|Year : 2017 | Volume
| Issue : 1 | Page : 1-6
Markers of coagulation activation in patients with hemoglobinopathy in Western Saudi Arabia
Soheir Adam MBBCh, FRCPath , Galila Zaher
Department of Hematology, King Abdulaziz University, Jeddah, Saudi Arabia
|Date of Web Publication||12-Apr-2017|
Department of Hematology, King Abdulaziz University, PO Box 80215, Jeddah 21589
Source of Support: None, Conflict of Interest: None
Objective: This study aims to examine markers of coagulation activation and their possible clinical associations in sickle cell disease (SCD) and thalassemia.
Materials And Methods: This study was conducted on patients with hemoglobinopathy followed up at King Abdulaziz University Hospital between October 2010 and November 2011. Demographic and clinical data were collected for all participants. The independent t-test was used to compare two group means, while the one-way analysis of variance test was used to compare more than two group means.
Results: The study included 122 hemoglobinopathy cases and 34 controls. Protein C, protein S, antithrombin, and activated protein C resistance (APCR) were significantly lower in patients with hemoglobinopathy than in the control group, while D-dimer levels were significantly higher (P < 0.001 for all comparisons). Patients with SCD had significantly higher protein C and D-dimer levels than those who had thalassemia (P < 0.01 for both). Arterial and venous thromboses were more prevalent in patients with SCD (12.5 and 18.7%), compared to patients with thalassemia (9.3 and 2.3%, respectively). Cases had significantly lower protein C, protein S, antithrombin, and APCR values (P < 0.001 for each), and higher D-dimer levels (P = 0.016) than male controls. There was no significant difference in markers of coagulation activation between patients who had undergone splenectomy compared with those who had intact spleens.
Conclusion: Natural anticoagulants were significantly lower, and D-dimer levels were higher in Saudi patients with SCD and thalassemia compared to healthy controls. The procoagulant phenotype was more pronounced in patients with SCD and was associated with a higher prevalence of clinical thrombosis, compared to patients with thalassemia in this population.
Keywords: Coagulation activation, sickle cell disease, thalassemia
|How to cite this article:|
Adam S, Zaher G. Markers of coagulation activation in patients with hemoglobinopathy in Western Saudi Arabia. J Appl Hematol 2017;8:1-6
|How to cite this URL:|
Adam S, Zaher G. Markers of coagulation activation in patients with hemoglobinopathy in Western Saudi Arabia. J Appl Hematol [serial online] 2017 [cited 2017 May 25];8:1-6. Available from: http://www.jahjournal.org/text.asp?2017/8/1/1/204426
| Introduction|| |
Sickle cell disease (SCD) and thalassemia are considered as hypercoagulable disorders., Available evidence for hypercoagulability in SCD includes abnormal levels of prothrombin fragment 1.2 and D-dimer, accelerated Factor VII turnover, increased levels of thrombin–antithrombin complexes, abnormal expression of tissue factor, and chronic platelet activation. Previous studies have reported complications due to hypercoagulability in patients with hemoglobinopathies., Evidence of thrombophilia ranges from subclinical disturbances in the levels of hemostatic factors to an increased incidence of a variety of thromboembolic events, such as pulmonary embolism and deep venous thrombosis. Furthermore, pulmonary hypertension (PHT) is common in patients with SCD, with an estimated prevalence rate of 30%., Chronic intravascular hemolysis has been reported to play a pivotal role in the pathogenesis of PHT.,, Other investigators have found that PHT in SCD may be caused by a variety of different factors, including continuous endothelial activation, in situ thrombosis, and thromboembolism of the pulmonary artery.
Saudi patients with SCD were found to have higher levels of markers of thrombin generation and inflammatory cytokines during painful crisis compared to those in steady state in a previous study, indicating that the painful vaso-occlusive crisis increases the inflammatory state in SCD.
While hemoglobinopathies are described as hypercoagulable states, little is known about the clinical associations of markers of hypercoagulability in the Saudi population. In this paper, we examine markers of coagulation activation and their possible association with the clinical complications seen in SCD and thalassemia.
| Materials and Methods|| |
This study was conducted between October 2010 and November 2011 at King Abdulaziz University Hospital (KAUH) and included 122 patients with hemoglobinopathy and 34 controls. The Research Ethics Committee at King Abdulaziz University approved this research study.
