Variation in fetal hemoglobin parameters and predicted hemoglobin S polymerization in sickle cell children in the first two years of life: Parisian Prospective Study on Sickle Cell Disease

Abstract Intracellular hemoglobin S (HbS) polymerization is most likely to be the primary determinant of the clinical and biologic manifestations of sickle cell disease (SCD). Fetal hemoglobin (HbF) does not enter the HbS polymer and its intracellular expression in sickle erythrocytes inhibits polymerization. HbF levels, high at birth but decreasing thereafter, protect the newborn from the clinical manifestations of this hemoglobinopathy. We have measured the sequential changes in HbF, F reticulocytes, and F cells in the first 2 years of life in 25 children with SCD and compared the results with those obtained in 30 normal children (AA). We have also calculated HbF per F cell (F/F cell), the preferential survival of F cells versus non-F cells, as measured by the ratio F cells versus F reticulocytes (FC/FR) and polymer tendency at 40% and 70% oxygen saturation. HbF levels decreased from about 80.4% +/- 4.0% at birth to 9.2% +/- 2.9% at 24 months. During this time, we observed a regular decrease of the F reticulocytes and the F cells. The kinetics of the decline of F/F cell was comparable with the decline of HbF, rapid from birth (mean, 27.0 +/- 3.6 pg) to 12 months of age (mean, 8.5 +/- 1.5 pg) and then slower from 12 to 24 months of age (mean, 6.2 +/- 1.0 pg) in the SCD children. In the AA children, the decrease in HbF, due to changes in both numbers of F cells and F/F cell, was more precipitous, reaching steady-state levels by 10 months of age. Calculated values for mean polymer tendency in the F-cell population showed that polymerization should begin to occur at 40% oxygen saturation at about 3 months and increase progressively with age, whereas polymerization at 70% oxygen saturation would not occur until about 24 months. These values correspond to HbF levels of 50.8% +/- 10.8% and 9.2% +/- 2.9%, respectively, and F/F cell levels of 15.6 +/- 4.5 pg and 6.2 +/- 1.0 pg, respectively. In the non--F-cell population, polymerization was expected at birth at both oxygen saturation values. Three individuals had significantly greater predicted polymerization tendency than the remainder of the group because of early decreases in HbF. These individuals in particular, the remainder of the cohort, as well as other recruited newborns, will be studied prospectively to ascertain the relationship among hematologic parameters, which determine polymerization tendency and the various clinical manifestations of SCD.

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Variation in fetal hemoglobin parameters and predicted hemoglobin S polymerization in sickle cell children in the first two years of life: Parisian Prospective Study on Sickle Cell Disease

Blood ◽

10.1182/blood.v84.9.3182.bloodjournal8493182 ◽

1994 ◽

Vol 84 (9) ◽

pp. 3182-3188 ◽

Cited By ~ 7

Author(s):

M Maier-Redelsperger ◽

CT Noguchi ◽

M de Montalembert ◽

GP Rodgers ◽

AN Schechter ◽

...

Keyword(s):

Sickle Cell Disease ◽

Oxygen Saturation ◽

Cell Population ◽

Sickle Cell ◽

Clinical Manifestations ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Normal Children ◽

