation in whole blood was evaluated at high shear to determine the relationship between PAR4 variant expression and response to antiplatelet drugs. Ex vivo thrombus formation was enhanced in blood from subjects expressing PAR4-Thr120 in the presence or absence of antiplatelet therapy., Conclusions: Together, these data support that the signaling difference by the PAR4-120 variant results in the enhancement of both G q and G 13 activation and an increase in thrombus formation resulting in a potential resistance to traditional antiplatelet therapies targeting COX-1 and the P2Y 12 receptor., (© 2018 American Heart Association, Inc.)

Objective: Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis., Approach and Results: DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets., Conclusions: This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner., (© 2016 American Heart Association, Inc.)

Objective: Black individuals are at an increased risk of myocardial infarction and stroke, 2 vascular diseases with strong thrombotic components. Platelet activation is a key step in platelet clot formation leading to myocardial infarction and stroke, and recent work supports a racial difference in platelet aggregation through the thrombin protease-activated receptors (PARs). The underlying mechanism for this racial difference, however, has not been established. Determining where in the signaling cascade these racial differences emerge will aid in understanding why individuals of differing racial ancestry may possess an inherent difference in their responsiveness to antiplatelet therapies., Approach and Results: Washed human platelets from black volunteers were hyperaggregable in response to PAR4-mediated platelet stimulation compared with whites. Interestingly, the racial difference in PAR4-mediated platelet aggregation persisted in platelets treated ex vivo with aspirin and 2MeSAMP (2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate), suggesting that the racial difference is independent of secondary feedback. Furthermore, stimulation of platelets from black donors with PAR4-activating peptide showed a potentiated level of activation through the Gq pathway compared with platelets from white donors. Differences in signaling included increased Ca(2+) mobilization, Rap1 (Ras-related protein 1) activation, and integrin αIIbβ3 activation with no observed difference in platelet protein expression between the groups tested., Conclusions: Our study is the first to demonstrate that the Gq pathway is differentially regulated by race after PAR4 stimulation in human platelets. Furthermore, the racial difference in PAR4-mediated platelet aggregation persisted in the presence of cyclooxygenase and P2Y12 receptor dual inhibition, suggesting that current antiplatelet therapy may provide less protection to blacks than whites., (© 2014 American Heart Association, Inc.)