Abstract 14183: Oxidized Phospholipids Promote NETosis and Arterial Thrombosis in LNK(SH2B3) Deficiency

Abstract

Background and Objective: GWAS have shown association of a common SNP (R262W) in LNK with neutrophilia, thrombocytosis and coronary artery disease (CAD). We have shown that LNK(R262W) 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 neutrophil extracellular traps (NETs) in atherosclerosis and thrombosis in mice with hematopoietic Lnk deficiency and to evaluate the role of LNK in atherothrombosis in humans and mice bearing a gain of function variant in JAK2 (JAK V617F ). Methods and Results: We bred mice with combined deficiency of Lnk and the NETosis-essential enzyme peptidylarginine deiminase4 (PAD4); their bone marrow was transplanted into Ldlr-/- mice. There was accelerated carotid artery thrombosis with prominent NETosis in Lnk deficient mice with complete reversal by PAD4 deficiency. In contrast, PAD4 deficiency abrogated increased NETosis in plaques but did not alter plaque size or morphology. Thrombin-activated Lnk -/- platelets promote NETosis when incubated with Lnk -/- neutrophils compared to WT platelets or WT 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. Using data from UK Biobank we showed that individuals with the JAK2 VF mutation only showed increased CAD when also carrying the LNK R262W allele. Mice with hematopoietic Lnk +/- and Jak2 VF clonal hematopoiesis, showed accelerated arterial thrombosis but not atherosclerosis compared to Jak2 VF Lnk +/+ controls. Conclusions: Increased NETosis in hematopoietic LNK deficiency promotes arterial thrombosis in an oxPL-dependent fashion. Reduced LNK function may increase CAD in humans carrying JAK2 VF mutations possibly reflecting increased arterial thrombotic risk.