Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Dou H

PROVIDER: S-EPMC8663540 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:RationaleHuman genome-wide association studies have revealed novel genetic loci that are associated with coronary heart disease. One such locus resides in LNK/SH2B3, which in mice is expressed in hematopoietic cells and suppresses thrombopoietin signaling via its receptor myeloproliferative leukemia virus oncogene. However, the mechanisms underlying the association of LNK single-nucleotide polymorphisms with coronary heart disease are poorly understood.ObjectiveTo understand the functional effects of LNK single-nucleotide polymorphisms and explore the mechanisms whereby LNK loss of function impacts atherosclerosis and thrombosis.Methods and resultsUsing human cord blood, we show that the common TT risk genotype (R262W) of LNK is associated with expansion of hematopoietic stem cells and enhanced megakaryopoiesis, demonstrating reduced LNK function and increased myeloproliferative leukemia virus oncogene signaling. In mice, hematopoietic Lnk deficiency leads to accelerated arterial thrombosis and atherosclerosis, but only in the setting of hypercholesterolemia. Hypercholesterolemia acts synergistically with LNK deficiency to increase interleukin 3/granulocyte-macrophage colony-stimulating factor receptor signaling in bone marrow myeloid progenitors, whereas in platelets cholesterol loading combines with Lnk deficiency to increase activation. Platelet LNK deficiency increases myeloproliferative leukemia virus oncogene signaling and AKT activation, whereas cholesterol loading decreases SHIP-1 phosphorylation, acting convergently to increase AKT and platelet activation. Together with increased myelopoiesis, platelet activation promotes prothrombotic and proatherogenic platelet/leukocyte aggregate formation.ConclusionsLNK (R262W) is a loss-of-function variant that promotes thrombopoietin/myeloproliferative leukemia virus oncogene signaling and platelet and leukocyte production. In mice, LNK deficiency is associated with both increased platelet production and activation. Hypercholesterolemia acts in platelets and hematopoietic progenitors to exacerbate thrombosis and atherosclerosis associated with LNK deficiency.

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