|Year : 2017 | Volume
| Issue : 2 | Page : 64-69
Candidate markers for thromboembolic complications in adult Egyptian patients with β-thalassemia
Hadeer A Abbassy MD 1, Omar M Ghallab2
1 Department of Clinical Pathology, Faculty of Medicine, Alexandria University, Alexandria, Egypt
2 Department of Internal Medicine, Faculty of Medicine, Alexandria University, Alexandria, Egypt
|Date of Submission||24-Feb-2017|
|Date of Acceptance||14-Mar-2017|
|Date of Web Publication||6-Oct-2017|
Hadeer A Abbassy
Department of Clinical Pathology, Faculty of Medicine, Alexandria University, 34, Ahmed Yehia Street, Zezenia, Alexandria, 11111
Source of Support: None, Conflict of Interest: None
Background Increased risk for thromboembolic complications in patients with thalassemia major has been attributed to the chronic hypercoagulable state. However, the underlying mechanisms are multifactorial.
Objectives To investigate the candidate markers of possible tendency to thrombophilia in adult patients with thalassemia major.
Methods Genetic polymorphisms of prothrombotic factors have been analyzed in 30 splenectomized patients (group I) and 30 nonsplenectomized patients (group II) as well as 30 age matched and sex matched healthy controls. In addition to routine hematological and coagulation parameters, estimation of protein C and protein S free antigen, antithrombin III activity, factor VIII, and factor IX levels was done. Plasma soluble endothelial protein C receptor levels were measured by ELISA, and factor V Leiden1691G-A, factor II polymorphism (G20210A), and methylenetetrahydrofolate reductase mutation (C677T) were detected by real-time PCR.
Results A significant increase of soluble endothelial protein C receptor was observed in patients (in group I more than group II), whereas protein C and protein S were significantly reduced together with more frequent congenital thrombophilic mutations.
Conclusion With the prolonged life expectancy of patients with β-thalassemia, more caution should be considered toward thromboembolic complications. Thus, after the first thrombotic event or even when they are exposed to thrombotic risk factors, they should be investigated for congenital thrombophilia, and prophylactic antithrombotic agents may be recommended.
Keywords: protein C, protein S, soluble endothelial protein C receptor, thalassemia, thrombophilia
|How to cite this article:|
Abbassy HA, Ghallab OM. Candidate markers for thromboembolic complications in adult Egyptian patients with β-thalassemia. Egypt J Haematol 2017;42:64-9
|How to cite this URL:|
Abbassy HA, Ghallab OM. Candidate markers for thromboembolic complications in adult Egyptian patients with β-thalassemia. Egypt J Haematol [serial online] 2017 [cited 2018 Feb 21];42:64-9. Available from: http://www.ehj.eg.net/text.asp?2017/42/2/64/216114
| Introduction|| |
One of the most common hereditary hemolytic anemias is thalassemia. It results from partial or complete lack of synthesis of one of the α or β globin chains of hemoglobin . The commonest type in Egypt is β-thalassemia, with a carrier rate ranging from 5.3 to more than 9% and a gene frequency of 0.03% . Numerous reports of thromboembolic complications have been recently associated with thalassemia. Venous thromboembolic events such as deep venous thrombosis, portal venous thrombosis, pulmonary embolism, postsplenectomy thrombosis, and cerebral thromboembolism have been observed in patients with thalassemia ,. Hypercoagulability in thalassemia is multifactorial, implicating thrombophilic DNA mutations , alterations in markers of coagulation activation and natural anticoagulant proteins , reactive oxygen species in the oxidized red cell membrane lipids  and loss of their organization  as well as endothelial, monocytic, and granulocyte activation . These clinical findings and hemostatic anomalies suggest the existence of a chronic hypercoagulable state. Signs of in-vivo platelet activation, shortened platelet survival, and decreased levels of naturally occurring anticoagulants such as protein C, protein S, and antithrombin (AT) III are evidenced in patients with thalassemia ,. Endothelial protein C receptor (EPCR) is a type I transmembrane protein, mainly expressed on the luminal endothelial cell surface of large blood vessels . It plays an anticoagulant role by binding with high affinity to protein C and it increases the generation of activated protein C by more than 10-fold. It also binds to factor VIIa and inhibits its coagulant activity . The soluble endothelial protein C receptor (sEPCR) is released from EPCR by thrombin, vascular injury, or regulated proteolytic cleavage by metalloproteinases .