Patients with hemoglobinopathy attending hematology clinics at KAUH were approached consecutively to participate in the study. Patients were enrolled in the study after signing in an informed consent. Blood samples were collected for laboratory studies, and data were collected by interview and review of medical records. Citrated plasma was collected, and 0preparation was completed in accordance with the manufacturers’ instructions. Functional assays for antithrombin, protein C, protein S, and activated protein C resistance ratio (APCR ratio) were estimated using the commercial kit (Dade Behring, Marburg, Germany). D-dimer was estimated using Advanced D-dimer (Dade Behring, Marburg, Germany). All the previous laboratory tests were performed routinely in the hematology laboratory at KAUH, where samples were run in batches and assayed on Blood Coagulation System (BCS®XS) analyzer. Non-routine measurements of plasma concentration of Fibrinopeptide A (FPA) and Fibrinopeptide B (FPB) were performed by Enzyme Linked Immunosorbent Assay (ELISA) technique using the commercial kit IMUCLONE by BioMEDICA.
Study inclusion criteria were:
- Patients able and willing to participate in the study after signing in their informed consent.
- Patients with a diagnosis of SCD or thalassemia receiving care at KAUH.
- Patients with SCD, thalassemia intermedia (TI), and thalassemia major (TM).
Study exclusion criteria were:
- Pregnant patients.
- Patients with SCD in acute vaso-occlusive crisis.
- Patients transfused within one month from sample acquisition.
- Patients on anticoagulant medications.
A data collection tool was designed to assist with the collection and collation of demographic and clinical data. This instrument was used to collect information from the study patients’ electronic medical records including age, gender, clinical signs and symptoms, as well as results of laboratory investigations.
The Statistical Package for the Social Sciences (SPSS Inc., IBM, New York, US), version 22, was used to analyze the data. Demographic and coagulation factor data are described in absolute numbers and percentages; and continuous variables are presented as means and standard deviations (SDs). The independent t-test was used to compare two group means, while the one-way analysis of variance (ANOVA) test with the least significant difference (LSD) was used to compare more than two group means. These tests were done with the assumption of normal distribution. Otherwise, Welch’s t-test for two group means and Games Howell method for multiple groups were used instead of the LSD test. To correlate variables which were represented by means, Pearson’s correlation coefficient was used. Lastly, a conventional P-value of <0.05 was the criterion for rejecting the null hypothesis.
| Results|| |
The study included 122 hemoglobinopathy cases and 34 controls, of whom 83 (53.2%) were males. The mean age was 26.7 (8.9) years (range, 13–48 years). Patients with TM comprised the majority of the hemoglobinopathy cases (86 patients, 70.5%), followed by those with SCD (32 patients, 26%) and TI (4 patients, 3.3%). A total of 20 cases of venous thrombosis and 26 arterial events were present. Venous thromboembolism (VTE) was clinically suspected in 12 patients (9.8%) and a radiological diagnosis of VTE was confirmed in eight patients (6.5%) whereas there was no evidence of VTE in 102 of the cases (82.9%). Embolic stroke was present in 26 patients. The prevalence of VTE was higher among patients with SCD compared to patients with thalassemia, with frequencies of arterial and venous thrombosis of 12.5 and 18.7%, respectively, in patients with SCD, and 9.3 and 2.3%, respectively, in patients with thalassemia.
On ultrasound examination, approximately 39.3% of the cohort had splenomegaly, while 34.4% were found to have an absent spleen, either due to autosplenectomy or a history of surgical removal of the spleen. In approximately one-third of the patients (26.2%), spleen size was normal.
Protein C, protein S, antithrombin, and APCR were significantly lower in patients with hemoglobinopathy than in the control group [Figure 1], while D-dimer levels were significantly higher (P < 0.001 for all comparisons). On the other hand, values for fibrinopeptide A and lupus anticoagulant did not differ significantly between cases and controls.
|Figure 1: Comparison of laboratory markers of coagulation activation in patients with hemoglobinopathy and controls|
Click here to view
When compared with patients who had thalassemia, those with SCD had significantly higher protein C and D-dimer levels [P < 0.01 for both; [Figure 2]]. Although patients with SCD had higher protein S, antithrombin, fibrinogen, and platelet levels, these differences did not reach statistical significance. Similarly, patients with SCD and thalassemia did not differ in terms of hemoglobin, and white blood cells (WBC) values or the prevalence of lupus anticoagulant and APCR. Differences in coagulation markers were present both overall and when stratified by gender. Among males, cases had significantly lower protein C, protein S, antithrombin, and APCR values (P < 0.001 for each), and higher D-dimer levels (P = 0.016), than male controls. Values were similar between female cases and controls (differing only in the actual P-value for the D-dimer level comparison, which was P = 0.006 for the females).
