ABSTRACT
Varicose veins (VV) are enlarged veins of the subcutaneous tissue, usually caused by faulty or damaged venous valves leading to impaired blood flow. Blood stasis, excessive clotting disorder and alterations in the vein walls are symptoms of Virchow's triad which may affect the morphotic elements of blood, including erythrocytes. The aim of this study was to investigate alterations in the properties of the erythrocytes taken from varicose veins in comparison to those from antecubital vein of patients with chronic venous disease. The investigation was conducted on whole erythrocytes using spin labeling method in EPR spectroscopy and flow cytometry. The internal viscosity of cells was determined by Tempamine. The conformation state of internal proteins, mainly hemoglobin and membrane proteins was determined by maleimide spin label (MSL, 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl). The plasma membrane fluidity was measured using two spin labeled fatty acids (5- and 16-doxylstearic acid), while conformational state of membrane protein was measured using two covalently bound spin labels MSL and ISL [4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl]. The osmotic fragility and the shape and size of the erythrocytes were also determined. A decrease in internal viscosity of the erythrocytes from varicose vein was observed. A significant decrease in lipid membrane fluidity indicated by 5-DS, which is located at the polar region of lipid layer was found in the erythrocytes from varicose vein in comparison to normal vein. A significant decrease in the motion of MSL and ISL attached to erythrocyte membrane proteins from varicose vein was found. Changes in the plasma membrane of the erythrocytes from varicose vein were also confirmed by measuring osmotic fragility. These cells were more sensitive to hemolysis than red blood cells from the peripheral blood vein. Meanwhile, no significant differences in size and shape were observed between the erythrocytes taken from varicose veins and those from peripheral veins. In conclusion, the erythrocytes from varicose veins exhibited decreased intracellular viscosity and decreased plasma membrane fluidity. At the same time, conformational changes of membrane proteins and higher osmotic fragility of these cells were found in comparison to the erythrocytes obtained from peripheral veins in the same patients with chronic venous disease. Our findings strongly suggest that presented abnormalities in the erythrocyte plasma membrane may have significant pathophysiological implications, including shortened cell survival and alterations in the hemorheology of the varicose vein blood.
