Diagnostics, Science & Technology: Winning Abstract 2017
Proteomic investigation of extracellular vesicles obtained from ex vivo cultured reticulocytes
J. Kittivorapart, A. Toye, N. Siritanaratkul, V. Karamatic Crew
School of Biochemistry, University of Bristol
Bristol Institute for Transfusion Sciences (BITS), NHSBT, Bristol
Faculty of Medicine Siriraj Hospital , Bangkok
Background:
Thalassaemia is the most common monogenic gene disorder with a wide spectrum of clinical manifestations, from asymptomatic to severe, requiring regular transfusion. Thalassaemia intermedia is a milder form where patients do not need regular transfusions.
Many factors contribute to their transfusion requirements and clinical severity, including extracellular vesicles (EV) produced due to oxidative stress in thalassaemic erythrocytes. This study focuses on thalassaemia intermedia and aims to explore the differences in ex vivo erythropoiesis and constituents of extracellular vesicles released by thalassaemic and normal erythroid cells.
Methods:
CD34+ haematopoietic cells were isolated from peripheral blood of three thalassaemic patients and matched controls, and were cultured in three-staged medium with dexamethasone to monitor the proliferation and viability rates. At the end of the culture, EVs were isolated by ultra centrifugation and proteomic studies were performed by Duplex-Tandem Mass Tag mass spectrometry.
Results:
Two of the three of thalassaemic samples had higher proliferative growth than the matched controls (11500 vs 488 for P1/C1, and 3122 vs 63 folds increase for P3/C3). Increased cell death after polychromatophilic stage was not observed, contrary to the pathology of in vivo thalassaemic erythrocytes.The proteomic data of EVs isolated from the cultured reticulocytes demonstrated 1,706 differentially expressed proteins, of which 655 proteins contained >2 unique peptides. 286 proteins were identified across all thalassaemic and control samples, where 193 (72%) of these were known constituents of vesicles. Majority of these proteins are involved in metabolic process (78%) and biological regulation (72%), whilst 203 of them facilitate protein binding activity and cell homeostasis. Furthermore,there were no significant differences in protein expression levels between patient and control EVs samples. Thus, in vitro erythroid EVs were likely to be products of normal erythropoiesis, shed during cell maturation; unlike the in vivo EVs isolated from plasma of thalassaemic patient, which were abundant in antioxidant proteins as a response to increased oxidative stress (Kittivorapart et al, Haematologica, 2017, in press).
Conclusion:
In vitro produced thalassaemic reticulocytes did not have different EV composition and did not show a protein profile typical of increased hemolysis or oxidative stress, unlike their in vivo counterparts.