Hemoglobin S ◽

F Cells

Intracellular hemoglobin S (HbS) polymerization is most likely to be the primary determinant of the clinical and biologic manifestations of sickle cell disease (SCD). Fetal hemoglobin (HbF) does not enter the HbS polymer and its intracellular expression in sickle erythrocytes inhibits polymerization. HbF levels, high at birth but decreasing thereafter, protect the newborn from the clinical manifestations of this hemoglobinopathy. We have measured the sequential changes in HbF, F reticulocytes, and F cells in the first 2 years of life in 25 children with SCD and compared the results with those obtained in 30 normal children (AA). We have also calculated HbF per F cell (F/F cell), the preferential survival of F cells versus non-F cells, as measured by the ratio F cells versus F reticulocytes (FC/FR) and polymer tendency at 40% and 70% oxygen saturation. HbF levels decreased from about 80.4% +/- 4.0% at birth to 9.2% +/- 2.9% at 24 months. During this time, we observed a regular decrease of the F reticulocytes and the F cells. The kinetics of the decline of F/F cell was comparable with the decline of HbF, rapid from birth (mean, 27.0 +/- 3.6 pg) to 12 months of age (mean, 8.5 +/- 1.5 pg) and then slower from 12 to 24 months of age (mean, 6.2 +/- 1.0 pg) in the SCD children. In the AA children, the decrease in HbF, due to changes in both numbers of F cells and F/F cell, was more precipitous, reaching steady-state levels by 10 months of age. Calculated values for mean polymer tendency in the F-cell population showed that polymerization should begin to occur at 40% oxygen saturation at about 3 months and increase progressively with age, whereas polymerization at 70% oxygen saturation would not occur until about 24 months. These values correspond to HbF levels of 50.8% +/- 10.8% and 9.2% +/- 2.9%, respectively, and F/F cell levels of 15.6 +/- 4.5 pg and 6.2 +/- 1.0 pg, respectively. In the non--F-cell population, polymerization was expected at birth at both oxygen saturation values. Three individuals had significantly greater predicted polymerization tendency than the remainder of the group because of early decreases in HbF. These individuals in particular, the remainder of the cohort, as well as other recruited newborns, will be studied prospectively to ascertain the relationship among hematologic parameters, which determine polymerization tendency and the various clinical manifestations of SCD.

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Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1612075113 ◽

2016 ◽

Vol 113 (38) ◽

pp. 10661-10665 ◽

Cited By ~ 79

Author(s):

Lin Ye ◽

Jiaming Wang ◽

Yuting Tan ◽

Ashley I. Beyer ◽

Fei Xie ◽

...

Keyword(s):

Sickle Cell Disease ◽

Genome Editing ◽

Sickle Cell ◽

Globin Gene ◽

Autologous Transplantation ◽

Clinical Manifestations ◽

Fetal Hemoglobin ◽

Compound Heterozygous ◽

Cell Disease ◽

Hematopoietic Stem

Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher γ-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous β-thalassemia and SCD.

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Hydroxyurea as an Alternative to Blood Transfusions for the Prevention of Recurrent Stroke in Children With Sickle Cell Disease

Blood ◽

10.1182/blood.v94.9.3022 ◽

1999 ◽

Vol 94 (9) ◽

pp. 3022-3026 ◽

Cited By ~ 111

Author(s):

Russell E. Ware ◽

Sherri A. Zimmerman ◽

William H. Schultz

Keyword(s):

Sickle Cell Disease ◽

Iron Overload ◽

Sickle Cell ◽

Recurrent Stroke ◽

Fetal Hemoglobin ◽

Hemoglobin Concentration ◽

Stroke Recurrence ◽

Hematologic Toxicity ◽

Cell Disease ◽

F Cells

Abstract Children with sickle cell disease (SCD) and stroke receive chronic transfusions to prevent stroke recurrence. Transfusion risks including infection, erythrocyte allosensitization, and iron overload suggest a need for alternative therapies. We previously used hydroxyurea (HU) and phlebotomy in two young adults with SCD and stroke as an alternative to transfusions. We have now prospectively discontinued transfusions in 16 pediatric patients with SCD and stroke. Reasons to discontinue transfusions included erythrocyte alloantibodies or autoantibodies, recurrent stroke on transfusions, iron overload, noncompliance, and deferoxamine allergy. HU was started at 15 mg/kg/d and escalated to 30 mg/kg/d based on hematologic toxicity. Patients with iron overload underwent phlebotomy. The children have been off transfusions 22 months, (range, 3 to 52 months). Their average HU dose is 24.9 ± 4.2 mg/kg/d, hemoglobin concentration is 9.4 ± 1.3 g/dL, and mean corpuscular volume (MCV) is 112 ± 9 fL. Maximum percentage fetal hemoglobin (%HbF) is 20.6% ± 8.0% and percentage HbF-containing erythrocytes (%F cells) is 79.3% ± 14.7%. Fourteen patients underwent phlebotomy with an average of 8,993 mL (267 mL/kg) removed. Serum ferritin has decreased from 2,630 to 424 ng/mL, and 4 children have normal ferritin values. Three patients (19%) had neurological events considered recurrent stroke, each 3 to 4 months after discontinuing transfusions, but before maximal HU effects. These preliminary data suggest some children with SCD and stroke may discontinue chronic transfusions and use HU therapy to prevent stroke recurrence. Phlebotomy is well-tolerated and significantly reduces iron overload. Modifications in HU therapy to raise HbF more rapidly might increase protection against stroke recurrence.

