Circulating Microparticles in Lebanese Patients with beta-Thalassemia Intermedia.
Authors: Habib Aida , Kunzelmann Corinne , Shamseddeen Wael , Isma’eel Hussain , Zobairi Fatiha , Freyssinet Jean-Marie , Taher Ali 
Published: October 2019

Abstract
Introduction: Beta-thalassemia is a genetic disease with a prevalence that varies in the world reaching 3% in Lebanon. One-third of these patients have beta-thalassemia intermedia. Patients with thalassemia intermedia have complications not present in those with thalassemia major, mainly leg ulcers, extramedullary hematopoiesis and thrombosis. The beta-thalassemia intermedia patients not receiving transfusions and who are splectomized, are at higher risk of thrombosis as well as the other complications. Microparticles (MP) are shed submicrometric plasma membrane fragments (~0.1–1μm) harboring negatively charged phosphatidylserine (PS) in their extracellular membrane leaflet. They mainly stem from apoptotic or activated cells, and generally present a procoagulant potential. Increased levels of MP were described in disorders with major vascular and thrombotic symptoms such as myocardial infarction, diabetes, renal failure or sickle cell disease.
Methods: In this study we determined circulating MP levels in platelet-free plasma samples of patients and healthy donors using a solid-phase capture assay on insolubilized streptavidin. The presence of circulating MP was first evaluated using annexin V-coupled biotin that binds the negatively charged PS in MP. Independent sample t-test was conducted to examine significant difference between patients and controls. Thirty-two patients with beta-thalassemia intermedia and 17 healthy controls were included. P-values less than 0.05 were considered to be statistically significant.
Results: Fifty percent of the patients and 81.3% of the control group were males. The average age of the patients was 26.5 ± 9.5 years. Around 77% of the patients had splenectomy. Shed membrane MP were elevated to near statistical significance in platelet-free plasma of patients with beta-thalassemia intermedia but not in control individuals (7.3 ± 5.7 vs 4.7 ± 2.9 nmol/L PS equivalent, p=0.09). The cell origin of MP was further investigated after capture onto specific biotinylated antibodies used instead of annexin V. MP of red blood cell origins were elevated, as expected in patients with beta-thalassemia intermedia vs controls (6.05 ± 3.8 vs 2.54 ± 3.8 nmol/L PS equivalent, p<0.01 for patients and controls, respectively). Interestingly, both platelet and endothelial-derived MP were elevated in patients vs controls (of platelet origin: 3.02 ± 2.06 vs 1.91 ± 1.15 nmol/L PS equivalent, p=0.05; and of endothelial origin: 1.58 ± 0.87 vs 0.41 ± 0.3 nmol/L PS equivalent, p<0.01). Leukocyte and neutrophil-derived MP were also elevated in patients vs control (p=<0.01).
Conclusion: These results indicate that circulating membrane MP are increased in beta-thalassemia intermedia and testify to increased platelet, endothelium and leukocyte activation, in probable association with the increased thrombotic risk. Further investigations are required to assess the significance of these enhanced markers of cell activation, with respect to a possible deleterious potential towards vascular or blood cell functions.