Benserazide racemate and enantiomers induce fetal globin gene expression in vivo: Studies to guide clinical development for beta thalassemia and sickle cell disease

Abstract Phenotypic improvement of hemoglobinopathies such as sickle cell disease and beta-thalassemia (beta-thal) has been shown in patients with high levels of fetal hemoglobin (HbF). In sickle cell disease (SCD) the beneficial effects of HbF are due to the inhibition of HbS polymerization and to the dilution of HbS determining the reduction of sickling and vascular occlusion. Moreover, in beta-thal, high levels of gamma-chains combined with the redundant alpha-chains, lead to a reduction of dyserythropoiesis and of the requirement for blood transfusions. The only drug approved for the treatment of adult patients with SCD and that has been entered in clinical practice of patients affected by beta-thal is hydroxyurea (HU); however there is a great variability in the responses of patients to HU, in fact some patients are good responder, while others exhibit little or no change in HbF levels after HU treatment; moreover a decrease in the efficacy during long term treatment was observed. Other pharmacological compounds, including 5-azacytidine and thalidomide have been shown to increase HbF production. Due to concerns about the safety of this agents, their use was limited to severe cases for whom conventional therapy was unfeasible. For this reason the search of new inducers of HbF production is important. Ruxolitinib is a JAK inhibitor and decreases the phosphorilation of STAT (Signal transducers and activators of transcription) family proteins, in particular STAT5 and STAT3. Phosphorylation of STAT5 is essential for basal erythropoiesis and for its acceleration during stress erythropoiesis. STAT3 plays an essential role in regulating gene expression of several genes involved in cell growth and apoptosis, in particular it was demonstrated to inhibit gamma-globin gene expression. The decrease of STAT3 phoshorilation could decrease the inhibition of gamma-globin gene expression; for this reason we considered ruxolitinib a candidate as inducer of HbF production. In our laboratory an ex vivo system was developed predictive of the in vivo response to hydroxyurea treatment by using liquid erythroid cultures, an in vitro culture system that recapitulates the process of human erythropoiesis. To evaluate the efficacy of ruxolitinib in increasing gamma-globin gene expression we carried out a study in vitro using liquid erythroid cultures. In this study we developed and exposed to ruxolitinib liquid erythroid precursors from 4 SCD and 17 beta-thal intermedia (beta-TI) patients. The use of quantitative Real-Time-polymerase chain reaction allowed us to determine the increase in gamma-globin mRNA expression in human erythroid cells treated with ruxolitinib compared to untreated cells. The results are summarized in Table 1 and showed that ruxolitinib at 200nM is able to determine a significant increase of gamma-globin gene expression (3.4±0.1)compared to HU (2.0± 0.2). In conclusion our study suggests that ruxolitinib could be considered an inducer of HbF and could be used in vivo for the treatment of hemoglobinopathies, particularly in patients who do not respond to HU therapy or who show a decreased response after long-term treatment. Table 1. Fold increase of Gamma-globin gene expression in presence of Ruxolitinib in erythroid cultured cells. Patient Sex Genotype gamma-globin mRNA fold increasein the presence of ruxolitinib #1 M b039/aaa +1 #2 F b039/aaa +1.65 #3 F b039/b039 +1.9 #4 F b039/IVS1,110 +1.5 #5 M IVS1,1/aaa +2.5 #6 M IVS1,110/IVS1,1 +9.2 #7 M b039/bs +6 #8 F bs/b039 +1.6 #9 F b039/IVS1,6 +1.7 #10 M IVS1,6/frcd6 +3 #11 M IVS1,6/bs +2.5 #12 M IVS1,6/frcd6 +8 #13 F IVS1,6/b039 +9 #14 M IVS1,1/b039 +2.2 #15 M db/IVS1,110 +8 #16 F db/IVS1,110 +1.8 #17 F IVS2,1/aaa +3.9 #18 M b039/-101 +1.4 #19 M IVS1,6/b039 +1 #20 M bs/IVS1,110 +1.4 #21 M IVS1,6/IVS1,6 +1.9 Disclosures No relevant conflicts of interest to declare.

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Delta‐globin gene expression improves sickle cell disease in a humanised mouse model

British Journal of Haematology ◽

10.1111/bjh.17561 ◽

2021 ◽

Author(s):

Susanna Porcu ◽

Michela Simbula ◽

Maria F. Marongiu ◽

Andrea Perra ◽

Daniela Poddie ◽

...

