Defibrotide Inhibits Antiphospholipid Antibody-Mediated Neutrophil Extracellular Trap Formation and Venous Thrombosis

Abstract

Objective: Defibrotide is a heterogenous mixture of polyanionic oligonucleotides currently approved for treatment of transplant-associated venoocclusive disease. While defibrotide has a known role in limiting endothelial cell activation, some studies have also demonstrated anti-leukocyte properties. In a recent study, we found that neutrophil extracellular traps (NETs) play a role in the thrombotic complications of antiphospholipid syndrome (APS). In the present study, we investigated the hypothesis that defibrotide might act to mitigate APS-relevant NET formation in vitro and in mouse models.

Methods: We used in vitro assays and a mouse model to determine the mechanisms by which defibrotide inhibits NET formation and venous thrombosis in APS.

Results: At doses ranging from 1 to 10 μg/ml, defibrotide significantly suppressed NET formation from control neutrophils stimulated with IgG isolated from patients with APS. Defibrotide increased levels of intracellular cyclic AMP in neutrophils, and its suppressive effects on NET formation were mitigated by blocking adenosine A_2A receptor or by inhibiting the cyclic AMP-dependent kinase protein kinase A. Defibrotide at doses ranging from 15 to 150 mg/kg/day inhibited NET formation and venous thrombosis in a model of antiphospholipid antibody-accelerated thrombosis-an effect that was reduced in adenosine A_2A receptor-knockout mice.

Conclusion: This study is the first to demonstrate mechanisms by which defibrotide counteracts neutrophil-mediated thrombotic inflammation inherent to APS.

Figure 1:. Defibrotide suppresses NET formation in response to various stimuli through adenosine A_2A receptor agonism.

A-B, Human neutrophils were isolated from healthy volunteers and then treated with PMA (A) or APS IgG (B) for 3 hours in the presence of different concentrations of defibrotide. NET formation was quantified by measuring the enzymatic activity of nuclease-liberated myeloperoxidase (MPO). C, Representative images of NET formation assessed qualitatively by immunofluorescence microscopy. Neutrophils were treated with APS IgG in the presence or absence of defibrotide. Defibrotide=1 μg ml⁻¹. Green=extracellular neutrophil elastase and blue=DNA. Scale bar=100 microns. D, Human neutrophils were treated with forskolin (10 minutes), CGS21680 (30 minutes), or defibrotide (30 minutes), and cyclic AMP (cAMP) levels were measured. E-F, Neutrophils were treated with APS IgG in the presence or absence of defibrotide (1 μg ml⁻¹). Some samples were additionally treated with a protein kinase A (PKA) inhibitor (10 μM), an adenosine A₁ receptor antagonist (10 μM), or an adenosine A_2A receptor antagonist (10 μM). NET formation was quantified by measuring the enzymatic activity of nuclease-liberated MPO. Throughout the figure, mean and standard error of the mean (SEM) are presented for 3 independent experiments; *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by one-way ANOVA corrected with Dunnett’s test; ns=not significant.

Figure 2:. Defibrotide prevents antiphospholipid antibody-mediated venous thrombosis in wild-type mice but not in adenosine A_2A receptor knockout mice.

A, Schematic depiction of the electrolytic inferior vena cava model of venous thrombosis. The application of direct current to a copper wire results in the release of free radicals. This activates endothelial cells and triggers a thrombogenic environment. Blood flow remains constant. B-C, C57BL/6J mice were treated with control IgG or APS IgG in the presence or absence of defibrotide. Thrombus formation was determined after 24 hours. Thrombus length (B) and myeloperoxidase (MPO)-DNA complexes in serum (C) were quantified. D, Cyclic AMP (cAMP) levels in neutrophils isolated from Adora2a^fl/fl MRP8-Cre⁺ as compared with Adora2a^fl/fl MRP8-Cre⁻ mice in the presence or absence of defibrotide (1 μg ml⁻¹) for 30 minutes. E-F, Either Adora2a^fl/fl MRP8-Cre⁺ or Adora2a^fl/fl MRP8-Cre⁻ mice were treated with control IgG or APS IgG in the presence or absence of defibrotide. Thrombus formation was assessed at 24 hours. Thrombus length (E) and MPO-DNA complexes in serum (F) were quantified. Each dot represents a unique mouse. Throughout the figure, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 by one-way ANOVA corrected with Dunnett’s test; ns=not significant.