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1. Introduction

Thalassemia, an inherited disease, is characterized by deficient or absent α- or β-globin chains within red blood cells, leading to imbalanced globin chains and reduced red cell survival. Excess globin chains form hemichromes, causing oxidative stress and phosphatidylserine (PS) exposure on red cell membranes (1,2). Thalassemia varies in severity: Thalassemia minor, thalassemia intermedia (TI) and thalassemia major (TM). Patients with TM require regular blood transfusions commencing within the first 2 years of life. Untreated or inadequately transfused patients display jaundice, growth retardation, splenohepatomegaly and skeletal malformation from extramedullary hematopoiesis. Excessive iron from transfusions may lead to irreversible organ damage, with death often resulting from cardiovascular diseases, iron overload and infections (3-5).

Of note, patients with β-thalassemia have an increased risk of hypercoagulability and thrombosis, significantly impacting morbidity and mortality (6). This phenomenon involves interactions between damaged red cells, activated leukocytes and platelets, adhesive endothelial cells and coagulation factor dysregulation (7). The incidence of thrombosis may be minimized by regular blood transfusion, while splenectomy significantly potentiates the risk (8,9). Increased circulating cell-derived vesicles have been reported in splenectomized thalassemic patients, suggesting the role of the spleen in the clearance of these vesicles (10).

Extracellular vesicles (EVs) are submicron-sized, bioactive membrane vesicles released by cells under stress, activation or apoptosis. They are classified into three types: Apoptotic bodies, microparticles (MPs), which are large EVs, and exosomes (which are small EVs), each differing in their biogenesis, properties and molecular markers. They have received extensive interest due to their important role as a conveyer of biological cargos. EVs are critical in cell communication, carrying proteins, lipids and nucleic acids to target cells (11,12). Their elevated levels in cardiovascular and hematological disorders make them potential diagnostic and prognostic biomarkers (12-14). Our group in Thailand was the first to characterize MPs in patients with β-thalassemia/hemoglobin (Hb)E following bone marrow transplantation. Lower levels of circulating PS-externalizing red blood cells, their MPs and procoagulant platelets were found in transplanted patients than in those receiving regular transfusions (15). These circulating MPs, along with leukocyte-platelet aggregates, may contribute to hypercoagulability in patients with β-thalassemia/HbE who have undergone bone marrow transplantation (16). Understanding the role of EVs in altering target cell phenotypes and inducing functional changes may provide insight into the disease's pathogenesis and complications.

2. β-Thalassemia

β-Thalassemia, an autosomal recessive anemia, arises from reduced (β+) or absent (β0) synthesis of the β-globin chain. Its severity varies, primarily depending on the excessive degree of α-globin chains, which accumulate in red blood cell precursors, causing ineffective erythropoiesis (17). Ineffective erythropoiesis features rapid expansion of early erythroid precursors and apoptosis of late-stage ones, resulting in low production of reticulocytes and mature red blood cells. This mechanism is primarily responsible for inherited anemia disorders such as β-thalassemia (18).

β-Thalassemia is categorized into β-TM, β-TI, thalassemia minor and the β-thalassemia trait. Patients with TM require regular blood transfusions for survival. Untreated or poorly transfused individuals exhibit growth retardation, pallor, jaundice, skin pigmentation, weak musculature, hepatosplenomegaly, leg ulcers, extramedullary hematopoiesis masses and skeletal changes due to marrow expansion.

Patients who do not receive blood transfusions usually succumb to heart failure. However, those receiving transfusions may develop iron overload, depending on their adherence to iron chelation therapy. Complications related to iron overload in children include growth retardation and failure of sexual maturation. In adults, complications may include liver fibrosis and cirrhosis, endocrine gland involvement (e.g., diabetes mellitus, and insufficiency of hormones released from the parathyroid, thyroid and pituitary glands), and cardiovascular diseases such as dilated myocardiopathy and arrhythmias (19).