Keyword(s):

Gene Expression ◽

Sickle Cell Disease ◽

Mouse Model ◽

Sickle Cell ◽

Globin Gene ◽

Cell Disease ◽

Globin Gene Expression

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Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34 + cells

Experimental Hematology ◽

10.1016/j.exphem.2015.01.009 ◽

2015 ◽

Vol 43 (5) ◽

pp. 346-351 ◽

Cited By ~ 24

Author(s):

Fabrizia Urbinati ◽

Phillip W. Hargrove ◽

Sabine Geiger ◽

Zulema Romero ◽

Jennifer Wherley ◽

...

Keyword(s):

Gene Expression ◽

Bone Marrow ◽

Gene Transfer ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Globin Gene ◽

Cell Disease ◽

Cd34 Cells ◽

Globin Gene Expression

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A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Erythrocytes Cultured from CD34+ Cells with Sickle Cell Disease

Blood ◽

10.1182/blood.v128.22.3506.3506 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3506-3506

Author(s):

Jianqiong Zhu ◽

Kyung Chin ◽

Wulin Aerbajinai ◽

Chutima Kumkhaek ◽

Hongzhen Li ◽

...

Keyword(s):

Gene Expression ◽

Sickle Cell Disease ◽

Fusion Protein ◽

Sickle Cell ◽

Globin Gene ◽

Erythroid Cell ◽

Cell Disease ◽

Cd34 Cells ◽

Long Form ◽

Globin Gene Expression

Abstract Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in CD34+ cells from healthy and sickle trait donor's blood. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from CD34+ cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 2.0-fold, δ-globin gene expression 4.3-fold, and γ-globin gene expression 2.6-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.3-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2+HbF was increased from 8.1 % in vector-transduced cells to 19.7% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 14.4% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 79.8% in medium-form Eklf-GATA-transduced cells as compared with 89.8% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. Our results shown that long form Eklf-GATA1 fusion protein has major effects on d- and g-globin induction than β-globin; the medium form Eklf-GATA1 elevated δ- and γ-globin expression without an effect on β-globin expression. Our results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.

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A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Cultured Mouse Erythrocytes

Blood ◽

10.1182/blood-2018-99-115209 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3479-3479

Author(s):

Jianqiong Zhu ◽

Hongzhen Li ◽

Wulin Aerbajinai ◽

Chutima Kumkhaek ◽

Kyung Chin ◽

...

Keyword(s):

Gene Expression ◽

Sickle Cell Disease ◽

Fusion Protein ◽

Sickle Cell ◽

Fusion Proteins ◽

Globin Gene ◽

Globin Chain ◽

Cell Disease ◽

Long Form ◽

Globin Gene Expression

Abstract β-hemoglobinopathies are inherited disorders caused by mutations/deletions in the β-globin chain that lead to structurally defective β-globin chains or reduced (or absent) β-globin chain production. These diseases affect multiple organs and are associated with considerable morbidity and mortality, representing a major public health challenge. Current gene therapy approaches for the treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in human CD34+ cells. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from mouse hematopoietic progenitor cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 1.8-fold, δ-globin gene expression 3.3-fold, and γ-globin gene expression 1.7-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.6-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2 was increased from 2.1 % in vector-transduced cells to 8.9% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 6.3% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 30.6% in medium-form Eklf-GATA-transduced cells as compared with 40.7% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. ChIP-sequencing analysis has demonstrated that Eklf-GATA1 fusion proteins and GATA1 having a similar protein-DNA binding pattern at a global level. Our results have found that the long form Eklf-GATA1 fusion protein has a major effect on δ-globin induction than β-globin; the medium form Eklf-GATA1 is able to elevate δ-globin expression without having an effect on β-globin expression. The above findings indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. Disclosures No relevant conflicts of interest to declare.

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Differences in Fetal Hemoglobin Induction in Sickle Cell Disease and beta-Thalassemia.

Blood ◽

10.1182/blood.v108.11.555.555 ◽

2006 ◽

Vol 108 (11) ◽

pp. 555-555 ◽

Cited By ~ 2

Author(s):

Hassana Fathallah ◽

Ali Taher ◽

Ali Bazarbachi ◽

George F. Atweh

Keyword(s):

Gene Expression ◽

Sickle Cell Disease ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Mrna Levels ◽

