Phosphatidylserine and the thrombin–antithrombin complex as markers for hypercoagulability in Egyptian beta-thalassemia patients :Marwa M Sadek, Amal S Ahmed, Samar M El Fiky, Shady I Tantawy, Amany M Hassan, The Egyptian Journal of Haematology

ORIGINAL ARTICLE
Year : 2022 | Volume : 47 | Issue : 3 | Page : 210--216

Phosphatidylserine and the thrombin–antithrombin complex as markers for hypercoagulability in Egyptian beta-thalassemia patients

Marwa M Sadek¹, Amal S Ahmed¹, Samar M El Fiky², Shady I Tantawy³, Amany M Hassan¹,
¹ Department of Clinical and Chemical Pathology, Suez Canal University, Ismailia, Egypt
² Department of Pediatric Medicine, Suez Canal University, Ismailia, Egypt
³ Department of Internal Medicine, Faculty of Medicine, Suez Canal University, Ismailia, Egypt

Correspondence Address:
Amany M Hassan
Department of Clinical and Chemical Pathology, Faculty of Medicine, Suez Canal University, 4.5 Km the Ring Road, Ismailia 41522
Egypt

Abstract

Background Hypercoagulability is a known complication of thalassemia, in particular, thalassemia intermedia. Several factors contribute to this hypercoagulability, including chronic platelet activation and the presence of other comorbid conditions. In addition, the oxidation of globin subunits in thalassemic red blood cells (RBCs) triggers the formation of reactive oxygen species. These factors lead to the exposure of negatively charged phospholipids like phosphatidylserine (PS), which ultimately causes increased thrombin generation, particularly in splenectomized patients. Aim This study aimed to assess the risk for hypercoagulability in thalassemic patients using PS expression on RBCs and the thrombin–antithrombin (TAT) complex in plasma. Patients and methods This study included 50 thalassemic patients (19 patients with splenectomy and 31 patients without splenectomy) and 30 apparently healthy individuals as a control group. Patients were subjected to assessment of history for deep venous thrombosis or pulmonary hypertension by echocardiography. Annexin V was used to detect PS expression on RBCs by flow cytometry, while the enzyme-linked immunosorbent assay was used to detect the TAT complex in plasma. Results Erythrocyte PS expression was significantly higher (P<0.001) in thalassemic patients than in the control group. The TAT complex level revealed no significant difference between thalassemia patients and the control group (P=0.468). Patients with pulmonary hypertension showed a statistically significant higher PS expression and TAT complex level. However, there was no significant increase in PS expression or TAT level in patients with a history of deep venous thrombosis only. Conclusion Increased PS expression and TAT complex level may be a risk factor for pulmonary hypertension in thalassemia patients with splenectomy.

How to cite this article:
Sadek MM, Ahmed AS, El Fiky SM, Tantawy SI, Hassan AM. Phosphatidylserine and the thrombin–antithrombin complex as markers for hypercoagulability in Egyptian beta-thalassemia patients.Egypt J Haematol 2022;47:210-216

How to cite this URL:
Sadek MM, Ahmed AS, El Fiky SM, Tantawy SI, Hassan AM. Phosphatidylserine and the thrombin–antithrombin complex as markers for hypercoagulability in Egyptian beta-thalassemia patients. Egypt J Haematol [serial online] 2022 [cited 2023 May 29 ];47:210-216
Available from: http://www.ehj.eg.net/text.asp?2022/47/3/210/366866

Full Text

Introduction

Thalassemias are inherited blood disorders where there is abnormal globin synthesis leading to decreased production of one or more globin chains [1]. Beta-thalassemia major (β-TM) (Cooley’s anemia) is the most severe form of thalassemia. It is characterized by a severe transfusion-dependent microcytic hypochromic anemia. It is due to the mutation of both β-globin genes. However, β-thalassemia minor results from a mutation in only one β-globin gene, resulting in mild disease [2].

Although there is a marked improvement in the life expectancy of thalassemia due to regular blood transfusion and iron-chelation therapy, some fatal complications are recognized like thromboembolic events (TEE) that may occur in thalassemic patients [3]. In a study carried out by Youssry et al. [4], TEE was found in 41 (47.1%) out of 87 thalassemia patients. In this study, a higher incidence of splenectomy was found among patients with TEE than patients without TEE.

