Loss of 12-Lipoxygenase Improves the Post-Transfusion Function of Stored Platelets

Abstract

BACKGROUND: Platelets for transfusion are stored for 5 to 7 days. Previous studies have shown that HETE levels in the storage bag negatively correlate with platelet performance in vivo, suggesting that the dysregulation of bioactive lipid mediators may contribute to the storage lesion. In the current study, we sought to understand how genetic deletion and pharmacological inhibition of 12-LOX (12-lipoxygenase) affects platelets during storage and after transfusion. METHODS: Platelets from 12-LOX +/+ (wild-type [WT]) and 12-LOX −/− mice were stored for 24 and 48 hours and profiled using liquid chromatography-tandem mass spectrometry–multiple reaction monitoring or transfused into thrombocytopenic hIL4R (human interleukin 4 receptor)-transgenic mice. Platelet function was assessed by flow cytometry and in vivo thrombosis and hemostasis models. To test the role of the COX-1 (cyclooxygenase-1) pathway, donor mice were treated with acetylsalicylic acid. Human platelets were treated with the 12-LOX inhibitor, VLX-1005, or vehicle, stored, and transfused to NOD/SCID (nonobese diabetic/severe combined immunodeficiency) mice. RESULTS: Polyunsaturated fatty acids increased significantly in stored platelets from 12-LOX −/− mice, whereas oxylipin concentrations were significantly higher in WT platelets. After transfusion to thrombocytopenic mice, we observed significantly more baseline αIIbβ3 integrin activation in 12-LOX −/− platelets than in WT platelets. Stored platelets from 12-LOX −/− mice occluded vessels significantly faster than stored WT platelets. In hemostasis models, significantly more stored 12-LOX −/− than WT platelets accumulated at the site of venous injury leading to reduced blood loss. Inhibition of COX-1 abrogated both increased integrin activation and thromboxane generation in stored 12-LOX −/− platelets, highlighting the critical role of this pathway for improved post-transfusion function. Consistent with our mouse studies, human platelets stored with VLX-1005, showed increased integrin activation compared with vehicle-treated platelets after transfusion. CONCLUSIONS: Deleting 12-LOX improves the post-transfusion function of stored murine platelets by increasing thromboxane generation through COX-1–dependent arachidonic acid metabolism. Future studies should determine the feasibility and safety of 12-LOX–inhibited platelets transfused to humans.