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Combining Fetal Hemoglobin and Reticulocytosis to Index Clinical Severity of Sickle Cell Disease in Children.

Blood ◽

10.1182/blood.v114.22.2556.2556 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2556-2556

Author(s):

Emily Riehm Meier ◽

Colleen Byrnes ◽

Maxine Weissman ◽

Pierre Noel ◽

Naomi L.C. Luban ◽

...

Keyword(s):

Sickle Cell Disease ◽

Supportive Care ◽

Disease Severity ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Treatment Decisions ◽

Clinical Severity ◽

Acute Chest Syndrome ◽

Cell Disease ◽

F Cells

Abstract Abstract 2556 Poster Board II-533 Predictors of disease severity during infancy or childhood in patients with sickle cell disease (SCD) are needed to guide treatment decisions with therapies that have known toxicities [transfusion, hydroxyurea (HU), bone marrow transplant]. Erythrocyte fetal hemoglobin (HbF) expression levels above 20% reduce sickle hemoglobin (HbS) polymerization and decrease hemolysis. As a result of the decreased hemolysis, the survival of erythrocytes is prolonged, and the overall level of erythropoiesis is reduced. To determine if clinical markers of increased HbF production and decreased erythropoiesis may be combined to score disease severity, we developed a Fetal Hemoglobin-Reticulocytosis Index (FRI) defined as: [HbF (%) × non-transfused F-cells (%)] / [Absolute Reticulocyte Count (K/uL)]. For these studies, red cell lysates were analyzed by high power liquid chromatography (HPLC) to estimate HbA, HbS, and HbF fractions. F-cells were analyzed by flow cytometry using antibodies directed against HbF, while transfused cells were labeled with antibodies directed against HbA. Dual staining with both antibodies provided a method for accurately distinguishing transfused and non-transfused F-cells (NT F-cells). A minimum of 10,000 cells was analyzed in all samples. Absolute reticulocyte counts (ARC) were determined using a Sysmex XE 2100 hematology analyzer (Sysmex America, Mundelein, IL). Preliminary studies revealed FRI values near 100 at one month of age followed by a rapid drop before the age of 4 years. Blood from children between the ages of 4 and 21 years was also studied to determine if FRI correlates with therapeutic regimen. FRI values for three groups were compared: those treated with chronic transfusion (n=19, mean FRI=0.72±1.04), HU (n=19, mean FRI=5.61±6.24), versus supportive care alone that did not include recent transfusions (n=42, mean FRI=2.70 ±4.85). When the FRI values from each of these groups were placed in rank order, the slope of the line increased sharply from a linear to an exponential shape near the FRI value of 2. To determine if the FRI=2 inflection may be indicative of reduced disease severity, the number of SCD events were determined in the 42 study subjects treated with supportive care. Overall, twenty-eight (66.7%) patients had an FRI<2, and fourteen (33.3%) patients had an FRI≥2. Among those patients, SCD events were tallied (listed in descending order according to number of events): painful crises requiring hospitalization (FRI<2, n=128; FRI≥2, n=25), pneumonia /acute chest syndrome (FRI<2, n=74; FRI≥2, n=18), splenic sequestration (FRI<2, n=14; FRI≥2, n=0), conditional transcranial Doppler [(TCD), FRI<2, n=13; FRI≥2, n=1), silent stroke (FRI<2, n=4; FRI≥2, n=2), bacteremia (FRI<2, n=2; FRI≥2, n=1), cholecystectomy (FRI<2, n=3; FRI≥2, n=0), and nephropathy (FRI<2, n=1; FRI≥2, n=0). None of the supportive care group had an overt stroke, abnormal TCD, sickle cell retinopathy, or priapism. Age adjusted analysis showed that the FRI≥2 group had significantly fewer total events per year [events/year: FRI<2 (0.70±0.52) vs. FRI≥2 (0.38 ± 0.36), p=0.02]. These data suggest that combining the clinical parameters of fetal hemoglobin production and reticulocytosis provides a simple index for SCD severity. Based upon this retrospective data, prospective studies are underway to determine if the FRI decline during infancy or FRI levels in childhood are useful to predict clinical severity and treatment decisions in SCD patients. Disclosures: No relevant conflicts of interest to declare.

