|Year : 2015 | Volume
| Issue : 2 | Page : 64-69
Possible role of hemoglobin S in implicating hemostatic and inflammatory reactions: Study on Saudi Arabian population
Mohammed A Sorour1, Salwa A Dabbous2, Reham Abdel Aleem Mohamed Afify2
1 Department of Clinical Pathology, Alazhar University, Giza, Cairo, Egypt
2 Department of Clinical Pathology, Cairo University, Giza, Cairo, Egypt
|Date of Web Publication||7-Jul-2015|
Reham Abdel Aleem Mohamed Afify
Department of Clinical Pathology, Cairo University, Cairo
Source of Support: None, Conflict of Interest: None
Background: Sickle cell disease (SS), is one of the most common inherited hematologic disorders, presents with chronic hemolytic anemia that can be punctuated by crises, infarcts, organ damage and hypercoagulable state. Sickle cell trait (AS) is a benign disorder. However, there is an increased risk for several abnormalities such as hematuria and pulmonary embolism. Coagulation and inflammation cannot be considered as two separate processes. Protein C is an important link between coagulation and inflammation; activated protein C can modulate inflammatory activity. Objectives: To examine the extent to which the coagulation activation and inflammatory link processes occur in hemoglobin SS disease (HbSS) patients in chronic state and in sickle cell trait (AS) individuals comparable with normal individuals. Subjects and Methods: Twenty sickle cell anemia patients in the chronic state and 20 AS individuals were included in this study. Coagulation tests and some acute phase reactants (inflammatory markers) were measured in all cases. Results: Comparison of SCD patients and AS individuals altogether, showed significantly higher prothrombin time, C-reactive protein and WVF activity (P < 0.05, P < 0.01 and P < 0.05, respectively) and significantly lower protein C level and protein S activity (P < 0.01 and P < 0.05, respectively) in HbSS patients than AS. Other parameters showed no significant difference. Conclusion: Some hemostatic and inflammatory link changes are present in SCD even in the chronic state and in AS individuals. Together with the known sickling of red blood cells, thrombocytosis, and increased viscosity, they contribute to the hypercoagulable state present in these individuals.
Keywords: Antithrombin, C-reactive protein, fibrinogen, protein C, protein S, sickle cell anemia
|How to cite this article:|
Sorour MA, Dabbous SA, Afify RA. Possible role of hemoglobin S in implicating hemostatic and inflammatory reactions: Study on Saudi Arabian population. J Appl Hematol 2015;6:64-9
|How to cite this URL:|
Sorour MA, Dabbous SA, Afify RA. Possible role of hemoglobin S in implicating hemostatic and inflammatory reactions: Study on Saudi Arabian population. J Appl Hematol [serial online] 2015 [cited 2018 Oct 19];6:64-9. Available from: http://www.jahjournal.org/text.asp?2015/6/2/64/160202
| Introduction|| |
Sickle cell disease (SCD), (SCD; Online Mendelian Inheritance in Man No. 603903) one of the most common inherited hematological disorders, is an autosomal recessive genetic red cell disorder that results from a point mutation (βs , 6V) in codon 6, with the insertion of valine in place of glutamic acid, leading to the production of a defective form of hemoglobin (Hb) termed Hb S.  SCD, has a worldwide distribution among African, American Blacks, in the Mediterranean area, Southern Europe, South Asia, India, and the Arabian Peninsula including Saudi Arabia specially in the Southern part; Jazan.  SCD presents with chronic hemolytic anemia that can be punctuated by crises, infarcts, and multi-organ damage and hypercoagulable state. Sickle cell trait (AS) is a benign disorder most of the time. However, there is an increased risk for abnormalities such as hematuria, pulmonary embolism and sudden death in low-oxygen conditions.  Hb S, termed on account of the sickle-shaped red blood cells (RBCs), when polymerized in the deoxygenated state with subsequent red cell membrane structural and functional change and increased adherence to the vascular endothelium,  intravascular sickling in capillaries and small vessels leads to vaso-occlusion and impaired blood flow. , The persistent membrane damage associated with Hb S polymerization also favors the generation of distorted rigid cells and further contributes to vaso-occlusive events and cell destruction in the peripheral circulation. The pathogenesis of hypercoagulability is considered to be multifactorial. Altered components of hemostasis system in SCD have been suggested. Low plasma levels of protein C, protein S, and antithrombin III (ATIII), elevated plasma levels of thrombin-antithrombin (TAT) complexes, prothrombin fragment 1 + 2 (F1 + 2), D-dimer complexes, and circulating antiphospholipid antibodies, platelet activation during vaso-occlusive crisis, abnormal external exposure of phosphatidylserine and adherence of sickle erythrocytes to the vascular endothelium, reducing nitric oxide level in the presence of hemolytic anemia, and increased tissue factor expression, all have been detected in SCD patients. , These abnormalities of the hemostatic system in SCD are leading to increase the risk of thrombosis. Protein C and S are Vitamin K-dependent proteins with an essential natural anticoagulant functions. Protein C exists in an inactive form and is activated by the thrombin-thrombomodulin complex. It's activated form (activated protein C [APC]) controls the coagulation process by cleaving and inactivating factors VIIIa (FVIIIa) and FVa in the presence of protein S, which acts as a cofactor for APC, down-regulating clot formation and promoting fibrinolysis.  Coagulation and inflammation cannot be dealt with as two separate entities, protein C is an important link between both processes. Previous studies have revealed different hemostatic abnormalities in hemoglobin SS (HbSS) patients being intensively studied during crises, but sparingly during chronic state and in AS individuals.  Furthermore, no much attention was directed to the conduit between coagulation and inflammation. The purpose of this study was to examine the extent to which the coagulation activation and inflammatory link processes occur in individuals with HbSS in chronic state and AS comparable with normal individuals and with each other.
