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Hydroxyurea is associated with reductions in hypercoagulability markers in sickle cell anemia
Article first published online: 4 SEP 2012
© 2012 International Society on Thrombosis and Haemostasis
Journal of Thrombosis and Haemostasis
Volume 10, Issue 9, pages 1967–1970, September 2012
How to Cite
COLELLA, M. P., De PAULA, E. V., CONRAN, N., MACHADO-NETO, J. A., ANNICCHINO-BIZZACCHI, J. M., COSTA, F. F., SAAD, S. T. O. and TRAINA, F. (2012), Hydroxyurea is associated with reductions in hypercoagulability markers in sickle cell anemia. Journal of Thrombosis and Haemostasis, 10: 1967–1970. doi: 10.1111/j.1538-7836.2012.04861.x
- Issue published online: 4 SEP 2012
- Article first published online: 4 SEP 2012
- Accepted manuscript online: 20 JUL 2012 11:50AM EST
- Received 6 July 2012, accepted 11 July 2012
Several lines of evidence indicate that sickle cell anemia (SCA) is a state of increased hemostatic activation [1–4]. SCA patients present an elevated rate of thrombotic complications, including pulmonary embolism, stroke, and pregnancy-related venous thromboembolism . Hydroxyurea (hydroxycarbamide) is currently one of the main pillars of SCA management, improving both clinical complications and mortality in SCA , and has several well-defined beneficial effects that could contribute to inhibition of the hypercoagulability state in SCA. These include a reduction in phosphatidylserine exposure by erythrocytes, a reduction in the adhesive properties of erythrocytes and leukocytes, endothelial activation, nitric oxide depletion, platelet activation, and adhesive properties [7–9].
We hypothesized that hydroxyurea could modulate the hypercoagulable state observed in this condition, and evaluated its effects on the activation of coagulation in a cohort of SCA patients by measurements of tissue factor (TF) expression and final markers of thrombin generation. This was a cross-sectional study that included steady-state SCA patients with no history of painful crisis, hospitalizations or blood transfusions in the preceding 3 months. We also selected 25 healthy age-matched controls. The university’s ethics committee approved the study, and all patients signed an informed consent form.
Patients were subdivided into two groups according to the use (SS-HU group, n = 23) or not (SS group, n = 15) of hydroxyurea. The median duration of hydroxyurea therapy was 28 months (range: 3–196 months), and the median dose was 20 mg kg−1 d−1 (range: 11–34 mg kg−1 d−1). Patients using hydroxyurea clearly had a history of a more severe clinical phenotype than those not using it, as indicated by a higher frequency of clinical sickling complications recorded before the initiation of therapy. Hydroxyurea resulted in significant improvements in the laboratory and clinical parameters of the SCA phenotype in treated patients (see Table S1 for detailed clinical information).
TF expression was analyzed by real-time quantitative PCR on total leukocyte mRNA. ELISA kits were used to measure plasma levels of TF, soluble thrombomodulin (American Diagnostica, Stamford, CT, USA), thrombin–antithrombin (TAT) complex, and prothrombin fragment F1 + 2 (F1 + 2) (Siemens, Marburg, Germany), and serum levels of tumor necrosis factor-α (TNF-α) (Invitrogen, Camarillo, CA, USA). TF mRNA expression and TAT, F1 + 2, soluble thrombomodulin and TNF-α levels were compared between patient subgroups by use of the Mann–Whitney U-test. Fisher’s exact test was used to compare categorical variables and to compare plasma TF levels. Spearman’s rank correlation coefficient was used to analyze bivariate associations between the various markers. Statistical analyses were performed with prism 4 (GraphPad Software, La Jolla, CA, USA).
