Interaction between JAK2-Mutated Neutrophils and Platelets Initiates Thrombosis Via Neutrophil Extracellular Traps

Thrombosis is one of the most common complications of myeloproliferative neoplasms with JAK2-V617F mutation. Recently, individuals with clonal hematopoiesis (CH) harboring JAK2-V617F mutation were also shown to have 12-fold higher risk of thrombosis compared to JAK2 non-mutated individuals (N Engl J Med. 2017;377:111-121). The finding that individuals with JAK2-V617F CH do not have altered blood counts suggests the mechanisms other than increases in the hematopoietic cells which induce thrombosis associated with JAK2-V617F hematopoiesis.

JAK2-V617F neutrophils are primed to neutrophil extracellular traps (NETs), which can promote thrombosis (Sci Transl Med. 2018;10: eaan8292). NETs were initially reported as an immunological reaction against infection. However, NETs also play various roles in non-infectious (sterile) settings, and the mechanism how sterile NETs are initiated in vivo remains largely unknown. Here, we studied the mechanisms how NETs are initiated and promote thrombosis in the presence of JAK2-V617F hematopoiesis.

First, we isolated neutrophils and platelets from Jak2-V617F transgenic ( Jak2VF) and wild-type (WT) mice, and co-cultured in each cell/genotype combination (see Figure1) at a 1:50 ratio. Of note, Jak2VF neutrophils formed NETs with Jak2VF platelets, but not with WT platelets. Also, Jak2VF platelets slightly increased formation of NETs by WT neutrophils. However, culture supernatant of Jak2VF platelets did not induce formation of NETs by Jak2VF neutrophils. These findings indicated that direct interaction, between Jak2VF neutrophils and Jak2VF platelets, initiates sterile NETs in the presence of JAK2-V617F hematopoiesis. Surprisingly, platelet activation of Jak2VF platelets measured by P-selectin release was inferior to WT platelets. Thus, it was unlikely that overactivation of Jak2VF platelets fully initiate NETs.

Subsequently, we sought an initiator on Jak2VF platelets to form NETs in the presence of JAK2-V617F hematopoiesis. Unbiased proteomics analysis of WT and Jak2VF platelets identified several proteins significantly enriched in Jak2VF platelets, including a membrane protein PODXL, which is expressed on podocytes in kidney as well as platelets and other hematopoietic cells. Interestingly, PODXL on platelet is associated with aggregation with other hematopoietic cells depending on CD41, SLC44A2 and P-selectin. Therefore, we hypothesized that PODXL enhances aggregation of Jak2VF platelets with neutrophils and initiates formation of NETs (Figure 2). We confirmed overexpression of PODXL in Jak2VF platelets and in platelets of another murine model, hematopoietic cell-specific Jak2^V617F/+ mice by FACS and immunofluorescence imaging. The formations of NETs measured by intensity of citrullinated histone H3 (citH3) correlated with aggregation of PODXL-expressing Jak2VF platelets on cell surface. Presently, we are conducting to confirm the findings utilizing patients' samples with JAK2-V617F.

To test if a JAK inhibitor prevents PODXL-associated formation of NETs, we collected platelets from ruxolitinib-treated Jak2VF mice. Platelets from ruxolitinib-treated mice similarly expressed PODXL and induced formation of NETs with neutrophils from untreated Jak2VF mice, compared to platelets from untreated Jak2VF mice, suggesting that ruxolitinib does not alter platelets' ability to initiate formation of NETs.

Next, we tested if these mechanisms induce venous and arterial thrombosis in vivo, utilizing models of half ligation of inferior vena cava (IVC) and photochemically induced thrombosis (PIT) in femoral artery. In both models, Jak2VF or Jak2^V617F/+ mice formed thrombosis more quickly than WT controls. Jak2-mutated mice formed white thrombus presenting citH3-positive neutrophils aggregated with PODXL-expressing platelets at the core of the thrombus, while WT mice mainly formed red thrombus without any nucleated cells in these models. Therefore, we are generating platelet-specific knockout mice of PODXL to directly clarify if PODXL on Jak2VF-platelet is the initiator of NETs and thrombosis.

In conclusion, PODXL on JAK2-V617F platelets may be a key player in the pathogenesis of thrombosis associated with formation of NETs with JAK2-V617F neutrophils in myeloproliferative neoplasms.