Bloodworks Northwest Research Institute, Seattle, WA (H.J.L., D.B., T.Ö., A.C., S.L.B., N.R., F.R., R.A., X.F., M.S.).
Division of Hematology, Department of Medicine (M.C.S., X.F., M.S.), University of Washington Medical Center, Seattle.
Arterioscler Thromb Vasc Biol. 2023 Oct;43(10):1990-2007. doi: 10.1161/ATVBAHA.123.319021. Epub 2023 Aug 31.
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.
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.
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.
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.
血小板的保存时间为 5 至 7 天。先前的研究表明，储存袋中的 HETE 水平与血小板在体内的性能呈负相关，这表明生物活性脂质介质的失调可能导致储存损伤。在本研究中，我们试图了解 12-LOX（12-脂氧合酶）的基因缺失和药理学抑制如何在储存和输注后影响血小板。
使用液相色谱-串联质谱-多重反应监测或输注到血小板减少症 hIL4R（人白细胞介素 4 受体）转基因小鼠中，对来自 12-LOX（野生型 [WT]）和 12-LOX 小鼠的血小板进行 24 和 48 小时储存并进行分析。通过流式细胞术和体内血栓形成和止血模型评估血小板功能。为了测试 COX-1（环氧化酶-1）途径的作用，用乙酰水杨酸处理供体小鼠。用 12-LOX 抑制剂 VLX-1005 或载体处理人血小板，储存并输注到 NOD/SCID（非肥胖糖尿病/严重联合免疫缺陷）小鼠中。
多不饱和脂肪酸在 12-LOX 小鼠的储存血小板中显著增加，而氧化脂质的浓度在 WT 血小板中显著升高。输注到血小板减少症小鼠后，我们观察到 12-LOX 血小板的基线 αIIbβ3 整合素激活明显高于 WT 血小板。与 WT 血小板相比，12-LOX 小鼠的储存血小板更快地阻塞血管。在止血模型中，与 WT 血小板相比，12-LOX 小鼠的储存血小板明显更快地在静脉损伤部位聚集，导致失血量减少。COX-1 的抑制消除了储存的 12-LOX 血小板中整合素的过度激活和血栓素的生成，突出了该途径对改善输注后功能的关键作用。与我们的小鼠研究一致，用 VLX-1005 储存的人血小板在输注后与用载体处理的血小板相比，整合素的激活增加。
通过 COX-1 依赖性花生四烯酸代谢增加血栓素生成，删除 12-LOX 可改善储存的小鼠血小板的输注后功能。未来的研究应确定输注给人类的 12-LOX 抑制血小板的可行性和安全性。