Another study by Taher et al. [5] estimated the prevalence of thrombotic events in β-thalassemia in 9% of the patients in the Mediterranean area and Iran. TEEs were found to be more prominent in thalassemia intermedia (TI) than thalassemia major (TM) patients. This was attributed to the more frequent blood transfusions noted in TM patients. Thus, it was believed that blood transfusion is protective against TEE [6].

TEEs in thalassemic patients may be arterial or venous. For example, thalassemia patients may develop pulmonary embolism, transient ischemic attacks, or deep venous thrombosis (DVT). At the same time, it varies widely in severity. For instance, TEE may be significant and symptomatic, or it may be subclinical, detected only in the autopsy as platelet and fibrin thrombi in the microvasculature of the lung and the brain [5].

There is higher occurrence of TEE in thalassemic patients in splenectomized patients with infrequent blood transfusion. Possible etiologies include abnormal red blood cell (RBC) membrane resulting from oxidative damage, with the resultant increased exposure of phosphatidylserine (PS) that causes activation of factor V and X leading to thrombosis [7]. Oxidation of globin subunits in thalassemic RBCs causes exposure of some antigens such as PS and phosphatidylethanolamine, which make the RBCs rigid and deformed. This leads to the formation of RBC aggregates and their premature removal from the circulation [8].

The exposure of PS on the surface of RBCs can be detected by Annexin V, which is a protein with a high affinity for anionic phospholipids like PS. Binding of PS to thalassemic RBCs causes thrombin release and platelet activation, leading to increased coagulability [6]. Elevated levels of thrombin–antithrombin III (TAT) complexes were found in 50% of adult and child patients with β-TM, supporting the chronic hypercoagulable state in thalassemia. Also, low levels of antithrombin III were found in Italian and Turkish thalassemic patients [9].

In our study, we aim to assess the hypercoagulable state in thalassemia by studying the expression of PS on thalassemic RBCs and the TAT complex in plasma as markers of hypercoagulability risk in thalassemic patients.

Patients and methods

Our study is a case–control study that included 50 patients diagnosed with β-thalassemia compared with 30 healthy control individuals. Patients were recruited from adult and pediatric hematology clinics at Suez Canal University Hospital. Patients were divided into two groups: group 1, which included splenectomized β thalassemia patients (n=19 patients, including 15 β-TM and four β-TI) patients, and Group 2, which included nonsplenectomized β-thalassemia patients (n=31 patients, including 29 β-TM and two β-TI patients).

Exclusion criteria included thalassemia minor patients, patients with other hemoglobinopathies like sickle cell anemia, and patients who had received blood transfusion recently (<1 month). The entire study population was subjected to a brief assessment of history including name, age, sex, family history, history of splenectomy, time interval since splenectomy, history of previous thrombosis, that is, DVT or pulmonary embolism, as well as frequency and number of blood units received per year. Echocardiography data were reviewed to check for pulmonary hypertension. The Suez Canal University Ethical Committee approved our study. Informed consent was obtained from all patients after explaining the aim of the study.

Blood sampling

The blood sample required for the study was obtained immediately before blood transfusion. EDTA blood sample was used for CBC and flow cytometric assessment of PS expression on RBCs using Annexin V. Citrated blood sample was used for assessment of the TAT complex using the enzyme-linked immunosorbent assay technique.

(1) Diagnosis of thalassemia: The diagnosis was made by complete blood count and RBC morphology assessment. Hemoglobin F level was obtained from patients’ records.

(2) Assessment of markers of hypercoagulability in thalassemic patients:

(a) Assessment of surface expression of PS on RBCs: from EDTA blood samples using Annexin V FITC (fluorescein isothiocyanate) (Miltenyi Biotec GmbH, Germany, Friedrich-Ebert-strabe68, 51429 Bergisch Gladbach, Germany) by flow cytometry: The cell numbers (106) were determined using the blood cell counter after dilution of the whole-blood EDTA sample with phosphate buffer saline.