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Fetal hemoglobin in sickle cell disease: relationship to erythrocyte phosphatidylserine exposure and coagulation activation

Blood ◽

10.1182/blood.v96.3.1119.015k53_1119_1124 ◽

2000 ◽

Vol 96 (3) ◽

pp. 1119-1124 ◽

Cited By ~ 2

Author(s):

B. N. Yamaja Setty ◽

Surekha Kulkarni ◽

A. Koneti Rao ◽

Marie J. Stuart

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Thrombin Generation ◽

Fetal Hemoglobin ◽

Strong Relationship ◽

Cell Disease ◽

Red Cell Membrane ◽

Prothrombin Fragment ◽

F Cells

In sickle cell disease (SCD), loss of erythrocyte membrane phospholipid asymmetry occurs with the exposure of phosphatidylserine (PS), which provides a docking site for coagulation proteins. In vivo sickling/desickling, with resulting red cell membrane changes and microvesicle formation, appears to be one of the factors responsible for PS exposure. We evaluated children with SCD homozygous for sickle hemoglobin (SS disease) and controls (n = 65) and demonstrate that high levels of fetal hemoglobin (assessed as F cells) are associated with decreased microvesicle formation, PS exposure, and thrombin generation. F cells correlated inversely with both microvesicles and PS positivity (P < .000001) in SS disease. Multiple regression analyses using various hematologic parameters as independent variables, and either microvesicles or PS positivity as the dependent variable, showed a strong relationship only with F cells. Additionally, plasma prothrombin fragment F1.2 levels (a marker for thrombin generation) correlated with both PS positivity (P < .001) and F cells (P < .01). An F-cell level of approximately 70% was associated with normal levels of prothrombin fragment F1.2 and with microvesicle formation indistinguishable from control values. We suggest that the use of such surrogate biologic markers in conjunction with F-cell numbers may provide valuable insights into the biology and consequences of in vivo sickling.

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RN-1, a Potent and Selective LSD1 Inhibitor, Induces High Levels of Fetal Hemoglobin (HbF) in Anemic Baboons (P. anubis)

Blood ◽

10.1182/blood.v124.21.336.336 ◽

2014 ◽

Vol 124 (21) ◽

pp. 336-336 ◽

Cited By ~ 1

Author(s):

Angela Rivers ◽

Kestis Vaitkus ◽

Maria Armila Ruiz ◽

Vinzon Ibanez ◽

Tatiana Kouznetsova ◽

...

