Oxidized Phospholipids Promote NETosis and Arterial Thrombosis in LNK(SH2B3) Deficiency
Authors: Dou Huijuan , Kotini Andriana , Liu Wenli , Fidler Trevor , Endo-Umeda Kaori , Sun Xiaoli , Olszewska Malgorzata , Xiao Tong , Abramowicz Sandra , Yalcinkaya Mustafa , Hardaway Brian , Tsimikas Sotirios , Que Xuchu , Bick Alexander , Emdin Conor , Natarajan Pradeep , Papapetrou Eirini P. , Witztum Joseph L. , Wang Nan , Tall Alan R. 
Published: November 2021

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
VF
Lnk
+/+

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.