| Subjects and methods|| |
This study was conducted on 20 SCD patients who attended hematology clinic of King Fahad Hospital in Jazan, they were 10 males and 10 females, with age ranged from 14 to 31 years (mean age: 24 years). Twenty sickle cell trait individuals, 12 males and 8 females, with age ranged from 16 to 37 years (mean age: 26.5 years) were also involved and were selected from families of SCD patients and premarital screening tests. Twenty healthy volunteers, 11 males and 9 females with age ranged from 15 to 35 years (mean age: 27 years), who attended different general clinics within the hospital were conducted as a healthy control group. All individuals were subjected to full history, clinical examination, and full investigations including: Complete blood count (CBC), Hb lectrophoresis, prothrombin time (PT), activated partial thromboplastin time (aPTT), D-dimer, protein C level, protein S activity, ATIII, activated protein C resistance (APC-R), C-reactive protein (CRP), von Willebrand factor (VWF) activity and fibrinogen concentration.
The subjects neither experienced any crisis (excluded clinically and by CBC) nor received anticoagulants at the time preceding sample collection. They exhibited normal liver functions, except for the mild elevation of indirect bilirubin of SCD patients due to chronic hemolysis. This study was approved by the local ethical committee of the hospital.
After verbal informed consent, venous blood samples were collected with minimal stasis; 2 ml of blood were collected in ethylenediaminetetraacetic acid tube for the assay of CBC (ADVIA 2120, Siemens) and Hb electrophoresis (SEBIA, Sadier), one part of sodium citrate 3.2% was mixed with nine parts of blood, and immediate centrifugation at 3500 rpm for 10 min was done to obtain plasma, for assay of PT and aPTT (BCT) which should be done as rapidly as possible and the remaining plasma was stored at −20°C for the other coagulation tests. Serum samples were taken for CRP (ADVIA, Siemens).
Determination of antithrombin III concentration and protein C level
Quantitative determination of ATIII concentration and protein C were done by chromogenic assay, (BCT, Dade Behring), protein C in plasma sample is activated by specific snake venom activator and the resulting protein C a is measured.
Determination of protein S activity and activated protein
Determination of protein S activity and APC-R were done by clotting assay, (BCT, Dade Behring), protein S activity involves a modified aPTT in the presence of APC and protein S deficient plasma. APC-R involves measuring aPTT of plasma sample with and without the addition of a standardized amount of exogenous APC and the result is calculated as a ratio.
Measurement of fibrinogen, D-dimer and von Willebrand factor activity
Fibrinogen is measured by an automated clauss assay - thrombin time based - in a Dade Behring fibrinometer. The immunologic assay that detects D-dimer uses monoclonal antibodies to the cross-linked d regions of degraded fibrin and is measured turbidimetrically through increase in turbidity (BCT, Dade Behring). WVF activity (Ristocetin cofactor) uses ristocetin; an antibiotic that causes VWF and platelets to stick together via GPIb, This decreases the turbidity of the reaction suspension, (BCT, Dade Behring), measures the change in absorbance and automatically determines the test as percent of normal.