Two FACS tubes, with 100 μl of RBC suspension in each, were labeled unstained and Annexin V tubes. The cells were washed twice using 1 ml of the binding buffer in each tube and centrifuged at 300g for 10 min, the supernatant was aspirated completely, the cell pellet was resuspended in 100 μl of the binding buffer, and proper vortexing was performed.

Annexin V FITC (5 μl) was added to the Annexin V tube, and good mixing by vortexing was performed. Then, the tubes were incubated for 15 min in the dark at room temperature.

The cells were washed twice again with 1 ml of the binding buffer and centrifuged at 300g for 10 min, and then the supernatant was aspirated completely. The pellet was resuspended in 500 μl of the binding buffer and mixed well with vortexing for acquisition. Data analysis and interpretation: the acquisition and fluorescence analysis were carried out by FACS Calibur flow cytometry (Becton Dickinson Immunocytometry Systems, USA, Becton Dickinson GmbH, Rowa Germany) using Cell Quest software. RBCs were gated according to their light-scattering properties on the upper part of the log–log dot plot curve on the forward and side scatters. The percentage of Annexin V-positive RBCs and their mean fluorescence intensity were measured.

(b) Assessment of the TAT complex was performed using the human TAT complex enzyme-linked immunosorbent assay kit (Assaypro LLC, USA) according to the manufacturer instructions. This was a quantitative sandwich enzyme immunoassay technique. A standard log–log curve was used for interpretation of results, and the detection range of the kit ranged from 1.481 to 120 ng/ml. No interferences or significant cross-reactivity were reported during the assay.

Statistical analysis

Data were collected and coded, and then entered as a spreadsheet using Microsoft Excel for Windows Office 2007. Data were analyzed using Statistical Package for Social Science SPSS software (version 19.0; SPSS Inc., Chicago, Illinois, USA). Data were presented as tables and graphs as appropriate. Quantitative data were described in the form of mean±SD. Qualitative data were described as frequency and percentage. Significance within a group of quantitative variables was calculated using the analysis of variance test between two groups in the case of parametric variables. Between groups of qualitative data, the χ2 test was used. To identify relations between different variables, Pearson (r) and Spearman correlation (ρ) were used.

Results

The mean hemoglobin levels were significantly lower (P=0.000) in the two groups with the disease than in the healthy controls. However, the mean platelet count was significantly higher (P=0.000) in the groups with the disease compared with the healthy control group. The mean hemoglobin F was compared in both groups with the disease, and no significant difference was found. A positive history of thromboembolism was found in 16.6% of splenectomized β-thalassemia patients, while none of the nonsplenectomized β-thalassemia patients had a history of TEEs. Blood transfusion in nonsplenectomized β-thalassemia patients was higher than splenectomized β-thalassemia patients. However, this was statistically insignificant ([Table 1]).{Table 1}

In the current study, there was a statistically significant (P=0.000) increase in the PS expression on the RBC membrane detected by Annexin V in splenectomized ([Figure 1]) and nonsplenectomized ([Figure 2]) β-thalassemia patients compared with the normal control group ([Figure 3]). Also, Annexin V percentage was higher (10.45 ± 6.86) in splenectomized than in nonsplenectomized (7.54 ± 7.61) β-thalassemia patients, but the difference was not statistically significant ([Table 2], [Figure 1]).{Figure 1} {Figure 2} {Figure 3} {Table 2}

In contrast to previous publications, the mean level of the TAT complex showed no significant difference between all the groups combined ([Table 3]). Also, there was no significant correlation between Annexin V and the TAT complex in any of the study groups ([Table 4]).{Table 3} {Table 4}

In adult thalassemia patients with splenectomy, three patients showed evidence of DVT or pulmonary hypertension (one with a history of DVT, one with pulmonary hypertension, and the last one had both pulmonary hypertension and DVT). All were adults with TI. Interestingly, the patient who had only DVT did not show any significant increase in the level of PS (2.7%) or TAT (2.98 ng/ml) compared with the mean of the group of adult patients who did not have a history of DVT or pulmonary hypertension (PS=9.215, TAT=4.493), while the other two patients who showed evidence of pulmonary hypertension showed a statistically significant increase in PS (23.65, P=0.0465) and the TAT complex (9.110, P=0.0306), indicating that patients with higher PS expression and TAT complex level might be at risk for pulmonary hypertension ([Table 5]).{Table 5}