|Figure 2: Markers of coagulation activation in patients with sickle cell disease and patients with thalassemia|
Click here to view
Markers of coagulation activation did not differ significantly between patients who had a history of venous thrombosis, and those who did not [Table 1]. Similarly, results of laboratory investigations did not differ between patients with hemoglobinopathy with and without a history arterial thrombosis.
|Table 1: Comparison of laboratory values between patients with a history of arteriovenous thrombosis and those with no history of thrombosis|
Click here to view
Using the one-way ANOVA test, we found a significantly higher mean platelet count (P < 0.001) and mean WBC count (P = 0.003) among patients who had undergone auto- or surgical splenectomy compared with those who had intact spleens [Table 2]. However, the levels of the other markers of coagulation activation did not differ significantly between the two groups.
|Table 2: Comparison of laboratory parameters between patients with intact spleens and those with a history of splenectomy|
Click here to view
| Discussion|| |
There is substantial evidence in the literature that SCD and other chronic hemolytic anemias such as beta-thalassemia represent hypercoagulable states. This is reflected in the increased incidence of thrombotic complications seen in these patients, including VTE, in situ pulmonary thrombosis, and stroke. Both disorders have been associated with specific laboratory findings consistent with increased platelet and coagulation activation, and decreased levels of natural anticoagulants. For example, in one previous study of patients with SCD, markers of coagulation including plasma thrombin–antithrombin complex, prothrombin fragment 1.2, and D-dimer levels were shown to be increased at steady state (i.e., during asymptomatic periods). The same study reported increased levels of these markers during acute pain episodes and found that the frequency of pain episodes in the subsequent year correlated with D-dimer plasma levels. These findings suggest that vaso-occlusive events may be triggered by coagulation activation., The authors have previously found that D-dimer levels were increased in a sample of 40 Saudi patients with SCD, and protein C and S levels were lower, compared to sickle cell trait and unaffected controls. In this study, we explored a possible relationship between laboratory coagulation markers and thromboembolic manifestations, stroke, and splenectomy in a sample of Saudi patients with SCD and thalassemia. Studies in patients with thalassemia also point toward an underlying hypercoagulability related to platelet activation, increased erythrocyte endothelial adherence, decreased protein C and S levels, and increased thrombin generation. Furthermore, thrombophilic propensity in hemolytic anemias overall was found to be increased following splenectomy.
In this study, we explored a possible relationship between laboratory coagulation markers and clinical complications (including VTE, stroke, and splenectomy) in a sample of Saudi patients with SCD and thalassemia. We found significantly lower levels of the natural anticoagulants protein S, protein C, and antithrombin in patients with hemoglobinopathies compared with controls, both overall and when stratified by gender. These results are consistent with those from prior studies.,,, No previous study has specifically compared markers of coagulation activation between patients with SCD and thalassemia. In the current report, patients with SCD had significantly higher D-dimer values, suggesting that SCD is associated with a higher rate of thrombin generation. The finding that thrombotic events were seen more frequently in patients with SCD compared with patients with thalassemia also reflects this. Available clinical assays for measurement of D-dimer antigen, detect both the terminal digestion product for fibrin and high molecular weight soluble fibrin fragments that are yet to enter a fibrin gel or that have already been released before plasma degradation. This may explain why D-dimer assay was elevated in this study population, while other plasma markers were not significantly increased. Further, exposure of phosphatidyl serene in sickle red cell membranes leads to thrombin generation in whole blood. Recently, a study of global thrombin generation in SCD concluded that cellular contribution in SCD is pivotal in thrombin generation. Thus, global thrombin generation assays show an increase in thrombin generation in whole blood but not in plasma.