The aim of the present study was to evaluate the contribution of naturally occurring anticoagulants, thrombophilic mutation risk factors, and sEPCR to arterial and venous thromboembolic complications in adult patients with β-thalassemia.
| Patients and methods|| |
A total of 60 adult patients with established β-thalassemia major were enrolled in this study (30 splenectomized: group I and 30 nonsplenectomized: group II). Patients were selected from the outpatient clinic of Alexandria Main University Hospital between 2014 and 2016. Group I consisted of 12 (40%) males and 18 (60%) females, with age range from 19 to 59 years with a mean of 29.3±12.2 years, whereas group II had eight (26.67%) males and 22 (73.33%) females, with age ranging from 18 to 38 years with a mean of 23.4±5.6 years. Moreover, 30 normal healthy individuals with matched age and sex served as a control group. They had no history of venous thrombosis, renal dysfunction, or smoking. Patients received regular blood transfusions and subcutaneous desferrioxamine infusion at doses in keeping with the severity of iron overload. All splenectomized patients had been operated on at least 6 months before the study. History of epistaxis was found in 16 (26.7%) patients, whereas history of thrombotic attacks (deep vein thrombosis and pulmonary embolism) was found in only four (6.7%) patients. Thorough clinical examination revealed hepatomegaly in all patients, whereas splenomegaly was only evident in group II (nonsplenectomized). Hematological parameters were studied before transfusion.
Laboratory investigations included complete blood count using automated cell counter (Sysmex XT-1800i, Kobe, Japan), prothrombin time (PT) and international normalized ratio (INR) by Quick’s one-stage method using calcium thromboplastin partial PT with Kaolin cephalin clotting time using prothrombin, and d-dimer by semiquantitative latex agglutination kit (Dialab,Wiener Neudorf, Austria). A PT value 2 s more than that in controls and an activated partial thromboplastin time (aPTT) value 6 s more than that in controls were taken as abnormal.
Laboratory investigations included complete blood count using automated cell counter (Sysmex XT-1800i, Kobe, Japan), prothrombin time (PT) and international normalized ratio (INR) by Quick’s one-stage method using calcium thromboplastin partial PT with Kaolin cephalin clotting time using prothrombin, and d-dimer by semiquantitative latex agglutination kit (Dialab,Wiener Neudorf, Austria).
Serum ferritin, protein C and protein S free antigen, AT activity, factor VIII levels, and factor IX levels were studied. Protein C less than 70%, protein S less than 70%, and AT III less than 80% were taken as low values.
Measurements of plasma sEPCR levels by ELISA (Stago Diagnostic, Asniere, France) were performed 6 months after the first episode of stroke according to the manufacturer’s instructions. Factor V Leiden (FVL) 1691G-A, factor II 20210G-A, and methylenetetrahydrofolate reductase (MTHFR) 677 C-T mutations were identified by melting curve analysis using real-time PCR on Light Cycler (Roche Diagnostics, Mannheim, Germany).
The study was approved by the medical ethics committee, and informed consents were obtained from the patients to participate in the study. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2008.
Data were fed to the computer and analyzed using IBM SPSS software package, version 20.0 (SPSS Inc., Chicago, Illinois, USA). Comparisons between groups for categorical variables were assessed using χ2-test. Student’s t-test was used to compare two groups for normally distributed quantitative variables. Mann–Whitney U-test was used to compare two groups for abnormally distributed quantitative variables. Paired t-test and Wilcoxon signed-rank test were assessed for comparison between different periods. Spearman’s coefficient was used to correlate between quantitative variables. Significance of the obtained results was judged at the 5% level.
| Results|| |
There was a statistically significant difference between patients and controls regarding hemoglobin concentration, red blood cell (RBC) count, mean corpuscular volume, white blood cell count (P=0.000), platelet count (P=0.020), and reticulocytic count (P=0.000). A statistically significant difference was found between all patients with thalassemia and normal controls (P=0.000) regarding INR, where the mean was 1.2±0.2 in patients and 1.04±0.01 in controls as shown in [Table 1]. On comparing the hematological parameters of group I and group II, platelet count had a mean of 561.5±150.7 in group I which was significantly higher than group II mean of 294.6±130.4 (P=0.000). Likewise, on comparing both groups, the mean values of INR were higher in group I than that of group II but with no statistically significant difference.
|Table 1 Hematological and coagulation parameters in patients compared with control|
Click here to view
Protein C, protein S, and AT III values were low in 28.6, 38, and 4.8% of patients, respectively. Elevated factor VIII levels were found in three (10%) patients with thalassemia. Elevated factor IX levels were only seen in one patient.
The prevalence of heterozygosity was 15% (9/60) and homozygosity was 3.3% (2/60) for the FVL mutation; 1.7% (1/60) of patients were heterozygous for the PT 20210A mutation, and none were homozygous. The MTHFR genotype was 41.7% for CC, 53.3% for CT, and 8.3% for TT in patients.