Patients with TI have milder anemia, requiring infrequent or no transfusions. They may be asymptomatic until adulthood, with symptoms including pallor, mild jaundice, cholelithiasis, liver and spleen enlargement, bone changes, leg ulcers, extramedullary erythroid marrow masses, osteopenia, osteoporosis and thrombotic complications (17). Patients with TI often have an increased tendency to develop thrombosis, particularly when compared to those with TM, particularly in splenectomized patients. This increased risk may lead to thromboembolic conditions such as deep vein thrombosis, portal vein thrombosis, stroke and pulmonary embolism (20). The β-thalassemia trait is typically clinically asymptomatic, although mild anemia may occur in certain cases (17).

The high incidence of thromboembolic events in TI leads to a chronic hypercoagulable state, causing major morbidity and mortality. These events include cerebral thrombosis, deep venous thrombosis, pulmonary embolism and recurrent arterial occlusion (7). In addition, all studies of EVs in β-thalassemia classified the patients as TM, and TI according to severity, anemia, blood transfusion and complications due to the higher incidence of hypercoagulable state found in patients with TI. In the present review, the forms of β-thalassemia, such as TM, TI and β-thalassemia trait, were described.

3. α-Thalassemia

In α-thalassemia, the clinical phenotypes range from asymptomatic to lethal, depending on the number of non-functional copies of α-globin genes. Two main groups of α-thalassemia include α-thalassemia 1 and α-thalassemia 2. As a result of deficient production of α-globin, it leads to the formation of homotetramer of unaffected β-globin chains. One of the moderately severe symptomatic forms of α-thalassemia is HbH disease (β4 tetramers). The non-deletion (ND) form of HbH disease (ααND/-) and Hb constant spring, which is caused by mutation in α-globin gene termination, are common in Asian countries. Patients in this group are severely anemic and have a risk of iron overload and splenomegaly. Therefore, splenectomy is recommended for patients with ND HbH, but they have a high rate of counteracting with the pro-thrombophilic complication (2,21). Similarly, abnormal externalization of PS on the red cell surface and their shedding MPs can stimulate the disturbance of coagulation. However, the coagulation parameters tested by Sirachainan et al (22) were not different among pediatric patients with α-thalassemia and normal controls. Further investigations on the effect of other factors, such as activation of platelet and endothelial cells and shedding EV, are required to confirm their contribution of hemostatic alteration in α-thalassemia.

4. EVs in patients with thalassemia

In patients with thalassemia, various studies have examined the profile and quantity of circulating MPs, focusing on their cellular origin and procoagulant properties. Among these studies, those involving patients who have undergone splenectomy revealed a notable presence of plasma MPs, primarily released from platelets and damaged red cells, which express negatively charged PS (23). In patients with β-thalassemia/HbE who have been splenectomized, these MPs are known to trigger platelet activation and aggregation, contributing to the formation of thrombi (24). The notably elevated levels of these MPs may partly contribute to thromboembolic events, hypercoagulable states and other clinical complications commonly observed in patients with thalassemia (25).

Discovery of EVs in thalassemia

The etiology of thromboembolic risk in thalassemic patients involves several factors. They include hyperaggregation and oxidative damage of red cells, chronic platelet activation and increased circulating cell-derived EVs. This complex interplay requires further investigation to fully understand its underlying mechanisms. The initial functional exploration of the role of EVs in the pathogenesis of the hypercoagulable state in thalassemia involved coculturing recipient cells, such as endothelial cells and leukocytes, with isolated EVs. This was followed by analyzing the induced phenotypic changes in these target cells to gain insight into the impact of EVs.

Initial evidence of the procoagulant role of EVs and their influence on thromboembolic events and endothelial dysfunction came from observing endothelial cells interacting with PS-externalizing red cells. This exposure showed that PS-enriched lipid vesicles compete for PS-binding sites on endothelial cell monolayers, indicating the prothrombotic effects of both PS-exposed red cells and PS-enriched vesicles (26). Studies have documented significant evidence that red cells in thalassemic patients externalize PS on their outer surface (27,28). When these cells shed as PS-expressing red cell vesicles, they contribute to the initiation of chronic hypercoagulability, along with other procoagulant factors (29-31).

With advancements in flow cytometric analysis, Pattanapanyasat et al (32) and another study by the same group (33) successfully quantified the number of vesicles shed from red cells. These vesicles are phenotypically distinct, as identified by their reactivity to annexin-V coupled with glycophorin A (CD235a). They are also smaller than the platelet population, as observed in logarithmic forward- and side-scatter dot plots. These characteristics revealed that thalassemic patients have more circulating red cell vesicles than healthy individuals. Furthermore, patients who had undergone splenectomy were found to have significantly elevated levels of these vesicles.