Discussion

In the current study, there was a statistically significant (P=0.000) increase in the PS expression on the RBC membrane detected by Annexin V in splenectomized and nonsplenectomized β-thalassemia patients compared with the normal control group. Also, Annexin V percentage was higher (10.45 ± 6.86) in splenectomized than nonsplenectomized (7.54 ± 7.61) β-thalassemia patients. These results are supported by Pattanapanyasat et al. [10] and Ibrahim et al. [11], who found that PS expression is strikingly high in splenectomized β-thalassemia patients. Splenectomy leads to an increase in the circulating RBC vesicles and, as a result, increased PS exposure on their surface. Our results are also in agreement with Kamel [1], who found that the percentage of Annexin V-labeled RBCs in patients with TM and TI is significantly increased, which indicated PS exposure on the surface of RBCs in thalassemic patients that in turn explains the hypercoagulability in thalassemia. Zahedpanah et al. [7] also found that erythrocyte PS exposure in splenectomized β-TI patients using Annexin V-labeled RBCs was higher than healthy controls and was statistically significant (P=0.05). Annexin V is highly correlated with the hypercoagulable state in thalassemia as the RBCs undergo oxidative stress, which results in exposure of PS and rigidity of their membrane, leading to thrombin generation and acceleration of thrombotic events, particularly in thalassemic patients with splenectomy [12].

Moreover, hemichromes are formed and bind to RBC membrane proteins with exposure of the negatively charged anionic phospholipids like PS, leading to the early elimination of RBCs from the circulation [13]. The exposure of PS on the surface of RBCs can be detected by Annexin V, which is a protein with a high affinity for anionic phospholipids like PS [1]. Our study revealed that the mean level of the TAT complex showed no statistically significant difference between splenectomized β-thalassemia patients (5.64 ± 3.25), nonsplenectomized β-thalassemia patients (6.24 ± 5.37), and the control group (7.15 ± 3.34). Our results are in contrast with the results of a study carried out by Eldor and Rachmilewitz [9], who reported that TAT complex levels were increased in 50% of children and adult TM patients who had no signs of evident thrombosis. In another study in Egypt, the TAT complex was markedly elevated in both splenectomized and nonsplenectomized thalassemic patients. However, it was higher in splenectomized thalassemic patients, which indicated a low grade of hypercoagulability [14]. In another study by Tripodi et al. [15], who investigated the role played by cells in the hypercoagulability in β-thalassemia patients using thromboelastometry and thrombin generation tests, all the thromboelastometry parameters that were determined in whole blood, including shortened clotting time and clot formation time and increased maximum clot firmness, were in accordance with hypercoagulability, particularly in splenectomized patients, although thrombin generation determined in platelet-poor plasma was not significantly different from that of healthy individuals, signifying that RBCs, platelets, and possibly other cellular elements play a more significant role than plasma alterations in the hypercoagulability observed in thalassemic patients. Explanation of absence of correlation between annexin V on surface of RBCs and level of TAT complex may be due to small sample size or the decreased adult subjects in our sample because hypercoagulability is more common in adults. Studies with larger sample size and including more adults may permit this correlation to appear. In the present study, no significant correlation was found between Annexin V exposure on the surface of the RBC membrane and TAT complex levels in the plasma in all study groups. Our results are in agreement with a previous study in hemoglobin E/β thalassemia splenectomized and nonsplenectomized thalassemic patients in which no significant correlation was found between Annexin V and TAT complex levels in both groups [16].

In the thalassemia patients with splenectomy (group 1), patients with pulmonary hypertension showed a significantly higher PS expression and TAT complex level compared with those with no history of pulmonary hypertension or DVT. In contrast, patients with a history of DVT only did not show any increase in the expression of PS or TAT complex level.

