Increased Neutrophil Adhesive Properties In Patients with Venous Thromboembolism and Residual Vein Thrombosis and High D-Dimer Levels

Venous Thromboembolism (VTE) is a multifactorial disease that affects 1:1000 individuals worldwide, with a recurrence rate of about 25% in 10 years. Although many risk factors for VTE are well defined, first presentation and recurrence depend, at least in part, on as yet unknown etiologic factors. Studies in animal models show a tight relation between inflammation and hemostasis, as well as the infiltration of neutrophils in the venous wall after the induction of venous thrombosis. Neutrophils also participate in different stages in the formation and resolution of venous thrombosis. Methods: In this study, we investigated the adhesive properties of neutrophils in VTE patients. We hypothesized that increased adhesive properties of these cells, either as an individual baseline characteristic or as an acquired alteration after a previous VTE episode, could be associated with the thrombotic process. The patient population consisted of 22 VTE patients (14F:8M; median age: 46.1 years) that had completed at least 6 months of oral anticoagulation. Twenty-two healthy volunteers matched to VTE patients by age, gender and ethnic background were used as controls. Neutrophil adhesion was measured by a static adhesion assay in triplicate. Peripheral blood was collected with heparin and neutrophils were separated on Histopaque^® (Sigma-Aldrich, St. Louis, MO, USA). Isolated neutrophils (2.2×10⁶ cells/ml) were allowed to adhere to fibronectin (FN)-coated 96-well plates (30 min, 37°C, 5%CO2). Non-adherent cells were then removed by washing and adherent cells calculated as the percentage of cells adhered, compared to a standard curve of known cell concentrations and using a colorimetric enzyme assay. Results are expressed as means ± standard error of mean (SEM) and were compared using the Mann-Whitney test. Results: Overall, adhesion of neutrophils from VTE patients (25.40% ±2.35) was not increased when compared to the control group (21.25%±1.20 p=0.2). However when only patients at a higher risk of recurrence (n=13) - here defined as the presence of elevated D-dimer (higher than 0.5mg/L) and residual vein thrombosis - were analyzed, a statistically significant increase in cell adhesion compared to matched controls was observed (26.70%±2.08 and 21.36%±1.26, respectively, p = 0.04). When these patients (higher recurrence risk; n=13) were compared to the remaining VTE patients (standard recurrence risk, n=9), a non significant increase in neutrophil adhesion was observed (26.70%±2.08 vs 23.51%±5.03 respectively, p=0.1). Conclusions: We demonstrate that neutrophil adhesion is increased in patients with VTE with characteristics associated with increased recurrence risk. In addition, we also observed a non-significant difference in neutrophil adhesion in these patients compared to other VTE patients. Our results suggest that the increased adhesive properties of neutrophils in VTE patients could play a role in the exacerbation of inflammation, and in the pathophysiology of VTE. Further studies are warranted to study whether neutrophil adhesiveness could be used as a biomarker of VTE recurrence.

No relevant conflicts of interest to declare.