These results align with those reported by other research groups, who found that platelets, rather than red cell-derived EVs, are predominantly responsible for procoagulant activity in splenectomized patients receiving blood transfusions (34). In addition, a marked increase in platelet-derived EVs has been identified as a predictive biomarker for thromboembolic events in both patients with TM and TI (35,36).

In addition to red blood cells and platelets, other cell types also release EVs. Flow cytometry may be employed to identify the cellular origin of these EVs using fluorochrome-conjugated antibodies specific to their parent cells (37). Common surface markers used to determine the cellular origin of EVs are listed in Table I. Beyond flow cytometry, several other techniques are utilized for characterizing circulating EVs, including proteomic studies for specific marker identification. Several common biomarkers for exosomes, including major histocompatibility complex, flotillin and the heat-shock 70-kDa protein (HSP70) are similarly present in all EV types. Proteins particularly enriched in small EVs and showing varying expression levels in different EV populations have been documented. Western blot analysis is used to assess the differential expression of CD9, CD63 and CD81. These transmembrane proteins are frequently observed in EVs from various cell types, providing a framework for identifying subtypes of EVs (38,39).

Table I Surface antigens employed in the characterization of circulating extracellular vesicles using flow cytometry.

Table I

Surface antigens employed in the characterization of circulating extracellular vesicles using flow cytometry.

Antigen marker	Alternative name	Cell of origin
Phosphatidylserine	-	Apoptotic cell membrane
CD235a	Glycophorin A	Red cells
CD71	Transferrin receptor	Erythroid precursor
CD41a	Glycoprotein IIb	Platelets
CD42b	Glycoprotein Ibα	Platelets
CD31	Platelet endothelial cell adhesion molecule	Platelets, endothelial cells
CD144	Vascular endothelial cadherin (or cadherin-5)	Endothelial cells
CD146	Melanoma cell adhesion molecule	Endothelial cells
CD105	Endoglin	Mesenchymal stroma
CD45	Protein tyrosine phosphatase receptor type C	Leukocytes
CD11b	Integrin α-M	Activated leukocytes
CD62P	P-selectin or platelet activation-dependent granule membrane protein	Activated platelets
CD142	Coagulation factor III or tissue factor	Leukocytes and subendothelial cells, such as smooth muscle cells and fibroblasts

Transmission electron microscopy, a widely utilized technique (40), is crucial in assessing the quality and purity of samples containing EVs. This technique can distinguish individual EVs from particles of similar size that are not EVs (41).

In addition, microRNAs (miRNAs) associated with EVs have gained attention as potential biomarkers for various human diseases. To detect these EV-miRNAs, reverse transcription-quantitative PCR (RT-qPCR) is commonly employed (42). Recent advancements include the development of a simplified and cost-effective approach, single-step RT-qPCR, which allows for the direct detection of EV-miRNAs without the need for RNA purification (43).

Nanoparticle tracking analysis is another critical technology used for quantifying and determining the size of EVs. It offers reproducibility and accuracy in measuring the particle concentration and size distribution of EVs isolated from diverse sources through various methods. To enhance reproducibility in future EV research, standardization of nanoparticle tracking analysis methods is essential (44).

EVs as predictive biomarkers in thalassemia

Proteomic analysis of plasma vesicles from β-thalassemia/HbE patients has revealed an accumulative increase in proteins associated with oxidative damage in red blood cells and platelets. Notable among these proteins are peroxiredoxin 6 and HSP90(45). In addition, changes in the levels of specific proteins in EVs from thalassemic patients, such as increased α-Hb-stabilizing protein and decreased haptoglobin, hemopexin and cathepsin S, suggest their potential as biomarkers for hemolysis and inflammation. Mass spectrometry has been utilized to quantify these markers in patients with β-thalassemia/HbE (46). Ferru et al (47) explored hemichromes, composed of denatured α-globin within thalassemic red cell EVs, that bind to band 3 protein and trigger phosphorylation of p27Syk kinase. This process promotes red cell membrane instability and subsequent EV shedding. They also identified a set of HSP70 and peroxiredoxin 2 proteins that are enriched in these EVs (47). Levin et al (48) used nanoparticle tracking analysis to show increased levels of thalassemic EVs following splenectomy. These EVs have high concentrations of HSP70, which contributes to ineffective erythropoiesis, hemolysis and disease severity (48). Further studies indicate that stored thalassemic blood units show overexpression of caspase-3 and molecular chaperones such as HSP70 and DJ-1 in the EV fraction. This finding suggests that oxidative stress affects the physiology and aging of stored thalassemic red cells (49).