Data were expressed descriptively as percentages for qualitative values and mean ± standard deviation for quantitative parametric data. The compiled data were computerized and analyzed by SPSS software package, version 2, Echo Soft Corporation, USA. The following tests of significance were used: Analysis of variance test between more than 2 means, t-test between means was used to analyze the mean difference. Comparison of qualitative data was carried out using Chi-square test, crosstabs and least significant difference. A level of significance with P ≤ 0.05 was considered significant, P ≤ 0.01 was considered of high significance and P > 0.05 insignificant.
| Results|| |
Comparison of hematological parameters between HbSS, AS and normal controls [Table 1] showed that there was a high statistical significant difference between HbSS and AS and HbSS and controls as regards Hb, platelets, and reticulocytcic count with (P < 0.001), while there was no statistical significant difference between AS and controls for the same parameters (P > 0.05).
|Table 1: Comparison of hematological parameters of HbSS, AS and controls|
Click here to view
The results of coagulation (hemostasis) profile of HbSS, AS and controls are shown in [Table 2], the mean PT and the mean D-dimer levels were both significantly higher in SS individuals than in the control group (P < 0.01). In AS individuals, PT was within normal limits (P > 0.05) but D-dimer levels showed the same results as in HbSS patients. aPTT was within normal controls in both HbSS patients and AS individuals (P > 0.05). The coagulation inhibitors; protein C level, protein S activity and ATIII concentration (AT) as well as APC-R were all found to be significantly lower in HbSS patients than in the control group (P < 0.01). They were also significantly lower in AS individuals than in the control group (P < 0.05, P < 0.01, P < 0.05 and P < 0.01 respectively). SCD patients showed significantly higher PT (P < 0.05) and significantly lower protein C level and protein S activity (P < 0.01 and P < 0.05 respectively) than AS individuals [Table 2].
In acute phase reactants (inflammatory markers), the mean value of CRP was significantly higher in HbSS patients than in the control group (P < 0.01). In AS individuals, CRP was within normal limits (P > 0.05). VWF activity was significantly higher in HbSS patients than in the control group (P < 0.05), but AS individuals showed no significant difference from the controls. Fibrinogen concentration showed significantly decreasing the difference between HbSS patients and controls (P < 0.05) but AS individuals showed no significant difference from the controls (P > 0.05). Comparing HbSS patients and AS individuals with each other showed significantly higher CRP and WVF activity (P < 0.01 and P < 0.05 respectively), but fibrinogen levels showed no statistical significant difference (P > 0.05) [Table 3].
| Discussion|| |
In view of blood vessel occlusion and tissue infarction that can occur in the presence of Hb S responsible for the sickle-shaped RBCs, sickle cell disease is regarded as a hypercoagulable state. Sickle cell traits, however apparently normal, are not remote from this concept. The problem is not solely mechanical; several factors contribute to it. The intravascular consumption syndrome is more studied during the crisis periods than in the steady state. Coagulation and inflammation co-exist and cannot be separated from one another. Ischemic damage to tissues elicits an acute inflammatory response. The later in the turn promotes a prothrombotic state by increasing tissue factor expression that initiates the coagulation cascade, activating endothelial cells and increasing platelet production.  Fibrin deposition is a prominent feature of all inflammatory reactions in a step for tissue repair.
In this study, blood parameters of sickle cell traits were within normal values and showed highly significant difference from the sickle cell disease patients who have variable degree of anemia, increased reticulocytic percentage caused by chronic hemolysis and thrombocytosis due to hemolysis and functional asplenia. In our study, the mean PT was significantly higher in sickle cell anemia (SCA) patients than in the control group (P < 0.01) but there was no statistical difference in the mean PT between AS and controls. The aPTT showed no significant change in all groups. There were studies that showed that PT of SCD patients is either prolonged  or normal  in the steady state. Prolonged PT, without anticoagulant use, is probably due to chronic consumption of coagulation factors from enhanced pro-coagulant activity, moreover, liver dysfunction resulting from anoxia secondary to sinusoidal obstruction by sickling RBCs, iron overload and associated viral hepatitis (if present), can result in decrease synthesis of these factors.  The proposed mechanisms may not be a constant defect and coagulation studies may vary over time in the same patient.
Protein C is activated (APC) by binding of thrombin to thrombomodulin; a process more accelerated in the presence of endothelial protein C receptor, APC cleaves FVa and FVIIIa shutting down the coagulation pathway and inhibiting thrombin-induced pro-inflammatory activities including platelet activation and cytokine-induced chemotaxis for monocytes and neutrophils.  Besides, APC is antiapoptotic and provides an endothelial barrier protection.  In our study, protein C levels of SCA patients in steady state and AS were found to be significantly low compared to the control (P < 0.01 and P < 0.05 respectively). These are also reported in other studies in medical reviews. , Protein S is a protein C cofactor that binds it to the platelet surface. Sixty percent of total plasma protein S antigen circulates bound to C4b binding protein; an acute phase reactant. Only the free portion has anticoagulant activity. Protein S also exerts APC-independent anticoagulant activity through direct binding to FVa, Xa, and VIIIa. Protein S plays a role in enhancing phagocytosis of apoptotic cells thereby, limiting the development of inflammation. 
