Neutrophil activation and circulating neutrophil extracellular traps are increased in venous thromboembolism patients for at least one year after the clinical event

Abstract

Neutrophil activation and neutrophil extracellular traps (NETs) have been associated with the pathogenesis of venous thromboembolism (VTE). Considering VTE-associated chronic sequelae, which suggest that some pathological mechanisms remain after the acute episode, we investigated whether neutrophil activation is increased in patients with a prior VTE at least one year before this investigation. Thirty-seven patients with prior VTE and 37 individuals with no history of VTE were included. Neutrophil activity was evaluated by the expression of the adhesive molecule activation-specific epitopes LFA-1 (CD11a) and MAC-1 (CD11b), chemotaxis, reactive oxygen species (ROS) and by MPO-DNA complexes as markers of NETs. The adhesive molecules sICAM-1 and sVCAM-1, involved in the cross talk between neutrophil and endothelial cells, were also evaluated. Patient neutrophils presented increased CD11a expression before and after TNF-α stimulus, whereas increased CD11b expression was observed only after TNF-α stimulus, as compared to controls. Neutrophil chemotaxis on both, basal state and after IL-8 stimulus, on circulating levels of sICAM-1 and sVCAM-1, and on MPO-DNA complexes were also increased in VTE patients. ROS release was similar between patients and controls. This is, to our knowledge, the first study to investigate neutrophil inflammatory activity in VTE patients a long period after an acute event (approximately 2 years). The results showed altered neutrophil activation patterns in these patients. While activated neutrophils can cause endothelial activation and injury, the activated endothelium can induce the release of NETs with consequent endothelial cytotoxicity, creating a vicious cycle of activation between neutrophils and endothelium that can lead to thrombosis. VTE patients (approximately 2 years after the clinical event) present an altered neutrophil activation state evidenced by increased activity of the LFA-1 and Mac-1 adhesive molecules, as well as increased chemotaxis and circulating levels of NETs remnants. Circulating levels of ICAM-1 and VCAM-1, which are endothelial adhesive molecules, are also increased in VTE patients, suggesting not only an exacerbated endothelial activation and dysfunction, but also an interaction of the neutrophil adhesive molecules with their endothelial ligands, favoring the migration process of neutrophil.

Keywords: Adhesion; Chemotaxis; Endothelial dysfunction; Extracellular traps; Neutrophil; Reactive oxygen species; Venous thromboembolism.

Fig. 1

Fluxogram of VTE patients inclusion in the study

Fig. 2

Assessment of neutrophil activation status in VTE patients one year at least after thrombosis compared to controls. a, c Expression of CD11a (LFA-1) and CD11b (MAC-1) in their activated conformations on basal state and b, d after stimulation by TNF-a (flow cytometry). e Spontaneous and f IL-8-stimulated neutrophils chemotaxis. g Analysis of neutrophil oxidative stress by ROS production (flow cytometry). h NETs activity assessed by DNA complex and neutrophil-derived MPO protein. The p value were calculated by Mann–Whitney U

Fig. 3

Increased level of soluble endothelial adhesion molecules in serum in VTE patients: a sICAM-1 and b sVCAM-1. The p value were calculated by Student t and Mann–Whitney U test, respectively