Oxidized Phospholipids Promote NETosis and Arterial Thrombosis in LNK(SH2B3) Deficiency.

Abstract

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 <i>LNK</i> (R262W, T allele) with neutrophilia, thrombocytosis, and coronary artery disease. We have shown that <i>LNK(TT</i>) 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 <i>Lnk</i> deficiency. We bred mice with combined deficiency of <i>Lnk</i> and the NETosis-essential enzyme PAD4 (peptidyl arginine deiminase 4) and transplanted their bone marrow into <i>Ldlr^-/-</i> mice. We evaluated the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK2^V617F). <i>Lnk</i>-deficient mice displayed accelerated carotid artery thrombosis with prominent NETosis that was completely reversed by PAD4 deficiency. Thrombin-activated <i>Lnk^-/-</i> platelets promoted increased NETosis when incubated with <i>Lnk^-/-</i> neutrophils compared with wild-type platelets or wild-type neutrophils. This involved increased surface exposure and release of oxidized phospholipids (OxPL) from <i>Lnk^-/-</i> platelets, as well as increased priming and response of <i>Lnk^-/-</i> 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 <i>Lnk^-/-</i> mice. We also showed increased NETosis when human induced pluripotent stem cell-derived <i>LNK(TT</i>) neutrophils were incubated with <i>LNK(TT</i>) platelet/megakaryocytes, but not in isogenic <i>LNK(CC</i>) 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 <i>Lnk^+/-</i> and <i>Jak2^VF</i> clonal hematopoiesis showed accelerated arterial thrombosis but not atherosclerosis compared with <i>Jak2^VFLnk^+/+</i> controls. Hematopoietic <i>Lnk</i> 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 <i>Jak2^VF</i> mutations. Therapies targeting OxPL may be beneficial for coronary artery disease in genetically defined human populations.