Globin Genes ◽

Thalassemia Intermedia ◽

Cell Disease ◽

Globin Mrna ◽

Globin Gene Expression

Abstract A number of therapeutic agents including hydroxyurea, butyrate and decitabine have shown considerable promise in the treatment of sickle cell disease (SCD). However, the same agents have shown less clinical activity in β-thalassemia. As a first step towards understanding the molecular basis of the different clinical responses to these agents, we have studied the mechanisms of induction of fetal hemoglobin (HbF) by butyrate in BFU-E derived cells from 5 patients with SCD and 9 patients with β-thalassemia intermedia. Exposure to butyrate resulted in a dose-dependent augmentation of γ-globin mRNA levels in erythroid cells from patients with SCD. In contrast, induction of γ-globin expression in erythroid cells from patients with β-thalassemia intermedia was only seen at a high concentration of butyrate. The increase in γ-globin mRNA levels in patients with SCD and β-thalassemia intermedia was associated with opening of the DNA structure as manifested by decreased DNA methylation at the γ-globin promoters. Interestingly, butyrate exposure had markedly different effects on the expression of the β- and α-globin genes in the two categories of patients. Butyrate decreased the level of β-globin mRNA in 4 out of 5 patients with SCD (P = 0.04), while in β-thalassemia the levels of β-globin mRNA did not change in 7 patients and decreased in 2 patients after butyrate exposure (P = 0.12). Thus in patients with SCD, the effects of the induction of the γ-globin gene on the γ/(β+γ) mRNA ratios were further enhanced by the butyrate-mediated decreased expression of the β-globin gene. As a result, γ/(β+γ) mRNA ratios increased in all patients with SCD, with a mean increase of 31% (P = 0.002). In contrast, butyrate increased γ/(β+γ) mRNA ratios only in 4 out of 9 patients with β-thalassemia, with a more modest mean increase of 12% (P = 0.004). Interestingly, the decreased β-globin expression in patients with SCD was associated with closing of the DNA configuration as manifested by hypermethylation of DNA at the promoter of the β-globin gene while methylation of the same promoter did not change following butyrate exposure in patients with β-thalassemia intermedia. More surprisingly, the expression of the α-globin genes increased following butyrate exposure in 4 out of 9 patients with β-thalassemia, while the levels of α-globin mRNA decreased in 4 out of 5 patients with SCD. As a result, the favorable effects of the butyrate-induced increase in γ-globin gene expression on the α: non-α mRNA imbalance in patients with β-thalassemia intermedia were partly neutralized by the corresponding increase in α-globin gene expression. These differences may explain, at least in part, the more favorable effects of inducers of HbF in SCD than in β-thalassemia. Further studies are necessary to fully understand the molecular bases of the different responses to agents that induce HbF in patients with these disorders.

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Mycobacterium aviumComplex Infection in a Patient with Sickle Cell Disease and Severe Iron Overload

Case Reports in Infectious Diseases ◽

10.1155/2014/405323 ◽

2014 ◽

Vol 2014 ◽

pp. 1-5 ◽

Cited By ~ 1

Author(s):

Kamal Shemisa ◽

Nasima Jafferjee ◽

David Thomas ◽

Gretta Jacobs ◽

Howard J. Meyerson

Keyword(s):

Sickle Cell Disease ◽

Iron Overload ◽

Sickle Cell ◽

Unknown Origin ◽

Mycobacterial Infection ◽

In Vivo Studies ◽

Recurrent Pain ◽

Cell Disease

A 34-year-old female with sickle cell anemia (hemoglobin SS disease) and severe iron overload presented to our institution with the subacute presentation of recurrent pain crisis, fever of unknown origin, pancytopenia, and weight loss. A CT scan demonstrated both lung and liver nodules concerning for granulomatous disease. Subsequent biopsies of the liver and bone marrow confirmed the presence of noncaseating granulomas and blood cultures isolatedMycobacterium aviumcomplex MAC. Disseminated MAC is considered an opportunistic infection typically diagnosed in the immunocompromised and rarely in immunocompetent patients. An appreciable number of mycobacterial infection cases have been reported in sickle cell disease patients without immune dysfunction. It has been reported that iron overload is known to increase the risk for mycobacterial infection in vitro and in vivo studies. While iron overload is primarily known to cause end organ dysfunction, the clinical relationship with sickle cell disease and disseminated MAC infection has not been reported. Clinical iron overload is a common condition diagnosed in the sub-Saharan African population. High dietary iron, genetic defects in iron trafficking, as well as hemoglobinopathy are believed to be the etiologies for iron overload in this region. Patients with iron overload in this region were 17-fold more likely to die fromMycobacterium tuberculosis. Both experimental and clinical evidence suggest a possible link to iron overload and mycobacterial infections; however larger observational studies are necessary to determine true causality.

