Increased Adhesive Properties and Reactive Oxygen Species of Red Cells, Platelets and Leukocytes and Neutrophil Chemotaxis in IVS-1-6 (T → C) Homozygous β-Thalassemia Intermedia.

The pathophysiology of b-thalassemia intermedia (TI) is characterized by extravascular hemolysis, with the release into the peripheral circulation of damaged red blood cells and erythroid precursors. In these patients vascular complications and endothelial cell activation are more frequent and activation of vascular endothelium is considered an important aspect of inflammation, vasculitis and thrombosis in this disease. The mutation at position +6 of the IVS-1 5’ (donor) consensus sequence is particularly mild and is usually associated with TI owing to a relatively high level of normal mRNA splicing. Some authors have been suggesting that red blood cells (RBC) adhesion and reactive oxygen species (ROS) are significantly increased in b-thalassemia major, however few studies evaluated these alterations in TI. The aim of this study was to evaluate expression of adhesion molecules, adhesion proprieties of platelets, RBC and neutrophils, RBC and leukocytes ROS production and neutrophil chemotaxis in TI IVS-1–6 (T → C) homozygous. Neutrophils, RBC and platelets were isolated from the peripheral blood of healthy controls (n=16) and patients with TI (n=16). Basal adhesion was compared using static adhesion assays and surface protein expression using flow cytometry. Chemotaxis of control and TI neutrophils (4x10⁶ cells/mL in RMPI medium) were assessed using static adhesion assays and a 96-well chemotaxis chamber assay (ChemoTX, Neuroprobe). For ROS measurement, the isolated cells were incubated with 2’-7’-dichlorofluorescin diacetate (DCF) and analyzed by flow cytometry. TI neutrophils demonstrate a greater ability to adhere to fibronectin (FN)-coated plates than control individual (13.2±1.2 % neutrophils compared to 7.7±0.7 %; P=0.003 Mann-Whitney test). Similar results were found with TI red cell adhesion to FN and TI platelet adhesion to fibrinogen compared to respective cells in control individual (7.24±0.6%, 21.2±3.3% RBC and platelets adhered compared to 3.97±0.5% and 9.5 ± 1.0%, p=0.001; p= 0.005, respectively). The integrin α-subunits (CD11a and CD11b) surface expression did not differ on neutrophils from TI patients and control individual. However, the VLA-4 integrin subunit CD49d surface expression was higher on TI neutrophils compared to controls individuals (4.94±0.71% vs 2.31±0.46%; p=0.013). The surface expressions of CD36, CD49d and CD71 on the RBC of TI patients were significantly higher than on these cells in control individuals (10.93±1.83%, 1.44±0.30%, 14.3±1.83%; 2.31±0.46%; 0.63±0.10% vs 0.13±0.02% and 1.95±0.37%, p<0.0001, respectively). Chemotaxis assays showed that TI neutrophils demonstrate greater spontaneous migration when compared with control neutrophils (4.1±0.29x10⁵/mL and 2.46±0.3x10⁵/mL; p=0.012, respectively). ROS production was significantly increased in TI RBC, neutrophils and mononuclear cells compared to the same cells in control individuals (236.2±22.8, 2352±222.8, 1255.9±193.5 vs 53.7±5.3; 1459.8±143.8, 329.6±47.6 MFC, p<0.0001; p=0.010; p<0.0001, respectively). In addition, superoxide dismutase (SOD) plasma activity was significantly reduced in TI compared to control plasma (4.8±1.7 vs 12.4±0.8 P=0.003, respectively). Our results show that the adherence to extracellular matrix protein was markedly enhanced for red blood cells, neutrophils and platelets from TI. Leukocytes and red blood cells of TI patients produce higher baseline levels of free radicals and the anti-oxidant mechanism in plasma is diminished. These data suggest that enhanced adherence (neutrohils, RBC and platelets), neutrophil chemotaxis and ROS production (neutrophils, mononuclear cells and RBC) may contribute to several clinical complications of β-thalassemia intermedia, such as pulmonary hypertension.