Loss of splenic function has more recently been found to increase the risk for thromboembolic events, although the actual risk is related to the underlying disorder. The greatest risk is seen in patients with a primary disorder associated with chronic hemolysis, such as hemoglobinopathies. However, when we examined patients with hemoglobinopathy who had surgical or auto-splenectomy and compared them to those with an intact spleen, we did not see significant differences in the natural anticoagulant or D-dimer levels. Thus, unlike in prior studies,,, we did not find evidence that splenectomy is associated with an increase in hypercoagulability. However, a recent study found no evidence of increased global thrombin generation in patients with splenectomized thalassemia in platelet-poor plasma. Similarly, markers of coagulation activation did not differ in patients with splenectomized and non-splenectomized thalassemia in other populations., In a study of splenectomized Saudi SCD patients; there was evidence of reduction in the rate of hemolysis and post-splenectomy transfusion requirements were also reduced. Since hemolysis was found to be closely associated with a higher risk of coagulation activation; thus, down-regulation of hemolysis may explain the reduced prothrombotic markers in our population. Interestingly, the risk for post-splenectomy thromboembolic events in patients with thalassemia has been found to be significantly greater in TI than in TM. Unfortunately, the number of patients with TI in our cohort was very low (3.3%), making it difficult for us to draw any relevant conclusions. Not surprisingly, however, patients without a functional spleen had higher WBC counts and platelet levels.While patients with SCD and thalassemia have numerous complications, some patients experience a more severe phenotype compared with others. This is true in SCD when comparing the African haplotype to the Arab-Indian haplotype. The cohort in this study includes Saudi Arabian patients with the Arab-Indian SCD haplotype, which is phenotypically different from other SCD haplotypes. For example, leg ulcers and priapism are uncommon in this population. In addition, certain disease complications, including pain crises and silent cerebral infarcts, generally occur at later ages compared to African SCD haplotypes.,, This phenotypical variance may indicate that our results are specific for Saudi patients with SCD and are not necessarily generalizable to other populations with SCD. If so, comparison of the results of coagulation studies in Arab-Indian patients with SCD to those in other populations could provide important insights into the unique pathophysiological mechanisms underlying the different clinical phenotypes.
In conclusion, we found significantly lower levels of natural anticoagulants and higher levels of D-dimer in patients with SCD and thalassemia compared to healthy controls, supporting the theory that they represent hypercoagulable states. Patients with SCD had evidence of increased thrombin generation, as reflected by elevated D-dimer values compared to patients with thalassemia, which was paralleled by an increase in clinical thromboembolic events. Future research should ideally focus on the pathophysiology behind the phenotypic variation in patients with hemoglobinopathy in different populations.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Tomer A, Harker LA, Kasey S, Eckman JR. Thrombogenesis in sickle cell disease. J Lab Clin Med 2001;137:398-407. doi: 10.1067/mlc.2001.115450
Eldor A, Rachmilewitz EA. The hypercoagulable state in thalassemia. Blood 2002;99:36-43.
Kurantsin-Mills J, Ofosu FA, Safa TK, Siegel RS, Lessin LS. Plasma factor VII and thrombin-antithrombin III levels indicate increased tissue factor activity in sickle cell patients. Br J Haematol 1992;81:539-44.
Key NS, Slungaard A, Dandelet L, Nelson SC, Moertel C, Styles LA et al.
Whole blood tissue factor procoagulant activity is elevated in patients with sickle cell disease. Blood 1998;91:4216-23.
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;94:1520-7. doi: 10.3324/haematol.2009.010546
Bhattacharyya M, Kannan M, Chaudhry VP, Mahapatra M, Pati H, Saxena R. Hypercoagulable state in five thalassemia intermedia patients. Clin Appl Thromb Hemost 2007;13:422-7. doi: 10.1177/1076029607303539
Ataga KI, Sood N, De Gent G, Kelly E, Henderson AG, Jones S et al.
Pulmonary hypertension in sickle cell disease. Am J Med 2004;117:665-9. doi: 10.1016/j.amjmed.2004.03.034
Gladwin MT, Sachdev V, Jison ML, Shizukuda Y, Plehn JF, Minter K et al.
Pulmonary hypertension as a risk factor for death in patients with sickle cell disease. N Engl J Med 2004;350:886-95. doi: 10.1056/NEJMoa035477
Aslan M, Ryan TM, Adler B, Townes TM, Parks DA, Thompson JA et al.
Oxygen radical inhibition of nitric oxide-dependent vascular function in sickle cell disease. Proc Natl Acad Sci U S A 2001;98:15215-20. doi: 10.1073/pnas.221292098
Reiter CD, Wang X, Tanus-Santos JE, Hogg N, Cannon RO 3rd, Schechter AN et al.
Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease. Nat Med 2002;8:1383-9. doi: 10.1038/nm799.