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Conflicts Of Interest ◽

Large Fraction ◽

Fetal Hemoglobin ◽

In Vitro Assays ◽

Mrna Levels ◽

Cell Disease ◽

F Cells ◽

Globin Synthesis

Abstract Increased levels of fetal hemoglobin are associated with decreased symptoms and increased life span in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients and therefore new agents are currently needed. Recent evidence has shown that LSD1, an enzyme that removes monomethyl and dimethyl residues from the lys4 residue of histone H3, is a repressor of γ-globin expression. Tranylcypromine (TCP), an LSD1 inhibitor, was shown to increase γ-globin expression in human βYAC transgenic mice (Shi et al Nat Med 19:291, 2013). Because the arrangement and developmental stage-specific expression pattern of the β-like globin genes is highly conserved between man and other simian primates, the use of an simian primate animal model such as the baboon (P.anubis) is the best predictor of the activity of HbF-inducing agents in man. In this investigation we compared the effect of TCP and the more potent and selective LSD1 inhibitor, RN-1, on HbF expression in anemic baboons (P. anubis). In vitro assays have shown that the LSD1 IC50 of RN-1 is at least 1000 fold less than TCP. Animals were phlebotomized for 14d prior to drug treatment to attain an Hct=20 to induce reticulocytosis and establish baseline HbF levels and were maintained at this Hct by periodic phlebotomies during the course of the experiment. In four baboons treated with varying doses of TCP (2-6mg/kg/; 10-20d; sc) low levels of HbF (4.9-7.9% HbF) were induced that were only slightly higher than those observed at the pretreatment baseline (2.2-4.1% HbF). In contrast, treatment with varying doses of RN-1 (2.5-0.125 mg/kg/d; 5-10d) induced high levels of HbF, F reticulocytes, and F cells in 5 of 6 animals (see Table). At high doses of drug the ratio of 5'Iγ/3'Vγ synthesis was >2 demonstrating a near-complete reversion to the pattern of fetal stage expression. Peak levels of F reticulocytes and γ-globin synthesis were observed 8d and HbF and F cells 11d after the first day of drug administration. Increased γ-globin mRNA levels (γ/γ+β) in reticulocytes measured by RT-PCR showed that increased HbF levels were not due to translational effects. Bisulfite sequence analysis showed that levels of DNA methylation of the γ-globin promoter were similar in pre- and post-treatment BM erythroid precursors from two animals. Flow cytometry analysis using anti-α4-integrin and anti-baboon RBC antibodies showed that RN-1 treatment altered terminal BM erythroid differentiation by increasing the proportion of less differentiated precursors, however no changes in MCHC or total hemoglobin synthesis (α/γ+β) were observed. RN-1 treatment was associated with decreased ANC (290-870 X 103/μl nadir) and increased platelets (1092-1445 X 103/μl peak) and monocytes that were likely caused by effects on hematopoietic differentiation. The ANC nadir and peak platelet and monocyte counts were observed between d14-19 and resolved with 2-3 days. Similar changes in ANC and platelets, although not in monocytes, are observed following treatment with decitabine and can be controlled by modification of dose and schedule of administration (Lavelle et al Blood 119:1240, 2012). We conclude that RN-1, a more potent LSD1 inhibitor than TCP, is a powerful HbF-inducing drug with activity similar to decitabine and predict that LSD1 inhibitors may be useful drugs for the treatment of sickle cell disease. Table HbF, F-cells, and F-retics in Baboons Treated with RN-1 Animal RN-1 Dose (mg/kg/d) HbF (%) Globin synthesis (γ/γ+β) F-cells (%) F-retics (%) Pre Post Pre Post Pre Post Pre Post 8548 2.5 (4d) 5.8 27.3 ND 0.78 28.5 59.2 34.6 92.3 8549 0.5 (5d) 2.4 29.5 0.06 0.68 19.1 60.8 33.9 97.5 8000 0.25 (5d) 4.1 20.6 0.15 0.49 20.3 47.0 45.7 80.7 8548 0.20 (5d) 3.7 20.5 0.04 0.52 36.9 54.8 36.9 89.2 8001 0.125 (5d) 1.8 4.8 ND 0.06 13.5 21.8 22.9 31.7 8549 0.125 (10d) 3.6 16 0.04 0.22 17.6 42.7 21.0 70.0 Disclosures No relevant conflicts of interest to declare.

