Background:LNK/SH2B3 inhibits Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling by hematopoietic cytokine receptors. Genome-wide association studies have shown association of a common single nucleotide polymorphism in LNK (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that LNK(TT) reduces LNK function and that LNK-deficient mice display prominent platelet–neutrophil aggregates, accelerated atherosclerosis, and thrombosis. Platelet–neutrophil interactions can promote neutrophil extracellular trap (NET) formation. The goals of this study were to assess the role of NETs in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency.Methods:We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into Ldlr^–/– mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2^V617F).Results:Lnk-deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated Lnk^–/– platelets promoted increased NETosis when incubated with Lnk^–/– neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from Lnk^–/– platelets, as well as increased priming and response of Lnk^–/– neutrophils to OxPL. To counteract the effects of OxPL, we introduced a transgene expressing the single-chain variable fragment of E06 (E06-scFv). E06-scFv reversed accelerated NETosis, atherosclerosis, and thrombosis in Lnk^–/– mice. We also showed increased NETosis when human induced pluripotent stem cell–derived LNK(TT) neutrophils were incubated with LNK(TT) platelet/megakaryocytes, but not in isogenic LNK(CC) controls, confirming human relevance. Using data from the UK Biobank, we found that individuals with the JAK2^VF mutation only showed increased risk of coronary artery disease when also carrying the LNK R262W allele. Mice with hematopoietic Lnk^+/– and Jak2^VF clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with Jak2^VFLnk^+/+ controls.Conclusions:Hematopoietic Lnk deficiency promotes NETosis and arterial thrombosis in an OxPL-dependent fashion. LNK(R262W) reduces LNK function in human platelets and neutrophils, promoting NETosis, and increases coronary artery disease risk in humans carrying Jak2^VF mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.

中文翻译：

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氧化磷脂促进 LNK（SH2B3）缺乏症中的 NETosis 和动脉血栓形成

背景：LNK/SH2B3 通过造血细胞因子受体抑制 Janus 激酶/信号转导和转录激活因子 (JAK/STAT) 信号传导。全基因组关联研究表明，LNK（R262W，T 等位基因）中常见的单核苷酸多态性与中性粒细胞增多症、血小板增多症和冠状动脉疾病有关。我们已经证明LNK(TT ) 会降低 LNK 功能，并且 LNK 缺陷小鼠表现出显着的血小板-中性粒细胞聚集、加速动脉粥样硬化和血栓形成。血小板-中性粒细胞相互作用可以促进中性粒细胞胞外陷阱 (NET) 的形成。本研究的目的是评估 NET 在造血Lnk小鼠动脉粥样硬化和血栓形成中的作用方法：我们饲养Lnk和NETosis必需酶PAD4（肽基精氨酸脱亚胺酶4）联合缺乏的小鼠，并将其骨髓移植到Ldlr ^-/-小鼠体内。我们评估了 LNK 在具有 JAK2 功能变体 (JAK2 ^V617F ) 的人类和小鼠的动脉粥样硬化血栓形成中的作用。结果：Lnk缺陷小鼠表现出加速的颈动脉血栓形成和显着的 NETosis，而 PAD4 缺乏完全逆转了这种血栓形成。与Lnk一起孵育时，凝血酶激活的Lnk ^–/–血小板促进了 NETosis 的增加^–/–中性粒细胞与野生型血小板或野生型中性粒细胞相比。这涉及增加表面暴露和氧化磷脂 (OxPL) 从Lnk ^–/–血小板中的释放，以及增加Lnk ^–/–中性粒细胞对 OxPL 的启动和反应。为了抵消 OxPL 的影响，我们引入了表达 E06 (E06-scFv) 的单链可变片段的转基因。E06-scFv 逆转了Lnk ^–/–小鼠中加速的 NETosis、动脉粥样硬化和血栓形成。当人诱导的多能干细胞衍生的LNK(TT ) 中性粒细胞与LNK(TT ) 血小板/巨核细胞一起孵育时，我们还发现 NETosis 增加，但在同基因LNK(CC) 控制，确认人类相关性。使用来自英国生物银行的数据，我们发现具有 JAK2 ^VF突变的个体在同时携带 LNK R262W 等位基因时仅显示出冠状动脉疾病的风险增加。与Jak2 ^VF Lnk ^+/+对照相比，造血Lnk ^+/–和Jak2 ^VF克隆性造血的小鼠动脉血栓形成加速，但动脉粥样硬化不明显。结论：造血Lnk缺乏以 OxPL 依赖性方式促进 NETosis 和动脉血栓形成。LNK(R262W) 降低人血小板和中性粒细胞中的 LNK 功能，促进 NETosis，并增加携带者患冠状动脉疾病的风险Jak2 ^VF突变。针对 OxPL 的疗法可能对遗传定义的人群中的冠状动脉疾病有益。