Increased amounts of circulating EVs have been reported to be associated with clinical complications commonly observed in patients with β-thalassemia. This increase has been observed to positively correlate with the levels of hypoxia-inducible factor α, a marker for tissue hypoxia, particularly in pediatric patients with thalassemia (50). This relationship underscores the connection between oxidative stress and the formation of EVs, which in turn has a role in the thromboembolic phenomena frequently observed in these patients. Significant increases in circulating CD146+ endothelial EVs, endothelial progenitor cells and von Willebrand factor have also been identified. These changes are linked to the incidence of cardiovascular complications in young patients with β-thalassemia (51). However, elevated levels of circulating EVs have not been associated with pulmonary arterial hypertension in splenectomized thalassemic patients. This finding is attributed to the use of antiplatelet drugs aimed at reducing platelet activation levels (52).

Classifying β-thalassemia subtypes is crucial for effective treatment and EVs have emerged as potential biomarkers for screening the disease and differentiating among its subtypes. Research conducted by Li et al (53) has shed light on the proteomics profiles of plasma-derived EVs in patients with TM, TI and healthy donors. They identified the top six proteins for the diagnosis of patients with β-thalassemia: Complement C1s subcomponent (C1S), clusterin (CLU), lactotransferrin, complement C1r subcomponent, C4b-binding protein β chain and C4b-binding protein α chain. Their study revealed distinct proteomic patterns in patients with TI and TM. The top six proteins that showed potential in distinguishing patients with TM from those with TI were serotransferrin (TF), Hb subunit α (HBA1), immunoglobulin heavy constant γ 4 (IGHG4), HBB, plasma kallikrein B1 and apolipoprotein M. Furthermore, they proposed a model using five proteins (TF, C1S, IGHG4, CLU and HBA1) to discriminate among the three groups. The findings suggest the potential of EVs in subtyping and highlight the ability of plasma-derived EVs to aid in diagnosing β-thalassemia patients (53).

Placenta-derived EVs have emerged as promising tools for the diagnosis of thalassemia. These EVs are detectable in maternal circulation as early as 6 weeks of gestation, and their release can be induced by conditions such as hypoxia and hyperglycemia. Changes in the quantity and composition of EVs have been observed in placental-related complications such as preeclampsia and fetal growth restriction, highlighting their potential as a diagnostic tool for identifying such complications in asymptomatic pregnant women. Furthermore, in cases of Bart's hydrops fetuses, placental hypoxia may prompt alterations in the number or function of placenta-derived EVs. This characteristic may offer a valuable means for early, noninvasive prenatal diagnosis. However, standardized detection methods are needed to effectively use placenta-derived EV detection in diagnosing, monitoring and treating Bart's hydrops fetalis and similar placental disorders (54).

Pathologic function of thalassemic EVs

In thalassemic patients, an increased number of plasma EVs has been linked to a hypercoagulable state; however, their specific role in thalassemia is not well defined. Various research groups have examined the biological effects of plasma EVs isolated from thalassemic patients on different types of recipient cells. EVs, primarily MPs, from splenectomized patients have shown a significant capacity to activate platelets and cause microaggregation of leukocyte platelets compared to vesicles isolated from healthy individuals or non-splenectomized patients (24).

The prothrombotic function of thalassemic EVs is further evident in their contribution to endothelial cell dysfunction. When endothelial cells internalize EVs derived from splenectomized patients, there is an enhanced induction of endothelial cell activation markers and proinflammatory mediators, as well as an increased binding ability of endothelial cells to the human monocytic leukemia cell line THP-1 (55,56). In addition, thalassemic EVs, particularly exosomes containing ferritin and hemichrome, have been found to promote the hyperproliferation of cardiac cells, potentially leading to cardiomyopathic complications commonly seen in thalassemic patients (57).

