Subcellular localization of radioactively labelled defibrotide in cultured endothelial cells

• Published in: Thrombosis research Vol. 66; no. 4; pp. 385 - 390
• Main Authors: Bilsel, S., Erşahin, Ç., Taga, Y., Emerk, K.
• Format: Journal Article
• Language: English
• Published: New York, NY Elsevier Ltd 15.05.1992; Elsevier Science
• Subjects: Binding Sites; Biological and medical sciences; Blood. Blood coagulation. Reticuloendothelial system; Cell Membrane - chemistry; Cell Nucleus - chemistry; Cells, Cultured; Defibrotide; Endothelial cells; Endothelium, Vascular - chemistry; Endothelium, Vascular - cytology; Humans; Infant, Newborn; Medical sciences; Pharmacology. Drug treatments; Polydeoxyribonucleotides - analysis; Subcellular Fractions - chemistry; Subcellular localization; Umbilical Veins; Endothelial cells; Defibrotide; Subcellular localization; Cell culture; Endothelial cell; Cell nucleus; Plasma membrane; Anticoagulant; Binding site; In vitro; Umbilical vein; Fibrinolytic
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/0049-3848(92)90287-K

Defibrotide is a new antithrombotic and fibrinolytic drug which is obtained by controlled depolymerization of mammalian DNA. In various models of arterial and venous thrombosis, it has been shown that it induces tissue plasminogen activator [tPA] and prostacyclin [PGI 2] release from the vessel wall. We have previously shown the presence of specific binding sites with a K d of 4.2 μg/ml for radioactively labelled defibrotide. The present study was undertaken to identify the location of the binding site. Confluent cultures of endothelial cells from human umbilical vein were incubated with media containing 3H-acetyl-defibrotide for various intervals of time. Cells were then washed and harvested nonenzymatically. Subcellular location of 3H-defibrotide was investigated by fractionating cells on discontinous sucrose gradient and measuring the distribution of radioactivity. 5′-nucleotidase enzyme activity was also measured to ensure the location of membrane fraction. Our results suggest that the major location of 3H-defibrotide in endothelial cells is the plasma membrane. On the other hand, nuclei also contain a considerable amount of the drug which suggests a mechanism where binding to a membrane protein is followed by internalization.