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New Concepts in Genome Regulation

Blood ◽

10.1182/blood.v128.22.sci-47.sci-47 ◽

2016 ◽

Vol 128 (22) ◽

pp. SCI-47-SCI-47

Author(s):

Ann Dean ◽

Jongjoo Lee ◽

Ryan Dale ◽

Ivan Krivega

Keyword(s):

Sickle Cell Disease ◽

Long Range ◽

Control Region ◽

Sickle Cell ◽

Globin Gene ◽

Locus Control Region ◽

Beta Thalassemia ◽

Beta Globin ◽

Cell Disease ◽

Gamma Globin

Abstract Manipulating gene regulation to favor gamma-globin transcription over beta-globin transcription has been a goal of research in erythropoiesis for decades because of its relevance to amelioration of the pathophysiology of sickle cell disease and beta-thalassemia. A fundamental unanswered question in biology is how the unique pattern of gene expression, the transcriptome, of the many different individual mammalian cell types arises from the same genome blueprint and changes during development and differentiation. There is a growing appreciation that genome organization and the folding of chromosomes is a key determinant of gene transcription. Within this framework, enhancers function to increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding, or looping. These long range interactions are orchestrated by cell type specific proteins and protein complexes that bind to enhancers and promoters and stabilize their interaction with each other. We have been studying LDB1, a member of an erythroid protein complex containing GATA1, TAL1 and LMO2. The LDB1complex activates erythroid genes through occupancy of virtually all erythroid enhancers. LDB1engages in homo- and heterotypic interactions with proteins occupying the promoters of erythroid genes to bring them into proximity with their enhancers. We find that enhancer long range looping activity can be redirected. Both targeting of the beta-globin locus control region to the gamma-globin gene in adult erythroid cells by the tethering of LDB1 or epigenetic unmasking of a silenced gamma-globin gene lead to increased locus control region (LCR)/gamma-globin contact frequency and reduced LCR/beta-globin contact. The outcome of these manipulations is robust, pan-cellular gamma-globin transcription activation with a concomitant reduction in beta-globin transcription. These examples suggest that chromosome looping can be considered a therapeutic target for gene activation or gene silencing to ameliorate genetic diseases such as sickle cell disease and beta-thalassemia. Disclosures No relevant conflicts of interest to declare.

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Additive Effect of Butyrate and Hemin on Fetal Hemoglobin Induction in Erythroid Cells from Sickle Cell Disease and Beta-Thalassemia.

Blood ◽

10.1182/blood.v108.11.1224.1224 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1224-1224

Author(s):

Hassana Fathallah ◽

Ali Taher ◽

Ali Bazarbachi ◽

George F. Atweh

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Histone Deacetylases ◽

Globin Gene ◽

Fetal Hemoglobin ◽

Beta Thalassemia ◽

Erythroid Cells ◽

Cell Disease ◽

Globin Mrna ◽

In Erythroid Cells

Abstract High levels of fetal hemoglobin (HbF) are known to ameliorate the pathophysiology of β-globin disorders. The objective of this study is twofold: the first is to evaluate the efficacy of hemin as an inducer of HbF in erythroid cells from patients with sickle cell disease (SCD) and β-thalassemia (β-thal); the second is to determine if the combination of butyrate and hemin can induce higher levels of expression of HbF than either agent alone. BFU-E derived cells from the peripheral blood of two patients with homozygous SCD, three patients with β-thal, one patient with sickle β-thalassemia (S/β-thal) and one normal individual (AA) were cultured in the absence (control) or presence of butyrate (B), hemin (H) or butyrate and hemin (B+H). As expected, the levels of γ-globin mRNA [expressed as % γ/(β+γ)] increased upon butyrate exposure in progenitor-derived erythroid cells from SS and S/β-thal patients, and to a lesser extent in patients with β-thal (P = 0.01). In contrast, butyrate did not increase γ-globin expression in BFU-E derived colonies from the AA individual. Moreover, hemin exposure increased the γ/(β+γ) ratio in all subjects (P = 0.02). These findings confirm that hemin can be an effective HbF inducing agent in SCD and β-thal. Although the mechanism of induction of HbF by hemin is not known, unlike butyrate, hemin is clearly not a direct inhibitor of histone deacetylases and is likely to induce HbF by a different mechanism of action. Thus, we investigated the effect of the combination of hemin and butyrate on γ-globin gene expression. Interestingly, the combination of butyrate and hemin resulted in additive increases in the γ/(β+γ) ratios in all patients compared to butyrate alone (P = 0.03) or hemin alone (P = 0.01) (Table I). Just as importantly, exposure to both drugs resulted in a decrease in the α/(β+γ) mRNA imbalance in β-thal, which is the predominant pathophysiological feature of this disorder. In conclusion, combination therapy consisting of butyrate and hemin, which are two agents with different mechanisms of action and different toxicity profiles, may provide a more effective way of inducing HbF in patients with SCD and β-thal. Table I mRNA SCD β-Thal S/β-Thal AA n 2 3 1 1 %γ/(β+γ) Control 36 42 26 7.1 B 45 50 41 6.9 H 55 55 52 15 B+H 60 61 59 13 α/(β+γ) Control 3.1 8.9 1.8 1.9 B 2.0 7.7 2.9 1.7 H 3.0 7.5 1.7 1.0 B+H 2.9 6.4 2.2 1.3

