Deletion of 12-Lipoxygenase Improves Platelet Function after Storage and Transfusion in Thrombocytopenic Mice

Introduction Human platelets are stored for up to 7 days for transfusion. During storage, platelets undergo numerous detrimental functional changes. Results from two recent randomized clinical trials challenge the hemostatic efficacy of stored platelets for transfusion. In a recent study in healthy humans, increased 12-HETE levels were associated with reduced platelet performance in vivo. In the current study, we sought to understand how genetic deletion of 12-LOX (the enzyme that generates 12-HETE) affects platelets during storage, before, and after transfusion in vitro and in mouse models. Methods and Results: We obtained platelets from wild-type (WT) and 12-LOX-/- mice and performed storage studies for 24 and 48 hours. Using LC-MS/MS-MRM, we showed that ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) increased significantly in stored platelets from 12-LOX-/- mice, while oxylipins were significantly lower in 12-LOX-/- platelets than in WT platelets. The ω-6/ω-3 PUFA ratio decreased significantly in stored 12-LOX-/- compared to stored WT platelets, highlighting a pronounced increase in ω-3 PUFAs. The circulation time of stored 12-LOX-/- platelets did not differ from stored WT platelets. Baseline, pre-transfusion αIIbβ3 integrin activation was significantly lower before and after 24 hours of storage in 12-LOX-/- platelets than in WT platelets. Surprisingly, once stored platelets were transfused, we observed significantly increased baseline αIIbβ3 integrin activation in 12-LOX-/- platelets than in WT platelets. To test whether this translates into better in vivo function, we exposed 24-hour stored platelets to an in vivo model of thrombosis and hemostasis. In brief, hIL4R-TG mice express the receptor for human interleukin 4 instead of GPIbα on the platelet surface. hIL4R-TG mice were rendered thrombocytopenic with an anti-human IL4R antibody. We transfused 24-hour stored WT, or 12-LOX-/- platelets, to thrombocytopenic hIL4R-TG mice, injured the carotid artery by FeCl3, and recorded the arterial blood flow. Indeed, stored 12-LOX-/- platelets had better in vivo function, as evidenced by more frequent and significantly faster vessel occlusions with stored and transfused 12-LOX-/- platelets than transfusion of stored WT platelets. Importantly, stored and transfused 12-LOX-/- occluded vessels in a similar time as normocytic WT mice. Untransfused, thrombocytopenic hIL4R-TG mice and untransfused, normocytic hIL4R-TG mice did not show vessel occlusion. Conclusion Deleting 12-LOX improves the post-transfusion function of stored murine platelets. Pharmacologic inhibition of 12-LOX or dietary alterations of ω-3 and ω-6 PUFAs could significantly enhance human platelet quality and function after storage. Future studies must determine the feasibility and safety of 12-LOX inhibition in stored and transfused human platelets.