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Quantitative analysis of erythrocytes containing fetal hemoglobin (F cells) in children with sickle cell disease

American Journal of Hematology ◽

10.1002/(sici)1096-8652(199701)54:1<40::aid-ajh6>3.0.co;2-4 ◽

1997 ◽

Vol 54 (1) ◽

pp. 40-46 ◽

Cited By ~ 34

Author(s):

Stacy J. Marcus ◽

Thomas R. Kinney ◽

William H. Schultz ◽

Erin E. O'Branski ◽

Russell E. Ware

Keyword(s):

Quantitative Analysis ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Hemoglobin F ◽

F Cells

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Hydroxyurea as an Alternative to Blood Transfusions for the Prevention of Recurrent Stroke in Children With Sickle Cell Disease

Blood ◽

10.1182/blood.v94.9.3022.421k17_3022_3026 ◽

1999 ◽

Vol 94 (9) ◽

pp. 3022-3026 ◽

Cited By ~ 7

Author(s):

Russell E. Ware ◽

Sherri A. Zimmerman ◽

William H. Schultz

Keyword(s):

Sickle Cell Disease ◽

Iron Overload ◽

Sickle Cell ◽

Recurrent Stroke ◽

Fetal Hemoglobin ◽

Hemoglobin Concentration ◽

Stroke Recurrence ◽

Hematologic Toxicity ◽

Cell Disease ◽

F Cells

Children with sickle cell disease (SCD) and stroke receive chronic transfusions to prevent stroke recurrence. Transfusion risks including infection, erythrocyte allosensitization, and iron overload suggest a need for alternative therapies. We previously used hydroxyurea (HU) and phlebotomy in two young adults with SCD and stroke as an alternative to transfusions. We have now prospectively discontinued transfusions in 16 pediatric patients with SCD and stroke. Reasons to discontinue transfusions included erythrocyte alloantibodies or autoantibodies, recurrent stroke on transfusions, iron overload, noncompliance, and deferoxamine allergy. HU was started at 15 mg/kg/d and escalated to 30 mg/kg/d based on hematologic toxicity. Patients with iron overload underwent phlebotomy. The children have been off transfusions 22 months, (range, 3 to 52 months). Their average HU dose is 24.9 ± 4.2 mg/kg/d, hemoglobin concentration is 9.4 ± 1.3 g/dL, and mean corpuscular volume (MCV) is 112 ± 9 fL. Maximum percentage fetal hemoglobin (%HbF) is 20.6% ± 8.0% and percentage HbF-containing erythrocytes (%F cells) is 79.3% ± 14.7%. Fourteen patients underwent phlebotomy with an average of 8,993 mL (267 mL/kg) removed. Serum ferritin has decreased from 2,630 to 424 ng/mL, and 4 children have normal ferritin values. Three patients (19%) had neurological events considered recurrent stroke, each 3 to 4 months after discontinuing transfusions, but before maximal HU effects. These preliminary data suggest some children with SCD and stroke may discontinue chronic transfusions and use HU therapy to prevent stroke recurrence. Phlebotomy is well-tolerated and significantly reduces iron overload. Modifications in HU therapy to raise HbF more rapidly might increase protection against stroke recurrence.

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Fetal hemoglobin in sickle cell disease: relationship to erythrocyte phosphatidylserine exposure and coagulation activation

Blood ◽

10.1182/blood.v96.3.1119 ◽

2000 ◽

Vol 96 (3) ◽

pp. 1119-1124 ◽

Cited By ~ 63

Author(s):