Conclusion

In conclusion, we have shown a higher expression of PS on RBCs in thalassemia patients. Patients with pulmonary hypertension showed a statistically significantly higher PS expression and TAT complex levels. However, these markers did not increase in patients with DVT only. The data indicate that patients with a higher PS expression and TAT complex are at risk for pulmonary hypertension.

Acknowledgement

The authors thank Dr Omar Fathi El Desouky (Assistant Professor of Clinical and Chemical Pathology, Faculty of Medicine, Suez Canal University) for his great support and help in data analysis and interpretation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References

1	Kamel M The potential role of RBCs and activated platelets in the thalassemic hypercoagulable state. J Am Sci 2013; 9:518–524.
2	Galanello R, Origa R Beta-thalassemia. Orphanet J Rare Dis 2010; 5:1–15.
3	Lv R-Y, Wen F-Q, Yu J Advances in pathogenesis and correlated clinical research of hypercoagulability in β thalassemia. Zhongguo Dang Dai Er Ke Za Zhi 2014; 16:774–778.
4	Youssry I, Soliman N, Ghamrawy M, Samy RM, Nasr A, Mohsen MA, et al. Circulating microparticles and the risk of thromboembolic events in Egyptian beta thalassemia patients. Ann Hematol 2017; 96: 597–603.
5	Taher AT, Otrock ZK, Uthman I, Cappellini MD Thalassemia and hypercoagulability. Blood Rev 2008; 22:283–292.
6	Cappellini MD, Musallam KM, Marcon A, Taher AT Coagulopathy in beta-thalassemia: current understanding and future perspectives. Mediterr J Hematol Infect Dis 2009; 1:e2009029.
7	Zahedpanah M, Azarkeivan A, Aghaieepour M, Nikogoftar M, Ahmadinegad M, Hajibeigi B, et al. Erythrocytic phosphatidylserine exposure and hemostatic alterations in β-thalassemia intermediate patients. Hematology 2014; 19:472–476.
8	Cappellini MD, Motta I, Musallam KM, Taher AT Redefining thalassemia as a hypercoagulable state. Ann N Y Acad Sci 2010; 1202:231–236.
9	Eldor A, Rachmilewitz EA The hypercoagulable state in thalassemia. Blood 2002; 99:36–43.
10	Pattanapanyasat K, Noulsri E, Fucharoen S, Lerdwana S, Lamchiagdhase P, Siritanaratkul N, et al. Flow cytometric quantitation of red blood cell vesicles in thalassemia. Cytometry part B: clinical cytometry. J Int Soc Analy Cytol 2004; 57:23–31.
11	Ibrahim HA, Fouda MI, Yahya RS, Abousamra NK, Abd Elazim RA Erythrocyte phosphatidylserine exposure in β-thalassemia. Lab Hematol 2014; 20:9–14.
12	Cappellini MD, Poggiali E, Taher AT, Musallam KM Hypercoagulability in β-thalassemia: a status quo. Expert Rev Hematol 2012; 5:505–512.
13	Habib A, Kunzelmann C, Shamseddeen W, Zobairi F, Freyssinet J-M, Taher A Elevated levels of circulating procoagulant microparticles in patients with β-thalassemia intermedia. Haematologica 2008; 93:941–942.
14	Hassan TH, Elbehedy RM, Youssef DM, Amr GE Protein C levels in β-thalassemia major patients in the east Nile delta of Egypt. Hematol/Oncol Stem Cell Therapy 2010; 3:60–65.
15	Tripodi A, Cappellini MD, Chantarangkul V, Padovan L, Fasulo MR, Marcon A, et al. Hypercoagulability in splenectomized thalassemic patients detected by whole-blood thromboelastometry, but not by thrombin generation in platelet-poor plasma. Haematologica 2009; 94:1520–1527.
16	Atichartakarn V, Angchaisuksiri P, Aryurachai K, Onpun S, Chuncharunee S, Thakkinstian A, et al. Relationship between hypercoagulable state and erythrocyte phosphatidylserine exposure in splenectomized haemoglobin E/β-thalassaemic patients. Br J Haematol 2002; 118:893–898.