Gwozdzinski L, Pieniazek A, Bernasinska J, Grabowski M, Kowalczyk E, Gwozdzinski K
General Surgery Ward, K. Jonscher Municipal Medical Center, Lodz, Poland; Department of Pharmacology and Toxicology, Medical University of Lodz, Lodz, Poland.
Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland.
Microvasc Res. 2017 May;111:72-79. doi: 10.1016/j.mvr.2016.12.005. Epub 2016 Dec 21.
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.
静脉曲张（VV）是皮下组织中扩张的静脉，通常由静脉瓣膜功能异常或受损导致血流受损引起。血液瘀滞、过度凝血障碍和静脉壁改变是维勒三联征的症状，可能会影响血液的形态成分，包括红细胞。本研究的目的是调查与慢性静脉疾病患者肘前静脉的红细胞相比，取自静脉曲张的红细胞特性的改变。使用电子顺磁共振光谱法和流式细胞术的自旋标记法对全红细胞进行了研究。细胞的内部粘度由Tempamine测定。内部蛋白质（主要是血红蛋白和膜蛋白）的构象状态由马来酰亚胺自旋标记（MSL，4-马来酰亚胺基-2,2,6,6-四甲基哌啶-1-氧基）测定。使用两种自旋标记脂肪酸（5-和16-二氧硬脂酸）测量质膜流动性，同时使用两种共价结合的自旋标记MSL和ISL [4-（2-碘乙酰胺基）-2,2,6,6-四甲基哌啶-1-氧基]测量膜蛋白的构象状态。还测定了红细胞的渗透脆性以及形状和大小。观察到取自静脉曲张的红细胞内部粘度降低。与正常静脉相比，位于脂质层极性区域的5-DS表明静脉曲张红细胞的脂质膜流动性显著降低。发现与静脉曲张红细胞膜蛋白结合的MSL和ISL的运动显著降低。通过测量渗透脆性也证实了静脉曲张红细胞质膜的变化。这些细胞比外周血静脉的红细胞对溶血更敏感。同时，取自静脉曲张的红细胞与外周静脉的红细胞在大小和形状上未观察到显著差异。总之，静脉曲张的红细胞表现出细胞内粘度降低和质膜流动性降低。同时，与同一慢性静脉疾病患者外周静脉获得的红细胞相比，发现这些细胞的膜蛋白构象变化和更高的渗透脆性。我们的研究结果强烈表明，红细胞质膜中呈现的异常可能具有重要的病理生理意义，包括细胞存活时间缩短和静脉曲张血液流变学改变。