In our SCD patients and AS individuals, protein S levels were found to be significantly low (P < 0.01). However, some other studies revealed normal levels of protein S as opposed to low protein C levels that can be explained by the fact that protein S is also synthesized in the endothelium of blood vessels as well as in the liver. 
The anticoagulant activity of APC was reduced in both SCD and sickle cell traits in a modified aPTT assay. This defect was termed APC-R ratio. It cohorts with the hyper-coagulable state.  This contributes to vaso-occlusive complications of Hb S. This is in accordance with a previous study that displayed a significant reduction of the median APC-R ratio in SCD compared to controls. 
ATIII is a plasma inhibitor protein that blocks the enzymatic activity of some serine protease coagulation factors. ATIII was reported here to be significantly low in SCD patients and AS individuals in relation to the control (P < 0.01 and P < 0.05, respectively), probably through the formation of TAT complexes in plasma. In the reviews, ATIII activity is found at varying values among SCS patients in the asymptomatic period. , The coagulation inhibitors were much lower in SCA patients than AS individuals. The significantly decreased levels of the natural coagulation inhibitors might be the result of increased consumption due the continuous activation of the coagulation system. Liver dysfunction can be an additional factor, besides, if subclinical inflammation exists, the portion of protein S bound to C4b binding protein will increase on the extent of the free portion.
The mean D-dimer level; the cross-linked d regions of degraded fibrin was significantly higher in SCD patients and AS individuals than in the control group (P < 0.01); a finding that could be an indicator of activation of the coagulation system and continued fibrin turnover even in the chronic state of HbSS. This is in accordance with other results that suggest an increase in tissue factor expression and continuous thrombin generation in both SCD and AS individuals. 
VWF is important for platelet-platelet and platelet vessel hemostatic interaction, thereby is a marker of endothelial activation. VWF is also an acute phase reactant that helps trap invading microbes in blood clots. It is synthesized and released mainly from the vascular endothelial cells and to a lesser extent synthesized in the bone marrow megakaryocytes and stored in the alpha granules of circulating platelets. The VWF activity in the current study was significantly higher in SCD patients than normal controls (P < 0.05). Some reviews agreed with our results. 
Fibrinogen is an acute phase reactant that also traps micro-organisms in blood clots. This study, fibrinogen showed the significantly decreasing difference between SCA patients and controls (P < 0.05), and no statistical significant difference was obvious between AS and control.
However, there are many studies that showed the blood fibrinogen levels are normal or high among SCD patients when in chronic state, , the increased levels contribute to vaso-occlusion and ischemia by increasing blood viscosity and erythrocytes aggregation. In our study, decreased fibrinogen level can be explained by the intravascular use up of fibrinogen due to coagulation activation and increased fibrinolytic activity that occurs in SCD. CRP, a general marker of inflammation, known to enhance opsinin mediated phagocytosis was significantly higher in SCA individuals than in the control group (P < 0.01) and sickle cell traits did not vary from the controls. Nowadays studies show that CRP elevation is an important risk factor of coronary heart disease in the general population  and this provides an additional risk factor for ischemia and vaso-occlusion in SCD patients. In conclusion, prolonged PT, elevated D-dimer level, decreased natural coagulation inhibitors and decreased fibrinogen levels are all markers of intravascular clotting activation and continued clot lysis; chronic disseminated intravascular coagulation that is present in sickle cell disease patients even in the chronic state. Elevated CRP, VWF activity, reduced protein S and protein C with its anti-inflammatory role are markers of at least on going subclinical inflammation. The pro-coagulant changes described in sickle cell traits are of particular interest but the full picture is not achieved (elevated D-dimer level, decreased natural coagulation inhibitors while other parameters are normal). The hemostatic changes that are present in SCD patients in the chronic state and sickle cell traits together with, inflammatory response, mechanical sickling, the sickle red cell membrane with its altered structure and function and subsequent increased adherence to the vascular endothelium, free radicals, thrombocytosis, and increased viscosity, are all possible contributors to the hypercoagulable state present in these individuals. They may also explain why some athletes and army recruits (with sickle cell traits) die suddenly on intensive exercise that deprives their body of oxygen; a condition that provokes a debate about screening such people for Hb S exactly as we do in premarital program.