Regarding sEPCR level, it was significantly higher in patients than in control (P=0.008) ([Table 2]). Although group I had a higher mean level of plasma sEPCR compared with group II, yet the difference was not statistically significant (P=0.709). However, still there was a significant difference when each group was separately compared with the control (P=0.021 and 0.048, respectively) ([Table 3] and [Table 4]). D-dimer test result was negative in all the studied groups.
|Table 2 Soluble endothelial protein C receptor level in patients compared with controls|
Click here to view
|Table 3 Soluble endothelial protein C receptor level in group I compared with controls|
Click here to view
|Table 4 Soluble endothelial protein C receptor level in group II compared with controls|
Click here to view
A significantly positive correlation was observed between sEPCR and total leucocytic count as well as platelet count (P=0.015 and 0.020, respectively) as shown in [Figure 1] and [Figure 2]. However, no correlation was noticed between sEPCR and age or sex.
|Figure 1 Correlation between soluble endothelial protein C receptor (sEPCR) and white blood cell (WBC) count|
Click here to view
|Figure 2 Correlation between soluble endothelial protein C receptor (sEPCR) and platelet count|
Click here to view
| Discussion|| |
Variable history of bleeding and thromboembolic complications was observed in patients with β-thalassemia. Chronic hypercoagulability observed in these patients is multifactorial. Damage of the phospholipid structure of the RBC membrane occurs with translocation of the procoagulant phosphatidylserine to the outer surface of the RBC membrane . Changes of the red cell membrane may explain their enhanced aggregation and capacity to enhance thrombin generation. Platelets and endothelial cells are eventually activated and tissue factor released. All these factors enhance the thrombotic process.
Epistaxis was evident in 26% of our patients which coincides with Naithani et al.  study, in which 16 of 54 patients with thalassemia had bleeding manifestations.
In one study, it was reported that the prolongation of PT was 40.7% and prolongation of aPTT was 46.3% . In the present study, PT and aPTT were prolonged in 86.7% and 83.3% of the patients, respectively with a statistical significance in comparison to controls. The d-dimer test result was negative in all of the studied groups even in patients with the highest levels of sEPCR, which agrees with Setiabudy et al.  who reported normal d-dimer in all patients with thalassemia.
A higher platelet count was especially observed in splenectomized patients which coincides with Setiabudy et al.  who found a higher platelet count in splenectomized versus nonsplenectomized group. Hazards of thrombosis may range from portal vein thrombosis to pulmonary embolism and deep vein thrombosis especially following splenectomy . As platelets play an important role in the pathogenesis of thrombosis, autopsy-based studies in a large number of patients with thalassemia documented striking pulmonary artery occlusions . Moreover, another study by Taher et al.  had estimated the prevalence of thrombotic events in β-thalassemia in 9% of the patients. In addition, a higher mean platelet volume was observed in group I compared with group II, which reflects a state of platelet hyperactivity. The enhanced platelet function and the higher platelet number comprise a double risk for hypercoagulability in splenectomized patients. In agreement with our findings, other studies also reported thromboembolic complications in splenectomized patients ,.
In the current study, low levels of protein C, protein S, and AT III were detected, which is in agreement with Naithani et al.  who reported that protein C was low in 26.2% of patients, protein S was low in 28.6%, and AT III was low in 46.8% of cases. Furthermore, Singer et al.  reported that protein C and protein S levels were below normal in most their patients with thalassemia.
The present study detected a slight increase in frequency of congenital thrombophilic mutations, including the FVL, PT G20210A, and MTHFR C677T mutations. Finkelstein et al.  found five of 23 patients with specific mutations indicating hereditary thrombophilia in their patients with thalassemia. Another study of patients with thalassemia intermedia revealed high FVL mutation .
In the current study, sEPCR was significantly higher in patients with thalassemia than in controls which may reflect a state of hypercoagulability. This is possibly induced by endothelial injury secondary to oxidative stress and/or platelet activation with subsequent release of procoagulant proteins . These findings are evidenced by the significantly positive correlation between sEPCR and platelet count in the studied patients. They also coincide with Setiabudy et al.  highlighting the role of platelets in inducing endothelial damage which is followed by enhanced production of sEPCR.
In the present study, another significantly positive correlation was found between sEPCR and total white blood cells. This may be explained by the fact that activated neutrophils and monocytes release their proteolytic enzymes with the production of free oxygen radicals as well as inflammatory cytokines that damage the endothelium and eventually degrade EPCR into its soluble form. This agrees with another study that found a positive correlation between plasma levels of sEPCR and disease activity in cases of Wegener granulomatosis .
Moreover, no correlation was found between sEPCR and age as our patients were adults, which agrees with Orhon et al.  who found a negative correlation between sEPCR level and age of the pediatric group, but failed to establish this negative correlation in the adult group. They also reported higher levels of sEPCR in healthy children compared with healthy adults, suggesting a regulatory mechanism for the protein C system over the first years of life . Similarly, no correlation was found between sEPCR and sex, whereas higher levels were detected in males by Orhon et al.  and were attributed to environmental factors as smoking.
