A Study on Experimentally Induced Thrombus Formation in JAK2-V617F and Calr-del52 Mouse Models Using Ligation of the Inferior Vena Cava (IVC)

Introduction

Arterial and venous thrombosis represent significant causes of mortality and morbidity in patients with Myeloproliferative Neoplasms (MPNs). While aberrant platelet and leukocyte activity has been identified as important cellular switchboards, the precise molecular chronology of pathologic blood coagulation in MPNs remains elusive. Our previous research (Edelmann et al., JCI, 2018) has demonstrated that JAK2-V617F induced over-activation of β1/β2 integrins and aberrant neutrophil adhesion to VCAM-1 and ICAM-1 are critical in pathological thrombus formation in MPNs. However, there is limited molecular data on the role of neutrophil adhesion in venous thrombosis associated with CALR mutations. This study aims to elucidate the molecular mechanisms underlying thrombosis formation in CALR-del52 and JAK2-V617F mutated MPNs, with a particular focus on the role of neutrophils.

Methods

We employed the inferior vena cava (IVC) stenosis mouse model, using Vav1-Cre x JAK2^+/+ and Vav1-Cre x JAK2^VF/+ mice (Mullally et al., 2010), as well as Vav1-Cre x CALR^+/+ and Vav1-Cre x CALR^+/del52 mice (Li et al., 2018). Thrombus size and weight and plasma cytokines levels were assessed. The role of integrins was investigated by treatment with and without anti-VLA-4/β2 integrin and anti-isotype antibodies. Additionally, MPN patients harboring JAK2-V617F and CALR mutations, along with healthy volunteers, were enrolled in the study. Citrated plasma samples were collected from these participants to profile cytokine levels using a multiplex assay for human cytokines. Furthermore, neutrophil extracellular trap (NET) formation was investigated using immunofluorescence microscopy.

Results

Using the IVC stenosis model, we observed a significant increase in thrombus size (p<0.0001) and weight (p<0.0022) in CALR-mutated mice (n=5) compared to CALR-WT mice (n=6). The fold change in thrombus size compared to the respective WT control post-ligation was found to be 4-fold in JAK2-V617F mice (Edelmann et al., JCI 2018) and only 2-fold in CALR-del52 mice. Additionally, thrombus induction time was 4 hours in JAK2-V617F mice and 12 hours in CALR-del52 mice. Profiling inflammatory cytokines revealed a significant elevation of G-CSF (25,898 ± 2,665 pg/ml; p=0.0001), IL-6 (466.4 ± 123.3 pg/ml; p=0.0036), IL-1α (73.33 ± 19.86 pg/ml; p=0.0042), IL-10 (20.33 ± 7.3 pg/ml; p=0.0203), and CXCL-1 (509 ± 131.7 pg/ml; p=0.0031) in both CALR-WT and CALR-del52 mouse models post ligation of IVC. Anti-VLA-4/β2 integrin antibody treatment in CALR-del52 mice (n=5) resulted in marked suppression of thrombus size (p<0.0001) and weight (p<0.0001), alongside with a reduction in elevated cytokine levels (G-CSF, p=0.0631; IL-6, p=0.0724; IL-1α, p=0.0374; IL-10, p=0.0840; CXCL-1, p=0.1637) compared to the IgG control (n=5) treatment group. Similarly, increases in G-CSF, IL-6, CXCL-1, and CCL-2 were observed in JAK2-V617F mutated mice (n=5) following IVC ligation. Furthermore, plasma profiling of JAK2-V617F patients (n=6) with a history of thrombosis showed upregulation of IL-6 (p=0.0078), IL-8 (p=0.0176), IL-10 (p=0.0175), sVCAM-1 (p=0.0001), CXCL-12 (p=0.0027), and MPO (p=0.0431) compared with heathy donors. Additionally, we selected one of the elevated cytokines post-ligation, CXCL-1, and used it to stimulate JAK2-V617F-mutated murine neutrophils. This stimulation of isolated murine neutrophils induced NETosis, as evidenced by CitH3 and DAPI staining.

Conclusion

Our study highlights the crucial role of neutrophil adhesion and cytokine modulation in venous thrombosis associated with CALR-del52 and JAK2-V617F mutations in MPNs. The significant suppression of thrombus size and inflammatory cytokine levels following anti-VLA-4/β2 integrin antibody treatment in CALR-del52 and JAK2-V617F mice suggests a potential therapeutic target. The similar cytokine profiles in JAK2-V617F mouse models and MPN patients underscore the relevance of our findings to human disease. These insights advance our understanding of the molecular mechanisms of thrombosis in MPNs, potentially guiding future therapeutic strategies.

Mougiakakos:Beigene: Consultancy, Honoraria; Galapagos: Consultancy, Honoraria; Miltenyi: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.