Key Points• Specific interaction of DF with EC membranes is followed by its internalization mainly through macropinocytic mechanisms.• DF attachment to the cell membrane is sufficient to perform its antiinflammatory and antioxidant effects on the endothelium.Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization.Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations. (Blood. 2016;127(13):1719-1727 Introduction Defibrotide (DF) is a mixture of 90% single-stranded phosphodiester oligonucleotides (length, 9-80 mer; average molecular mass, 16.5 6 2.5 KDa) and 10% double-stranded phosphodiester oligonucleotides, derived from the controlled depolymerization of porcine intestinal mucosal DNA.1-3 Several functions, specially related to hemostasis, have been ascribed to DF. 4 In this regard, our group has demonstrated the protective effect of DF on the endothelium, by preventing the endothelial damage associated with hematopoietic cell transplantation (HCT) conditions, 5,6 and with the deleterious effect of immunosuppresants. 7 In our in vitro endothelial activation model, DF has exhibited reproducible effects on endothelial cells (ECs) from different origins. DF demonstrates antiinflammatory, antithrombotic, and antiapoptotic properties. However, although its effects are increasingly better understood, its precise mechanism of action remains to be elucidated.There is limited knowledge about DF pharmacokinetics, pharmacodynamics, and mechanisms of action. [8][9][10] However, 2 distinct properties of ...

The aim of the present study was to explore whether there is enhanced endothelial dysfunction in patients developing acute GvHD (aGvHD) after allogeneic hematopoietic cell transplantation (allo-HCT) and to identify biomarkers with predictive and/or diagnostic value. In in vitro experiments, endothelial cells (ECs) were exposed to serum from patients with (aGvHD, n=31) and without (NoGvHD, n=13) aGvHD, to evaluate changes in surface adhesion receptors, the reactivity of the extracellular matrix by measuring the presence of Von Willebrand factor (VWF) and platelet adhesion, and the activation of intracellular signaling proteins. Plasma levels of VWF, ADAMTS-13, TNF receptor 1 (TNFR1), soluble vascular cell adhesion molecule 1 and soluble intercellular adhesion molecule 1 were also measured. In vitro results showed a more marked proinflammatory and prothrombotic phenotype in ECs in association with aGvHD. Regarding circulating biomarkers, levels of VWF and TNFR1 above an optimal cutoff score, taken independently or combined, at day 7 after allo-HCT, would be able to positively predict that around 90% of patients will develop aGvHD. Our results demonstrate that endothelial damage is aggravated in those allo-HCT recipients developing aGvHD, and that VWF and TNFR1 are promising predictive aGvHD biomarkers. These findings could contribute to improve the understanding of the pathophysiology of aGvHD.