Endothelial Protein C Receptor (EPCR), Oxide Nitric Synthase (NOS) and Tissue Factor (Factor III) Gene Expression: The Role Like Promising Biomarkers In Sickle Cell Anemia

Sickle cell anemia is a severe monogenic disorder characterized by the homozygous state of a single beta globin gene mutation, with heterogeneous clinic characteristics, associated with pro-inflammatory profile, oxidant state and hypercoagulable state. Vessels occlusion is likely initiated by intimal proliferation and amplified by inflammation, excessive adhesion of cells to activated endothelium and vascular tone dysregulation, normally modulated by NFkB pathway both endothelium cells as leukocytes. Herein, we investigated the gene expression of tissue factor (Factor III), oxide nitric synthase (NOS) and endothelial protein C receptor (EPCR) associating with biomarkers of prognosis like hemolysis markers, pro-inflammatory and anti-inflammatory profile. Forty two steady-state sickle cell disease (SCD) patients (16.5 ± 13.5 years, 20 female) from Northeast Brazil were enrolled in this study and were diagnosed in attendance of the outpatients’ clinic of the Sickle Cell Disease Center of Itabuna (CERDOFI). The control group was compound by 20 healthy Brazilian individuals with hemoglobin AA pattern matched by age, years and ethnic origin. The study was approved by the UESC ethical committee and informed consents were signed by patients or official responsible. Using real time quantitative PCR, we analyzed tissue factor, NOS and EPCR gene expression. We also measured hematological and hemoglobin parameters by electronic cell counter and HPLC respectively, biochemical profile was evaluated by colorimetric methodology and cytokine by flow cytometry. The statistical analysis was performed using the Kolmogorov–Smirnov test to access distribution of quantitative variables. Mean values of quantitative variables between groups were compared using an unpaired t-test for data distributed normally and a Mann–Whitney test for non-normal data. Oxide nitric synthase gene expression was increased in SCD patients 1.58-fold compared with healthy controls and higher tissue factor and EPCR gene expression were detected in patients than healthy controls, 4.82-fold and 5.46-fold respectively. The SCD cohort comprised pediatric and adult patients, and the medical history data was search from patient’s records where 95% of patients presented painful crisis at least once. Tissue factor gene expression was positive correlated with expression of NOS (p=0.005) and EPCR (p=0.0001). The increase tissue factor gene expression was detected in patients with high serum levels of bilirubin (p=0.026). Tissue factor gene expression above 75^th percentile was associated high concentration of serum creatine kinase and serum calcium (p<0.005) in SCD patients. Our study reveals that genes associate to hemostasis and vascular integrity are upregulated in SCD patients, probably associated to chronic oxidative stress and pro-inflammatory state. Enhanced tissue factor, NOS and EPCR gene expression may influences in the pathophysiology of SCD. Studies tissue factor, NOS and EPCR should be carried out in order to explore mechanism, clarify participation and contributing to search of therapeutic strategies on prevention of vascular events among SCD patients.