Quantitative PCR analysis showed upregulation of TF mRNA expression in SS patients in comparison with healthy controls (5.0 [SS] vs. 1.16 [controls], P = 0.0004). Hydroxyurea therapy was associated with an important reduction in this expression (2.6 [SS-HU] vs. 5.0 [SS], P = 0.02) (Fig. 1A). The decrease in TF expression in patients using hydroxyurea was confirmed by measurements of detectable protein plasma levels. TF could be detected in the plasma of 79% (11/14) of patients not using hydroxyurea, as compared with only 28% (5/18) of the patients using the agent (P = 0.01) (Fig. 1B). The functional consequences of variations in TF expression were supported by measurements of indirect markers of thrombin generation. Levels of TAT and F1 + 2 were significantly higher in SS patients than in controls (TAT, 11.5 μg L–1 [SS] vs. 2.4 μg L–1 [controls], P < 0.0001; F1 + 2, 301.5 pmol L–1 [SS] vs. 145 pmol L–1 [controls], P < 0.0001). Treatment with hydroxyurea was accompanied by a significant reduction in TAT levels (11.5 μg L–1 [SS] vs. 7.5 μg L–1 [SS-HU], P = 0.03) and a reduction in F1 + 2 levels (301.5 pmol L–1 [SS] vs. 245 pmol L–1 [SS-HU], P = 0.09) (Fig. 1C–D).
The main finding of our study was that hydroxyurea therapy in SCA patients was associated with significant downregulation of TF expression and decreased levels of markers of thrombin generation (TAT and F1 + 2). These results provide biological evidence for the association between hydroxyurea therapy and inhibition of coagulation activation in SCA. TF is the main physiologic initiator of coagulation in vivo, and a recent study showed that, in SCA mice, activation of coagulation was dependent on TF activity .
We also evaluated the effects of hydroxyurea on endothelial activation (soluble thrombomodulin) and inflammation (TNF-α). Significant elevations of soluble thrombomodulin plasma levels (3.6 ng mL–1 [SS] vs. 2.6 ng mL–1 [controls], P = 0.0007) and TNF-α serum levels (2.7 pg mL–1 [SS] vs. 0 pg mL–1 [controls], P < 0.0001) were observed in SS patients in comparison with controls. Hydroxyurea was associated with important decreases in the levels of both markers (soluble thrombomodulin, 3.6 ng mL–1 [SS] vs. 2.5 ng mL–1 [SS-HU], P = 0.0007; TNF-α, 2.7 pg mL–1 [SS] vs. 0.3 pg mL–1 [SS-HU], P < 0.0001)(Fig. 1E–F).
Next, we analyzed the associations between hypercoagulability markers (TF mRNA expression, and TAT and F1 + 2 levels) and other relevant laboratory parameters. TF mRNA levels showed significant correlations with fetal hemoglobin levels, hemoglobin/hematocrit, soluble thrombomodulin levels, TNF-α levels, and absolute neutrophil counts (all P ≤ 0.05). TAT and F1 + 2 levels showed significant correlations with fetal hemoglobin, hemoglobin and lactate dehydrogenase (LDH) levels (all P ≤ 0.05) (Table S2). These results are in agreement with results from a recent study in children with sickle cell disease that also showed a significant association of TF expression and activity with hemolytic markers (reticulocyte counts and LDH levels), and it was suggested that this could support a role for early use of agents, such as hydroxyurea, to reduce the hemolytic activity in this disease . Overall, our findings demonstrate that downregulation of hypercoagulability markers is associated with higher levels of fetal hemoglobin, and decreases in the levels of markers of hemolysis, endothelial activation, and inflammation, which are pathways that are closely connected to each other and to the activation of coagulation. Hydroxyurea is a drug that is capable of modulating all of these pathways, and it is possible that all of these mechanisms contribute to the potential inhibitory effect of hydroxyurea on the activation of coagulation.
In summary, we have demonstrated that hydroxyurea is associated with a reduction in the levels of biomarkers of hypercoagulability in SCA patients. Our findings suggest a new clinical benefit for the use of hydroxyurea in SCA patients, beyond its traditional use. Future longitudinal prospective studies including a large number of patients and longer follow-up are needed to verify whether this biological effect of hydroxyurea may result in a decreased incidence of thrombotic complications in SCA patients.
Disclosure of Conflict of Interests
This study received financial support from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and INCT do Sangue.
Table S1. Clinical and laboratory characteristics of the Sickle Cell Anemia patients.
Table S2. Correlations of TF expression and coagulation markers with hemolysis, endothelial activation and inflammation markers.
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