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Contributions of Immune Cells to Vascular Occlusion in Sickle Cell Disease.

Blood ◽

10.1182/blood.v112.11.sci-44.sci-44 ◽

2008 ◽

Vol 112 (11) ◽

pp. sci-44-sci-44

Author(s):

Paul S. Frenette ◽

Andres Hidalgo ◽

Jungshan Chang ◽

Anna Peired

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

Blood Cells ◽

High Speed ◽

Globin Gene ◽

Clinical Manifestations ◽

Leading Edge ◽

Cell Disease ◽

Humanized Mouse Model

Abstract Sickle cell disease (SCD) results from a single missense mutation in the β-globin gene, making the gene product susceptible to polymerize in conditions of low O2 tension. The resulting polymers can disrupt the normal architecture of sickle red blood cells (sRBC), altering their membrane and promoting adherence. sRBC adhesion events within the vasculature produces the painful vaso-occlusive episodes that account for most of the morbidity and mortality of this disease.1 The mechanisms mediating vaso-occlusion (VOC) in SCD are not well understood. Several studies have shown that sRBC, most notably the immature reticulocytes, can bind to endothelial cells (EC) though multiple overlapping adhesion mechanisms. However, whether these interactions are directly involved in VOC is controversial. sRBC can clearly interact with other blood cells inside the vasculature. For example, in a humanized mouse model of SCD, sRBCs interact prominently with leukocytes that are adherent in inflamed venules.2 A role for leukocytes in VOC is consistent with clinical data that have linked leukocyte counts, but not reticulocyte counts, with clinical manifestations of SCD. High-speed multichannel fluorescence intravital microscopy (MFIM) using low doses of lineage-specific antibodies has shown that sRBCs interact specifically with adherent neutrophils.3 Most adherent neutrophils are not immobile but rather crawl along inflamed venules, and virtually all of them exhibit a polarized appearance in vivo with clustered L-selectin at the uropod. Inhibition of or deficiency in the endothelial selectins (E- and P-selectins) protects SCD mice from VOC. Recent studies into the individual role of each selectin have revealed, unexpectedly, a major function for E-selectin. Whereas E-selectin inhibition or deficiency does not significantly affect neutrophil adhesion in inflamed venules, it dramatically blocks the interactions of RBC with adherent neutrophils, suggesting that E-selectin signaling into an adherent leukocyte may induce RBC capture. We have found that RBCs carrying normal hemoglobin (nRBCs) also interact with adherent neutrophils in inflamed venules of wildtype mice. In the mouse, three glycoproteins, P-selectin glycoprotein ligand-1 (PSGL-1, encoded by the Selplg gene), CD44 (encoded by Cd44), and E-selectin ligand-1 (ESL-1, encoded by Glg1) mediate all E-selectin binding activity of leukocytes.4 Detailed analyses of Selplg−/− mice, Cd44−/− mice, or chimeric mice, in which Glg1 is knocked-down by RNA interference, revealed that E-selectin-induced signals are mediated specifically by Glg1. Using MFIM analyses, we have mapped the location of RBC captures on the leading edge of polarized neutrophils. Further investigations have revealed that RBC capture was mediated by the β2 integrin Mac-1 (αMβ2). We have developed a new method to assay Mac-1 activation on adherent leukocytes in live mice. These analyses have shown that Glg1-mediated signals activate αMβ2 regionally at the leading edge, allowing RBC capture. These results suggest a new pathway for the development of targeted therapies for VOC. The fact that genetically normal RBCs are captured by neutrophils through mechanisms similar to sRBCs also suggests broad functions for this paradigm in other thrombo-inflammatory diseases.

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