Fig. 1 illustrates the prothrombotic potential of thalassemic EVs in contributing to hyperthrombophilic phenomena. Thalassemic EVs, which are mainly released from activated platelets and damaged red cells, facilitate the formation of microemboli: Complexes of platelets, red cells, leukocytes and coagulation factors. These microaggregates adhere to activated endothelial cells, triggering a hypercoagulable state in thalassemic patients.

Figure 1 Role of thalassemic EVs in thromboembolic events. The schematic illustrates how thalassemic EVs, predominantly released by activated platelets and damaged red cells, contribute to the formation of microemboli. These microemboli, composed of platelets, red cells, leukocytes and coagulation factors, adhere to activated endothelial cells, thereby inducing a hypercoagulable state in thalassemic patients. The characterization of these circulating EVs involves various advanced techniques: Flow cytometry (to determine size and cell of origin); proteomic analysis (to identify biological content); reverse transcription-quantitative PCR (for EV miRNA profiling); transmission electron microscopy (to assess the quality and purity of EV-containing samples); nanoparticle tracking analysis (to measure size and concentration); and western blot analysis (to detect HSP70 and EV-specific protein markers, such as the tetraspanin proteins CD9 and CD63). The figure was designed using BioRender.com. EV, extracellular vesicle; miRNA/miR, microRNA; HSP, heat shock protein.

Exosomes have a critical role in intercellular communication by shuttling biological cargos, such as miRNAs, to recipient cells. This transfer leads to modifications in the functional properties of target cells. In the case of β-thalassemia, where genes that encode γ-globin are silent, thalassemic exosomes and miR-223-3p have been reported to suppress γ-globin expression by downregulating LIM-only protein 2, the globin gene regulator (58). Furthermore, differential expression of miRNA profiles in EVs from patients with β-thalassemia compared to those from healthy individuals has been observed.

When various recipient cells, such as endothelial cells, hepatocytes, pancreatic cells and mesenchymal stromal cells, are exposed to thalassemic EVs containing miR-144-3p, they exhibit dysfunctional phenotypes. These include reduced cell viability and induction of cell apoptosis. The mechanism behind β-thalassemia EV-induced apoptosis in endothelial cells is associated with the MAPK/JNK signal transduction pathway. By contrast, EVs from splenectomized patients with β-thalassemia induce the proliferation of bone marrow mesenchymal stromal cells. This finding suggests that EVs contribute to organ damage and complications in β-thalassemia (59).

EVs in bone marrow transplantation

Allogeneic hematopoietic stem cell transplantation (HSCT) is currently the only established, potentially curative therapy for patients with TM. However, it is limited by the availability of matched donors and each patient's medical condition. Human leukocyte antigen (HLA)-matched stem cells, sourced from cord blood or bone marrow, have yielded excellent outcomes in HSCT. Therefore, it is recommended that patients with TM, particularly young patients, should consider HSCT at an early stage of the disease if they have HLA-identical sibling donors. This approach aims to prevent the development of severe clinical complications due to iron overload and multiorgan failure. Anurathapan et al (60,61) have utilized haploidentical-related donors as a source of hematopoietic stem cells for pediatric patients with TM. Following matched-donor or haploidentical HSCT, certain patients with thalassemia exhibited increased endothelial activation and a heightened risk of thromboembolic events. These complications are attributed to several factors, including the conditioning regimen, high-dose chemotherapy, immune response and graft-versus-host disease (62). However, evidence indicates that HSCT can effectively correct hemostatic alterations in patients with TM. Sirachainan et al (63) found that plasma levels of coagulation markers (thrombin-antithrombin complex, prothrombin fragment and D-dimer) and anticoagulation factors (protein C, protein S and antithrombin activity) returned to normal levels following successful HSCT. This signified a positive therapeutic outcome.

