Hydroxycarbamide Is Related with Higher Expression of Band-3 and CD59 in Sickle Erythrocytes
Authors: Mecabo Grazielle , Yamamoto Mihoko , Kimura Eliza Y. S. , Guirao Fabio P. , Figueiredo Maria Stella 
Published: October 2019

Abstract
BACKGROUND: Sickle Cell Anemia (SCA) presents extra and intravascular hemolysis. The eryptosis depends on phagocytosis signs like CD47, which is known as a "don't eat me" signal. Other mechanisms associated to hemolysis, such as complement activation mediated by CD59, are poorly understood. One of the effects of hydroxycarbamide (HU) is the reduction of hemolysis, but little is known about the action of HU in these mechanisms.
OBJECTIVES: To evaluate markers related to hemolysis in patients with SCA at steady state, with and without HU.
METHODS: We studied 40 adult patients with SCA (n=36) or Sβ0-Thalassemia (n=4), 70% females, median age 30 years (range 18-70y). The patients were followed at the Outpatient Clinic of EPM /UNIFESP. Inclusion criteria: patients in use of HU in maximum tolerated dose for more than 1 year (G1, n=20); or without HU (G2, n=20). Exclusion criteria: pregnant women and chronic transfusion. Laboratory evaluation: CBC, reticulocyte (Ret), fetal hemoglobin (HbF), indirect bilirubin, lactate dehydrogenase (LDH), LDH isoforms, free Hb (free-Hb), haptoglobin (Hp), hemopexin, phosphatidylserine, microvesicles (Mcv), Howell-Jolly bodies (in erythrocytes and Ret), and expression of band-3, CD47 (in erythrocyte and Ret) and CD59 (in erythrocytes and Mcv). The last parameters were evaluated by flow cytometry (FACS Calibur®, BDB, San Jose, CA), using specific antibodies according to the manufacturer instructions. Data were analyzed by CellQuest® software (BDB), and the results expressed in median fluorescence intensity or percentage of positivity. Statistical analysis: Mann-Whitney test and Pearson's correlation, with significance level of 5%.
RESULTS: Themean corpuscular volume and HbF were higher in G1 than G2 (p=0.033, p=0.0001, respectively) (Table 1). Free-Hb, LDH and isoforms LDH-1, -2, and -3 were lower in G1 (p=0.023, p=0.003, p=0.005, p=0.001, p=0.002, respectively). There were correlation between free-Hb and LDH-1 (r=0.45, p=0.003), LDH-2 (r=0.80, p=0.001), and LDH-3 (r=0.71, p=0.001). As expected, Hp was diminished in all patients, however G1 patients showed higher values (p=0.040). The expression of band 3 and of CD59, in erythrocytes and Mcv, were higher in G1 (p=0.001, p<0.0001 and p<0.0001, respectively). Other variables showed no statistical difference.
DISCUSSION AND CONCLUSIONS: The beneficial effects of HU include decrease hemolysis, although the associated mechanisms are still unknown. Total LDH and isoforms levels were decreased in patients using HU. LDH-2 and LDH-3 showed an important correlation with free-Hb. Despite the fact that LDH-3 has been considered a nonspecific marker of tissue damage and frequently associated with lung injury, our results suggest that LDH-3 may be a valuable marker of hemolysis. The use of HU was also associated with higher levels of Hp, reinforcing its possible role on intravascular hemolysis. The higher band-3 and CD59 expression in HU patients support the protective action of this drug in sickle erythrocytes, since lower expression of these proteins have been associated with erythrocyte aging. These data present new and important insights on the effects of HU in sickle erythrocyte survival.
This work was supported by FAPESP (2011/17349-0) and CAPES-SUS.
Table 1: Laboratorial data. G1with HU G2without HU p Mean corpuscular volume (fL) 111.0 (86.6 - 125.0) 86.8 (71.9 - 106.6) < 0.0001 Fetal Hemoglobin (HbF, %) 12.6 (1.5 - 46.6) 6.9 (0.8 - 27.1) 0.0337 LDH (U/L) 351 (214 - 728) 526 (174 – 1,112) 0.0035 LDH -1 (U/L) 154.9 (72.1 - 361.0) 225.7 (54.1 -3,248) 0.0054 LDH -2 (U/L) 132.5 (86.5 - 361.4) 203.2 (71.9 - 421.4 ) 0.0013 LDH -3 (U/L) 33.7 (25.7 - 69.9) 56.0 (28.0 - 111.2) 0.0025 Free Hemoglobin (free-Hb, mcg/dL) 51.2 (15.1 - 248.1) 104.6 (24.9 - 286.1) 0.0239 Haptoglobin (Hp, mcg/dL) 0.6 (0 - 90.8) 0.1 (0 - 56.59) 0.0406 Microvesicles (Mcv, /µL) 21,643 (5,322 – 51,726) 32,185 (8,708 – 114,384) 0.0639 Howell Jolly in erytrocytes (x106) 5,350 (1,800 – 164,850) 2,750 (100 -75,850) 0.0829 Band-3 (MFI) 396.0 (291.6 - 461.5) 342.2 (217.7 - 388.9) 0.0010 CD47 in Ret (MFI) 164.7 (95.6 - 230.8) 146.6 (96.4 - 226.7) 0.2913 CD59 in erytrocytes (MFI) 1,039 (749.9 – 1,202) 808.0 (523.5 -1,032) <0.0001 CD59 in Mcv (MFI) 830.7 (636.4 - 1.023) 604.8 (495.9 - 802.2) <0.0001
HU = hydroxycarbamide; Ret= reticulocytes; LDH= Lactate Dehydrogenase ; MFI= median fluorescence intensity

Disclosures
No relevant conflicts of interest to declare.