Adedeji MO, Cespedes J, Allen K, Subramony C, Hughson MD. Pulmonary thrombotic arteriopathy in patients with sickle cell disease. Arch Pathol Lab Med 2001;125:1436-41. 10.1043/0003-9985(2001)125≺1436:PTAIPW≻2.0.CO;2
Qari MH, Dier U, Mousa SA. Biomarkers of inflammation, growth factor, and coagulation activation in patients with sickle cell disease. Clin Appl Thromb Hemost 2012;18:195-200. doi: 10.1177/1076029611420992
Ataga KI. Hypercoagulability and thrombotic complications in hemolytic anemias. Haematologica 2009;94:1481-4. doi: 10.3324/haematol.2009.013672
Singer ST, Ataga KI. Hypercoagulability in sickle cell disease and beta-thalassemia. Curr Mol Med 2008;8:639-45.
Lee SP, Ataga KI, Orringer EP, Phillips DR, Parise LV. Biologically active CD40 ligand is elevated in sickle cell anemia: Potential role for platelet-mediated inflammation. Arterioscler Thromb Vasc Biol 2006;26:1626-31. doi: 10.1161/01.ATV. 0000220374.00602.a2.
Adam SS, Zaher G, Ibrahim M, Ataga KI, De Castro LM, Key NS. Markers of coagulation activation and inflammation in sickle cell disease and sickle cell trait. Blood 2008;112.
Crary SE, Buchanan GR. Vascular complications after splenectomy for hematologic disorders. Blood 2009;114:2861-8. doi: 10.1182/blood-2009- 04-210112.
Westerman MP, Green DD, Gilman-Sachs AA, Beaman KK, Freels SS, Boggio LL et al.
Antiphospholipid antibodies, proteins C and S, and coagulation changes in sickle cell disease. J Lab Clin Med 1999;134:352-62.
Cappellini MD, Robbiolo L, Bottasso BM, Coppola R, Fiorelli G, Mannucci AP. Venous thromboembolism and hypercoagulability in splenectomized patients with thalassaemia intermedia. Br J Haematol 2000;111:467-73.
Adam SS, Key NS, Greenberg CS. D-dimer antigen: Current concepts and future prospects. Blood 2009;113:2878-87. doi: 10.1182/blood-2008-06-165845.
Tripodi A, Martinelli I, Chantarangkul V, Clerici M, Artoni A, Passamonti S et al.
Thrombin generation and other coagulation parameters in a patient with homozygous congenital protein S deficiency on treatment with rivaroxaban. Int J Hematol 2016;103:165-72. doi: 10.1007/s12185-015- 1898-6.
Abolghasemi H, Amid A, Zeinali S, Radfar MH, Eshghi P, Rahiminejad MS et al.
Thalassemia in Iran: Epidemiology, prevention, and management. J Pediatr Hematol Oncol 2007;29:233-8. doi: 10.1097/MPH.0b013e3180437e02.
Tripatara A, Jetsrisuparb A, Teeratakulpisarn J, Kuaha K. Hemostatic alterations in splenectomized and non-splenectomized patients with beta-thalassemia/hemoglobin E disease. Thromb Res 2007;120:805-10. doi: 10.1016/j.thromres.2007.02.006
Rashidi A, Alavi S, Arzanian MT, Ahmadinejad M. Anticoagulant system activity in splenectomized versus non-splenectomized Iranian patients with beta-thalassemia major. Pediatr Hematol Oncol 2007;24:645-7. doi: 10.1080/08880010701620798
Canatan D, Zorlu M, Bayır N, Ertürk C, Dorak A, Oğuz N et al.
Thrombosis after splenectomy in patients with thalassemia. Turk J Haematol 2001;18:259-63.
Ghmaird A, Alnoaiji MM, Al-Blewi S, Zaki S, El-Lewi A, Ahmad N. Splenectomy in patients with sickle cell disease in Tabuk. Open Access Maced J Med Sci 2016;4:107-11. doi: 10.3889/oamjms.2016.034
Padmos MA, Roberts GT, Sackey K, Kulozik A, Bail S, Morris JS et al.
Two different forms of homozygous sickle cell disease occur in Saudi Arabia. Br J Haematol 1991;79:93-8.
Adekile A, Al-Kandari M, Haider M, Rajaa M, D’Souza M, Sukumaran J. Hemoglobin F concentration as a function of age in Kuwaiti sickle cell disease patients. Med Princ Pract 2007;16:286-90. doi: 10.1159/000102151
Adekile AD, Yacoub F, Gupta R, Sinan T, Haider MZ, Habeeb Y et al.
Silent brain infarcts are rare in Kuwaiti children with sickle cell disease and high Hb F. Am J Hematol 2002;70:228-31. doi: 10.1002/ajh.10143.
[Figure 1], [Figure 2]
[Table 1], [Table 2]