Several studies have noted an increase in the number of circulating EVs following HSCT in hematological disorders. Trummer et al (64), for instance, reported a rise in plasma P-selectin glycoprotein ligand-1-expressing MPs and linked this increase to the risk of refractory disease and relapse in patients. Research by our group focusing on EV profiling in HSCT for thalassemia, flow cytometric analysis was utilized to measure plasma MPs in children with TM undergoing HSCT. The randomized study by our group specifically measured PS-expressing red cells and CD235a+ red cell MPs. The levels of these two factors decreased after transplantation (15). Conversely, in another study by our group, an increase in other MP populations from platelets (CD41a+ MP), leukocytes (CD45+ MP) and endothelial cells (CD146+ MP) in the plasma of transplanted patients was observed. These procoagulant MPs were associated with a higher incidence of monocyte-platelet microaggregates, suggesting a complex interaction and potential clinical implications in the post-transplant setting (16).

5. EVs in other hemoglobinopathies

In the realm of common hemoglobinopathies, sickle cell disease (SCD) serves as the prototype. This inherited disorder arises from a point mutation in the β-globin gene, leading to the formation of hemoglobin S. SCD is often linked to chronic vascular inflammation and hypercoagulability. These conditions result from the combined effects of procoagulant sickle red cells, activated platelets and cell-derived EVs. Collectively, these components induce endothelial cell activation, leading to cardiovascular complications (25,65,66). Two core mechanisms have been identified in this process: The transfer of heme to endothelial cells (67) and the increased adhesiveness of sickle red cells to endothelial cells (68). These interactions highlight the complex pathophysiology of SCD and the significant role of EVs in its progression and complications.

In SCD, numerous studies have reported the presence of large numbers of EVs, particularly those derived from red blood cells and platelets. EVs are found in patients both during steady-state and sickling crises, and their levels are significantly higher than those in healthy individuals (69,70). These sickle cell-derived EVs initiate hemostatic abnormalities, such as prolonged thrombin generation (71). With regard to abnormal thrombin generation in SCD, the amplification was presumably mediated by PS exposed on sickle cell MPs that activates the intrinsic pathway of the coagulation cascade through factor XI (69), and the extent of prothrombotic PS relies on the size of red cell MPs, particularly macrovesicles (72). Of note, treatments with hydroxyurea (73) or hydroxycarbamide (74) have been shown to ameliorate these effects.

Beyond their role as bioeffectors in SCD, these sickle cell-derived EVs also serve as biomarkers for predicting disease-related complications. These complications include oxidative stress related to vaso-occlusive crises (75), osteonecrosis (76), leg ulcers and pulmonary hypertension (77). A notable aspect of SCD-EVs is their content of functional miRNAs, specifically miR-124-3p, miR-2278 and miR-4763-5p. These miRNAs are reported to be enriched in SCD-plasma EVs and could contribute to the initiation and progression of the disease (78). This finding highlights the multifaceted role of EVs in the pathophysiology and potential diagnostic and therapeutic approaches in SCD.

6. Conclusions and future perspectives

In both normal and disease conditions, EVs, including MPs and exosomes, are produced by most, if not all, cell lineages. The study of MPs has garnered increased attention in exploring the pathogenesis of common complications in patients with thalassemia. While certain studies have demonstrated the regulatory role of exosomes and exosomal miRNAs in contributing to physiological and pathophysiological processes in thalassemia biology, further research is needed. The phenotypic characterization and functional study of EVs require precise and accurate methodologies and techniques due to the challenges posed by their small size. Understanding the molecular mechanisms that govern the production and clearance of EVs, as well as those involved in intercellular communication, is crucial. This knowledge could pave the way for novel therapeutic approaches to reduce clinical complications in thalassemia, offering new insight and potential breakthroughs in the treatment of thalassemia.

Acknowledgements

Not applicable.

Funding

Funding: The authors were funded by the National Research Council of Thailand: High-Potential Research Team Grant Program (grant no. N42A650870), the Talented Young Researcher Scholarship Program (grant no. N41A640122) and the Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation (grant no. RGNS 63-161). The funders had no role in the design of the study; the collection, analysis, or interpretation of data; the decision to publish; or the writing of this manuscript.

Availability of data and materials

Not applicable.

Authors' contributions

Conceptualization: PK, KP and PP. Writing-original draft preparation: PK and PP. Writing-review and editing: KP and PP. Figure preparation and visualization: PK. Supervision: KP and PP. Funding acquisition: KP, PK and PP. All authors have read and agreed to the published version of the manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References