RESUMO
Splenectomised ß-thalassaemia/haemoglobin E (HbE) patients have increased levels of circulating microparticles or medium extra-cellular vesicles (mEVs). The splenectomised mEVs play important roles in thromboembolic complications in patients since they can induce platelet activation and endothelial cell dysfunction. However, a comprehensive understanding of the mechanism of mEV generation in thalassaemia disease has still not been reached. Thalassaemic mEVs are hypothesised to be generated from cellular oxidative stress in red blood cells (RBCs) and platelets. Therefore, a proteomic analysis of mEVs from splenectomised and non-splenectomised ß-thalassaemia/HbE patients was performed by liquid chromatography with tandem mass spectrometry. A total of 171 proteins were identified among mEVs. Interestingly, 72 proteins were uniquely found in splenectomised mEVs including immunoglobulin subunits and cytoskeleton proteins. Immunoglobulin G (IgG)-bearing mEVs in splenectomised patients were significantly increased. Furthermore, complement C1q was detected in both mEVs with IgG binding and mEVs without IgG binding. Interestingly, the percentage of mEVs generated from RBCs with IgG binding was approximately 15-20 times higher than the percentage of RBCs binding with IgG. This suggested that the vesiculation of thalassaemia mEVs could be a mechanism of RBCs to eliminate membrane patches harbouring immune complex and may consequently prevent cells from phagocytosis and lysis.
Assuntos
Hemoglobina E , Proteômica , Talassemia beta , Humanos , Talassemia beta/sangue , Talassemia beta/metabolismo , Hemoglobina E/metabolismo , Proteômica/métodos , Feminino , Masculino , Adulto , Vesículas Extracelulares/metabolismo , Esplenectomia , Imunoglobulina G/sangue , Membrana Eritrocítica/metabolismo , Proteoma/análise , Adolescente , Eritrócitos/metabolismo , Micropartículas Derivadas de Células/metabolismo , Adulto JovemRESUMO
Pulmonary arterial hypertension (PAH) is a serious complication in ß-thalassemia. The mechanism of PAH development is believed to be through chronic platelet activation and red cell (RBC) membrane abnormality contributing to a hypercoagulable state and thrombosis, which consequently leads to the development of PAH. Extracellular vesicles (EVs) shed from the plasma membrane of platelets and RBCs are found to be associated with thrombotic risk. This study aimed to investigate the involvement of phosphatidylserine (PS)-bearing cells and EVs in accelerating the progression of the hypercoagulable state in transfusion-dependent thalassemia (TDT) patients. Fresh whole blood samples from splenectomized TDT-ß-thalassemia/HbE patients (11 with PAH and 14 without PAH) and 15 normal subjects were analyzed for platelet activation by measuring P-selectin expression using flow cytometry and the number of dense granular using an electron microscope. The amounts of PS-bearing RBCs, large RBC-EVs, platelets, and medium EVs were determined by flow cytometry. Platelet activation in PAH patients was not significantly different from other groups; however, the amounts of PS-bearing large RBC-EVs, platelets, and medium platelet-derived EVs were significantly increased in PAH patients as compared to normal subjects, but they were not different from patients without PAH. This could be affected by antiplatelet therapy that reduced the levels of platelet activation and the amount of PS-bearing cells, including EVs, in PAH patients as well as in patients without PAH.
Assuntos
Plaquetas , Micropartículas Derivadas de Células/metabolismo , Eritrócitos , Hipertensão Pulmonar/sangue , Talassemia beta/sangue , Adulto , Biomarcadores/sangue , Transfusão de Sangue , Feminino , Hemoglobina E , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/terapia , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária , Esplenectomia , Talassemia beta/complicações , Talassemia beta/terapiaRESUMO
Thromboembolic events including cerebral thrombosis, deep vein thrombosis, and pulmonary embolism are major complications in ß-thalassemia. Damaged red blood cells and chronic platelet activation in splenectomized ß-thalassemia/HbE patients were associated with increased microparticles (MPs) releases into blood circulation. MPs are small membrane vesicles, which play important roles on coagulation. However, the role of MP in thalassemia is poorly understood. In this study, the effects of splenectomized-MPs on platelet activation and aggregation were investigated. The results showed that isolated MPs from fresh platelet-free plasma of patients and normal subjects directly induce platelet activation, platelet aggregation, and platelet-neutrophil aggregation in a dose-dependent manner. Interestingly, MPs obtained from splenectomized patients are more efficient in induction of platelet activation (P-selectin+) when compared to MPs from normal subjects (P < 0.05), tenfold lower than pathophysiological level, at 1:0.1 platelet MP ratio. Co-incubation of splenectomized-MPs with either normal-, non-splenectomized- or splenectomized-platelets at 1:10 platelet MP ratio increased platelet activation up to 5.1 ± 2.2, 5.6 ± 3.7, and 9.5 ± 3.0%, respectively, when normalized with individual baseline. These findings suggest that splenectomized patients were proned to be activated by MPs, and splenectomized-MPs could play an important role on chronic platelet activation and aggregation, leading to thrombus formation in ß-thalassemia/HbE patients.
Assuntos
Coagulação Sanguínea/fisiologia , Micropartículas Derivadas de Células/metabolismo , Hemoglobina E/metabolismo , Esplenectomia , Trombose/sangue , Talassemia beta/sangue , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/fisiologia , Esplenectomia/tendências , Trombose/cirurgia , Adulto Jovem , Talassemia beta/cirurgiaRESUMO
Thromboembolic complication occurs frequently in ß-thalassaemia/HbE patients, particularly in splenectomised patients. Endothelial cells play an important role in thrombosis. There is strong evidence of endothelial cell activation and dysfunction in ß-thalassaemia. Microparticles (MPs) are associated with thrombosis and endothelial cell dysfunction in many diseases including ß-thalassaemia. However, the effect of thalassaemic-MPs on endothelial cells mediating thrombus formation has not been elucidated. In this study, the effects of circulating MPs from ß-thalassaemia/HbE patients on endothelial cell functions were investigated. The results showed that MPs directly induce tissue factor, interleukin (IL)-6, IL-8, intracellular adhesion molecule-1, vascular cell adhesion molecule-1 and E-selectin expression in human umbilical vein endothelial cells (HUVECs). Notably, the levels of these endothelial cell activation markers were significantly increased in HUVECs treated with MPs obtained from splenectomised ß-thalassaemia/HbE patients when compared to MPs from non-splenectomised patients or normal subjects. The increased endothelial cell activation ultimately lead to increased monocyte-endothelial cell adhesion. THP-1 and HUVECs adhesion induced by MPs from normal subjects, non-splenectomised and splenectomised patients increased to 2.0 ± 0.4, 2.3 ± 0.4 and 3.8 ± 0.4 fold, respectively when compared to untreated cells. This finding suggests that MPs play an important role on thrombosis and vascular dysfunction in ß-thalassaemia/HbE disease, especially in splenectomised cases.