Future use of human recombinant APC may be of value in the management of sickle cell disease patients, more studies are recommended to further identify the nature of interaction between the altered red cell membrane, the coagulation system and the endothelium to define the natural history of sickle cell traits.
Financial Support and Sponsorship
Conflicts of Interest
There are no conflicts of interest.
| References|| |
De Franceschi L, Cappellini MD, Olivieri O. Thrombosis and sickle cell disease. Semin Thromb Hemost 2011;37:226-36.
Piel FB, Patil AP, Howes RE, Nyangiri OA, Gething PW, Williams TN, et al.
Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis. Nat Commun 2010;1:104.
Bender MA, Seibel GD. Sickle Cell Disease. Gene Reviews®
. Initial Posting: September 15, 2003. Available form: http:// www.ncbi.nlm.nih.gov
. [Last update on 2014 Oct 23].
Eaton WA, Hofrichter J. Sickle cell haemoglobin polymerization. Adv Protein Chem 1990;40:63-279.
Steinberg MH. Management of sickle cell disease. N Engl J Med 1999;340:1021-30.
Solovey AA, Solovey AN, Harkness J, Hebbel RP. Modulation of endothelial cell activation in sickle cell disease: A pilot study. Blood 2001;97:1937-41.
Ataga KI. Hypercoagulability and thrombotic complications in hemolytic anemias. Haematologica 2009;94:1481-4.
Ataga KI, Orringer EP. Hypercoagulability in sickle cell disease: A curious paradox. Am J Med 2003;115:721-8.
Attvall E, Frigyesi A, Sternby B. What is the impact of resistance to activated protein C (Leiden mutation to factor V) in inflammatory bowel disease? Int J Colorectal Dis 2006;13:1-6.
Bayazit AK, Kilinç Y. Natural coagulation inhibitors (protein C, protein S, antithrombin) in patients with sickle cell anemia in a steady state. Pediatr Int 2001;43:592-6.
Makis AC, Hatzimichael EC, Mavridis A, Bourantas KL. Alpha-2-macroglobulin and interleukin-6 levels in steady-state sickle cell disease patients. Acta Haematol 2000;104:164-8.
Kotila T, Adedapo K, Adedapo A, Oluwasola O, Fakunle E, Brown B. Liver dysfunction in steady state sickle cell disease. Ann Hepatol 2005;4:261-3.
Minami T, Sugiyama A, Wu SQ, Abid R, Kodama T, Aird WC. Thrombin and phenotypic modulation of the endothelium. Arterioscler Thromb Vasc Biol 2004;24:41-53.
Sarangi PP, Lee HW, Kim M. Activated protein C action in inflammation. Br J Haematol 2010;148:817-33.
Anderson HA, Maylock CA, Williams JA, Paweletz CP, Shu H, Shacter E. Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells. Nat Immunol 2003;4:87-91.
Liesner R, Mackie I, Cookson J, McDonald S, Chitolie A, Donohoe S, et al.
Prothrombotic changes in children with sickle cell disease: Relationships to cerebrovascular disease and transfusion. Br J Haematol 1998;103:1037-44.
Sheppard DR. Activated protein C resistance: The most common risk factor for venous thromboembolism. J Am Board Fam Pract 2000;13:111-5.
Wright JG, Cooper P, Malia RG, Kulozik AE, Vetter B, Thomas P, et al.
Activated protein C resistance in homozygous sickle cell disease. Br J Haematol 1997;96:854-6.
Corrigan JJ. Hemorrhagic and thrombotic diseases. In: Behrman RE, Kliegman RM, Arvin AM, editors. Nelson Text Book of Pediatrics. Philadelphia: W. B. Saunders; 1996. p. 1422-8.
Westerman MP, Green D, Gilman-Sachs A, Beaman K, Freels S, Boggio L, et al.
Coagulation changes in individuals with sickle cell trait. Am J Hematol 2002;69:89-94.
Schnog JB, Mac Gillavry MR, van Zanten AP, Meijers JC, Rojer RA, Duits AJ, et al.
Protein C and S and inflammation in sickle cell disease. Am J Hematol 2004;76:26-32.
Obiefuna PC, Photiades DP. Sickle discocytes form more rouleaux in vitro
than normal erythrocytes. J Trop Med Hyg 1990;93:210-4.
Ridker PM, Rifai N, Rose L, Buring JE, Cook NR. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med 2002;347:1557-65.
[Table 1], [Table 2], [Table 3]