B. N. Yamaja Setty ◽

Surekha Kulkarni ◽

A. Koneti Rao ◽

Marie J. Stuart

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Thrombin Generation ◽

Fetal Hemoglobin ◽

Strong Relationship ◽

Cell Disease ◽

Red Cell Membrane ◽

Prothrombin Fragment ◽

F Cells

Abstract In sickle cell disease (SCD), loss of erythrocyte membrane phospholipid asymmetry occurs with the exposure of phosphatidylserine (PS), which provides a docking site for coagulation proteins. In vivo sickling/desickling, with resulting red cell membrane changes and microvesicle formation, appears to be one of the factors responsible for PS exposure. We evaluated children with SCD homozygous for sickle hemoglobin (SS disease) and controls (n = 65) and demonstrate that high levels of fetal hemoglobin (assessed as F cells) are associated with decreased microvesicle formation, PS exposure, and thrombin generation. F cells correlated inversely with both microvesicles and PS positivity (P < .000001) in SS disease. Multiple regression analyses using various hematologic parameters as independent variables, and either microvesicles or PS positivity as the dependent variable, showed a strong relationship only with F cells. Additionally, plasma prothrombin fragment F1.2 levels (a marker for thrombin generation) correlated with both PS positivity (P < .001) and F cells (P < .01). An F-cell level of approximately 70% was associated with normal levels of prothrombin fragment F1.2 and with microvesicle formation indistinguishable from control values. We suggest that the use of such surrogate biologic markers in conjunction with F-cell numbers may provide valuable insights into the biology and consequences of in vivo sickling.

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Mechanisms of NRF2 activation to mediate fetal hemoglobin induction and protection against oxidative stress in sickle cell disease

Experimental Biology and Medicine ◽

10.1177/1535370219825859 ◽

2019 ◽

Vol 244 (2) ◽

pp. 171-182 ◽

Cited By ~ 3

Author(s):

Xingguo Zhu ◽

Aluya R Oseghale ◽

Lopez H Nicole ◽

Biaoru Li ◽

Betty S Pace

Keyword(s):

Oxidative Stress ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Hemoglobin S ◽

Nrf2 Activation ◽

Chronic Hemolysis ◽

Abnormal Hemoglobin ◽

Small Chemical

Individuals with sickle cell disease have severe anemia due to the production of abnormal hemoglobin S, chronic red blood cell hemolysis, and increased oxidative stress leading to endothelial cell dysfunction, vasculopathy, and progressive organ damage. The transcription factor NRF2 (erythroid-derived 2)-like 2) is a master regulator of antioxidant proteins; under low oxidative stress, NRF2 is sequestered in the cytoplasm by Kelch-like ECH-associated protein 1, β-transducin repeat-containing protein or HRD1, and directed to the proteasome for degradation. When cells are exposed to oxidative stress, NRF2 is released from these repressor proteins, translocates to the nucleus, and activates antioxidant genes to suppress cellular reactive oxidant species and inflammation. In erythroid progenitors, NRF2 also modulates fetal hemoglobin expression through direct binding in the γ-globin promoter and modification of chromatin structure in the β-globin locus. In sickle erythroid cells, NRF2 provides unique benefits through fetal hemoglobin induction to inhibit hemoglobin S polymerization and protection against oxidative stress due to chronic hemolysis. Thus, development of small chemical molecules that activate NRF2 has the potential to ameliorate the clinical severity of sickle cell disease. In this review, we discuss progress towards understanding NRF2 regulation and strategies to develop agents for the treatment of sickle cell disease. Impact statement Sickle cell disease (SCD) is a group of inherited blood disorders caused by mutations in the human β-globin gene, leading to the synthesis of abnormal hemoglobin S, chronic hemolysis, and oxidative stress. Inhibition of hemoglobin S polymerization by fetal hemoglobin holds the greatest promise for treating SCD. The transcription factor NRF2, is the master regulator of the cellular oxidative stress response and activator of fetal hemoglobin expression. In animal models, various small chemical molecules activate NRF2 and ameliorate the pathophysiology of SCD. This review discusses the mechanisms of NRF2 regulation and therapeutic strategies of NRF2 activation to design the treatment options for individuals with SCD.

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