Hydroxyurea, which is a cytotoxic, antimetabolic and antineoplastic agent, is the only disease-modifying therapy approved for sickle cell disease [14]. Hydroxyurea has been shown to be partially effective in reducing the frequency of vaso-occlusive events; but, there is no evidence that it prevents organ damage [15]. One of the factors which restricts HU usage is that it undergoes renal clearance, and hence there is a need for careful dose adjustment and close monitoring of myelotoxicity in individuals with renal impairment [16]. This vital requirement is hardly possible to undertake in most developing countries where SCD is highly prevalent because of lack of functional facilities and expertise. Therefore, there is a need for safe, effective and easily manageable treatment(s) for children and adult patients with sickle cell disease. Clinical trials have provided evidence that omega-3 fatty acids are effective in reducing frequency and severity of vaso-occlusive episodes, severe anemia, blood transfusion rate, markers of inflammation and oxidative stress [17,18,19,20,21]. In the current study, patients treated with omega-3 showed a significant reduction in D-dimer. The findings of this study indicate that treatment with omega-3 may partially ameliorate SCD-associated coagulopathy.
The SCD patients and healthy controls who participated in this study were homogenous with respect to ethnicity and socio-economic background and the patients received similar quality of care under regular management protocols. The samples were collected in one clinic and at the same time of the year. Therefore, the observed findings are likely to be due to the effect of intervention with omega-3 fatty acids or hydroxyurea treatment rather than of extraneous confounding factors.
Consistent with previous studies, both the omega-3 fatty acid treated and untreated patients had elevated steady state white blood cell and platelet counts confirming that sickle cell disease is a chronic inflammatory disorder [22]. Similarly, Tomer et al. [18] have reported that treatment with omega-3 fatty acids do not effect significantly the blood cell count, MCV or MCHC.
There is evidence which indicates that HU mediates its beneficial effects in SCD, partially, by lowering leukocyte, reticulocyte and platelet counts [23]. In the current study, HU treatment reduced platelet count significantly but had no noticeable effect on WBC. This unexpected effect of HU on WBC among Sudanese children with SCD might be a reflection of the fact that HU is generally being administered mostly to the severely ill children with SCD [24], or a response peculiar to Sudanese SCD patients that warrant further research.
The PT, aPTT and INR levels of the untreated patients were significantly higher than the n-3 fatty acid and hydroxyurea treated groups. Similar findings have been observed on Americans [25] and Nigerians children [26] and adult Jamaicans [6] with sickle cell disease. The prolongation of PT in the adult patients was not as remarkable as in the children. In contrast, another study investigated 17 subjects did not find a difference in mean PT between SCD and healthy children [27]. These controversial findings might be a reflection of the study small sample size.
The mechanism behind the prolongation of PT in children with SCD is not fully understood. It is suggested that impaired liver function [25] and depletion of coagulation factors [28] play a role in the prolongation process. However, it is worth pointing out that a relationship between an abnormal liver function and coagulation prolongation is yet to be established.
In contrast to the findings of the current study, high omega-3 fatty acid intake did not have a significant effect on PT and aPTT in adult patients with sickle cell disease [18], and in adult carcinoma patients undergoing elective surgery [29]. The subjects in the latter two studies, [18, 29], were adults from different ethnic background clinical complications and normal coagulation parameter values at baseline. In addition, the n-3 fatty acids composition of the oil/supplement used in these two studies were different from the high DHA capsules given to the children in the current study. The observed reduction of coagulation parameters in treated SCD patients in this study could be the result of increased availability of the coagulation factors and the possible concomitant reduction in coagulation activation that could surpass its potential detrimental hypo-coagulant effect [30, 31]. HU treatment had a similar effect as n-3 fatty acids on PT and aPTT suggesting that children with abnormal coagulation profile are responsive to either therapy.
The low level steady-state proteins C and S in the Sudanese children with SCD, which agrees with previous findings [6, 32], is in line with the hypothesis that the chronic activation of both the inflammatory and coagulant pathways in SCD are partially due to the disease associated down regulation of anti-coagulant pathway [33]. Protein C, activated by thrombin in the presence of protein S, inhibits the clotting ability of factor V and VIII [34]. The underlying cause of the natural anti-coagulant deficiency is yet to be elucidated. However, it has been generally attributed to the known SCD-associated hemostatic abnormalities and hepatic dysfunction [27]. An earlier study [35] reported a decrease in proteins C in children with sickle cell disease treated with hydroxyurea. That study included 11 children, 5 of them were homozygous SCD patients. The discrepancy of HU effect on proteins C and S between this study and the previous study could be due to the fact that Koc et al. study [35] was relatively underpowered to detect the true HU effect on natural anti-coagulant system.
Despite the observed improvement in coagulation parameters and hypercoagulable state, treatment with n-3 fatty acids did not result in a significant change on the level of the natural anti-coagulant proteins C and S. These findings may indicate that the liver role on low natural anticoagulant in SCD might outweigh the effect of over-consumption due to SCD-associated hypercoagulable state.
The high levels of D-dimer in patients in the current study confirm that hypercoagulability state is one of the major elements of pathophysiology of the disease [33, 36]. Previous studies have reported that omega-3 fatty acid intake is inversely associated with the level of fibrinogen, factor VIII and von Willebrand factor (VWF) [37] and D-dimer [18]. Consistent with the latter study, the current investigation demonstrates high DHA omega-3 fatty acid, but not HU, treatment reduces plasma D-dimer concentration in patients with SCD. The decrease of D-dimer by omega-3 fatty acids has implications for clinical management of patients because plasma D-dimer level is associated with a history of stroke in SCD [36].
This study did not attempt to elucidate the mechanism through which omega-3 fatty acids, particularly DHA and EPA, mediate their anti-coagulant effect. Nevertheless, it is well established that some of the metabolites of these fatty acid are antithrombotic, antiaggregatory, antiinfalammrory and vasodilatory. EPA by competing with arachidonic acid (AA) for cyclooxygenase and lipooxygenase enzymes [38] inhibits the synthesis of the prothrombotic proaggregatory, pro-inflammatory and vasoconstrictor metabolites of AA. Recent animal and human studies suggest that DHA is more potent anti-aggregatory agent than EPA at high doses [39,40,41]. Interestingly, studies in SCD have demonstrated that endothelial tissue factor expression is specifically dependent upon the nuclear factor-kappa B (NFκB) component of blood mononuclear cells [42]. Our group and others have shown that treatment with high DHA omega-3 fatty acid was associated with down regulation of NFκB gene expression in mononuclear cell and amelioration of SCD-associated chronic inflammatory state [20, 43]. Hence, it is justifiable to attribute the observed improvements in the patient’s hypercoagulable state after high DHA intervention to its suppressive effect on NFκB gene expression and partial resolution of the chronic inflammatory state [44].
Besides the limitations of the observational studies, the current study did not assess liver function in order to have a better understanding of the observed abnormalities of coagulation system and the responses to HU and omega-3 treatments. In addition, the effect of omega-3 fatty acids and HU treatments on markers of thrombin generation such as prothrombin fragment F1, 2 and thrombin-anti-thrombin complexes, was not investigated. Due to the fact that only patients above 10 years of age are treated with HU and generally small fraction of patients were treated with HU in Sudan, we did not manage to recruit enough number of patients on HU matched by age and gender.