In conclusion, with the prolonged life expectancy of patients with β-thalassemia, more caution of thromboembolic complications should be considered owing to high sEPCR and low protein C and protein S levels together with more frequent congenital thrombophilic mutations. Thus, after the first thrombotic event or even when they are exposed to thrombotic risk factors, they should be investigated for congenital thrombophilia, and prophylactic antithrombotic agents may be recommended.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Thein SL, Rees D. Hemoglobin and the inherited diseases of globin synthesis. In: Hoffbrand AV, Catovsky D, Tuddenham EG, Green AR, editors. Postgraduate haematology
. 6th ed. London, UK: 2011. pp. 83–107.
Elbeshlawy A, Yossry I. Prevention of hemoglobinopathies in Egypt. Hemoglogin
Ataga KI, Cappellini MD, Rachmilewitz EA. Beta-thalassemia and sickle cell anemia as paradigms of hypercoagulability. Br J Haematol
Cappelini MD, Grespi E, Cassinerio E, Bignamini D, Fiorelli G. Coagulation and splenectomy: an overview. Ann N Y Acad Sci
Zalloua PA, Shbaklott H, Mourad YA, Koussa S, Tahert A. Incidence of thromboembolic events in Lebanese thalassemia intermedia patients. Thromb Haemost
Riewald M, Petrovan RJ, Donnr A, Ruf M. Activated protein C signals through the thrombin receptor PAR1 in endothelial cells. J Endotoxin Res
Tavazzi D, Ducal L, Graziadei G, Comino A, Fiorelli G, Capallini MD. Membrane-bound iron contributes to oxidative damage of beta-thalassemia intermedia erythrocytes. Br J Hematol
Gandrille S. Endothelial cell protein C receptor and the risk of venous thrombosis. Haematologica
Buthep P, Rummavas S, Wisedpanichkij R, Fucharoen S. Increased circulating activated endothelial cells, vascular endothelial growth factor, and tumour necrosis factor in thalassemia. Am J Hematol
Naithani R, Chandra J, Narayan S, Sharma S, Singh V. Thalassemia major on the verge of bleeding or thrombosis? Hematology
Ghosh S, Pendurthi UR, Steinoe A, Esmon CT, Mohan V, Rao L. Endothelial cell protein C receptor acts as a cellular receptor for factor VIIa on endothelium. J Biol Chem
Lopez-Sagaseta J, Montes R, Puy C, Diez N, Fukudome K, Hermida J. Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity. J Thromb Haemost
Qu D, Wang Y, Esmon NL, Esmon CT. Regulated endothelial protein C receptor shedding is mediated by tumor necrosis factor converting enzyme/ADAM17. J Thromb Haemost
Setiabudy R, Wahidiyat PA, Setiawan L. Platelet aggregation and activation in thalassemia major patients in Indonesia. Clin Appl Thromb Hemost
Fucharoen S, Winichagon P. Thalassemia and abnormal hemoglobin. Int J Hematol
Taher A, Isma’eel H, Mehio G, Bignamini D, Kattamis A, Rachmilewitz EA et al.
Prevalence of thromboembolic events among 8, 860 patients with thalassaemia major and intermedia in the Mediterranean area and Iran. Thromb Haemost
Mohran M, Markmann I, Dworochak V. Thromboembolic complications after splenectomy for hemolytic diseases. Am J Hematol
Vant Riet M, Burger JW. Diagnosis and treatment of portal vein thrombosis following splenectomy. Br J Surg
Singer ST, Kuypers FA, Styles L, Vichinsky EP, Foote D, Rosenfeld H. Pulmonary hypertension in thalassemia: association with platelet activation and hypercoagulable state. Am J Hematol
Finkelstein Y, Yaniv I, Berant M, Zilber R, Garty BZ, Epstein O et al.
Central venous line thrombosis in children and young adults with thalassemia major. Pediatr Hematol Oncol
Zalloua PA, Shbaklo H, Mourad YA, Koussa S, Taher A. Incidence of thromboembolic events in Lebanese thalassemia intermedia patients. Thromb Haemost
Eldor A, Rachmilewitz EA. The hypercoagulable state in thalassemia. Blood
Boomsma MM, Stearns-Kurosawa DJ, Stegman CA, Raschi E, Meroni PL, Kurosawa S et al.
Plasma levels of soluble endothelial cell protein C receptor in patients with Wegener’s granulomatosis. Clin Exp Immunol
Orhon FS, Frgun H, Egin Y, Ulukol B, Baskan S, Akar N. Soluble endothelial protein C receptor levels in healthy population. J Thromb Thrombol
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]