Hydroxyurea Reduces the Hypercoagulable State In Sickle Cell Anemia
Authors: Colella Marina P , Machado-Neto Joao , Quaino Susan K.P. , Conran Nicola , Annichino-Bizzacchi Joyce M , Costa Fernando Ferreira , Saad Sara Olalla , de Paula Erich V , Traina Fabiola 
Published: October 2019

Abstract
Abstract 1068
Sickle cell anemia (SCA) is associated with a hypercoagulable state, through mechanisms not yet clearly defined. SCA patients present an elevated rate of thrombotic complications and increased biological markers of coagulation activation. Currently one of the major pillars of SCA management is hydroxyurea (HU), a drug used primarily as an inductor of fetal hemoglobin (HbF), but with many other pleiotropic effects. Tissue factor (TF) is the major initiator of blood coagulation and has been shown to be up regulated in several inflammatory conditions. In the present study, we evaluated the effect of HU on coagulation activation by studying the expression of TF and final markers of coagulation activation: thrombin-antithrombin complex (TAT) and prothrombin fragment F 1+2 (F 1+2). We also correlated these measurements with HbF levels, lactate dehydrogenase (LDH), markers of endothelial activation (soluble thrombomodulin [sTM]) and inflammation (tumor necrosis factor-alpha [TNF-α] and white blood cell counts, including leukocyte, monocyte and neutrophil counts). We studied a cohort of 48 adult SCA patients (all with genotype SS), median age of 37 years (minimum: 20 – maximum: 50) and 25 healthy age and race matched controls. The patients included were all in steady state and 23 of them were receiving HU (SSHU). We analyzed leukocyte TF mRNA expression by real time quantitative RT-PCR and TF protein plasma levels by ELISA. TAT, F 1+2, sTM and TNF-α were all measured by ELISA. Statistical analyses were performed using Mann-Whitney's U test and Spearman's correlation test. Fisher's exact test was used to compare plasma TF levels, on the basis of detectable levels. Leukocyte TF mRNA expression was up regulated in SCA patients, in comparison to healthy controls (5.29 vs. 1.16; P = 0.0005). HU was effective in inhibiting this expression significantly (5.29 vs. 2.34; P = 0.0083). These results were confirmed by the measurements of protein plasma levels of TF. Only 27.8% (5/18) of SSHU patients had detectable plasma levels of TF, in comparison to 78.5% (11/14) in the group without the drug (P = 0.01). SCA patients also showed higher levels of TAT (11.34 vs. 2.44; P <0.0001), F 1+2 (301.5 vs. 145.2; P = 0.0003), sTM (3.11 vs. 2.58; P = 0.0008) and TNF-α (2.49 vs. 0; P < 0.0001) when compared to controls. HU therapy was able to effectively reduce all of these markers (TAT: 11.34 vs. 6.53, P = 0.019; F1+2: 301.5 vs. 216.7, P = 0.05; sTM: 3.11 vs. 2.52, P = 0.0075; TNF-α: 2.49 vs. 0.27, P = 0.0003). Levels of TF mRNA showed a strong negative correlation with HbF levels (r=−0.47) and hemoglobin (r=−0.54), and a positive correlation with sTM (r=0.6), TNF-α (r= 0.52), leukocyte (r=0.41), neutrophil (r=0.46) and monocyte (r=0.54) counts (P values ≤0.01). TAT and F 1+2 presented a positive association with LDH and TAT also presented a significant negative association with HbF levels (P values <0.05). sTM showed a significant negative correlation with HbF and a positive correlation with LDH and inflammation markers (P values <0.05). Additionally, TNF-α presented a negative association with HbF and a positive association not only with TF and sTM, but also with LDH and white blood cell counts (P values < 0.001). Our results clearly demonstrated that HU therapy reduces the hypercoagulability encountered in SCA. We showed that HU was capable of inhibiting TF expression and decreasing both TAT and F1+2 levels, final markers of thrombin generation. In addition, HU reduced the endothelial marker sTM, as well as the pro-inflammatory marker TNF-α. Correlation analyses indicated that TF inhibition was proportional to the increase in HbF levels and a reduction in LDH, a relevant marker of hemolysis and disease severity in SCA. In parallel, TF down-regulation was associated with a reduction in the endothelial marker sTM, the inflammatory markers TNF-α and white blood cell counts. Hemolysis, endothelial activation and inflammation are pathways closely connected to each other and to the activation of coagulation. Thus, as HU is a drug capable of modulating all of these pathways, most likely all of these mechanisms are involved in the inhibitory effect of HU on activation of coagulation.

Disclosures:
No relevant conflicts of interest to declare.