Erythropoietin-Driven Signaling Complements the Survival Defect of DMT1-Mutant Erythroid Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2104-2104
Author(s):  
Monika Horvathova ◽  
Katarina Kapralova ◽  
Zuzana Zidova ◽  
Dalibor Dolezal ◽  
Dagmar Pospisilova ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Post Treatment ◽  
High Dose ◽  
Erythroid Cells ◽  
Wild Type ◽  
Normal Range ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Pre Treatment

Abstract Abstract 2104 Mutations inactivating the divalent metal transporter 1 (DMT1) cause impaired erythroid iron utilization and lead to the development of hypochromic microcytic anemia associated with ineffective erythropoiesis (IE). The anemia can be ameliorated with high-dose erythropoietin (EPO) therapy (Pospisilova D, et al. Blood. 2006. 108:404–5). In contrast to β-thalassemia mouse model with dramatically elevated EPO levels which were proposed to protect erythroid cells from apoptosis (Libani IV, et al. Blood. 2008. 112:875–885), DMT1-mutant mice (mk/mk) have only 2.8-fold higher EPO levels when compared to the wild-type littermates. This corresponds to 2-fold elevation of serum EPO above the normal range for DMT-1-mutant patient before initiation of EPO therapy. Different mechanisms may therefore drive IE in anemia due to DMT1 mutation. In this study we analyzed the bases for the clinical success of high-dose EPO supplementation in a DMT1-mutant patient and mk/mk mice. EPO administration significantly increased hemoglobin levels (7.4 g/dL to 9.1 g/dL for the patient and 7.5±0.6 to 9.5±0.4 g/dL for mk/mk mice) and partially ameliorated IE. Colony forming assay using patient's cells showed significantly improved in vitro growth of post-treatment DMT1-mutant burst-forming unit erythroid (BFU-E) progenitors when compared to pre-treatment BFU-Es. In addition, the reduced plating efficiency and colony-forming capacity of pre-treatment DMT1-mutant BFU-Es can be corrected by the addition of the broad spectrum caspase inhibitor z-VAD-fmk to the cultures. This indicates involvement of caspase-dependent apoptosis in the defective survival of pre-treatment BFU-E progenitors and in their impaired capacity to form erythroid colonies. TUNEL assay on patient's bone marrow smears showed markedly decreased rate of apoptosis (from 4% to 1.5% of TUNEL-positive erythroblasts) after EPO supplementation. No profound changes in erythroblast maturation were noted in post-treatment bone marrow with the exception of additional expansion of polychromatophilic pool suggesting that inhibition of apoptosis rather than increased differentiation of DMT1-mutant erythroid cells predominantly accounts for amelioration of anemia and IE. In accordance with the patient's results, EPO administration to mk/mk mice did not alter the distribution of erythroblasts of different maturation stages. On the other hand, augmented STAT5 activation and enhanced expression of anti-apoptotic proteins BCL-XL and MCL-1 was detected in EPO treated mice. This correlated with decreased number of erythroid Ter119+ precursors undergoing apoptosis in EPO treated mk/mk bone marrow (12.4±2.3% to 5.4±0.9%) and spleen (7.3±0.7% to 3.1±0.9%). EPO supplementation also significantly reduced susceptibility of mk/mk erythrocytes to undergo stress-induced death that could reflect increased eryptosis (apoptosis of DMT1-mutant erythrocytes) in vivo and protective effect of EPO. Low to undetectable expression of hepcidin in mk/mk liver could be attributed to 16-fold increase in GDF15 expression in the bone marrow; the expression of TWSG1 was comparable to wild-type littermates. Also patient's urinary hepcidin is low (55.3 ng/mg creatinine; normal range 71–1762), however, in contrast to mk/mk mice and β-thalassemia patients the suppression of hepcidin seems to be only partly mediated by GDF15 as patient's GDF15 plasma levels are only 1.9-fold higher (548.4 pg/mL) in comparison to gender- and age-matched controls (288.4±56.9 pg/mL). These results indicate that mouse models may not fully mimic the human disease and suggest existence of additional bone marrow-derived regulator of hepcidin expression. In summary we present the bases for the clinically approved success of EPO treatment under condition of iron-deprived erythropoiesis. We conclude that EPO-driven signaling rescues the survival defect of DMT1-mutant erythroid cells. Grant support: Czech Grant Agency, grants No. P305/10/P210 and P305/11/1745; Internal Grant of Palacky University Olomouc (LF_2011_011), and Ministry of Health Czech Republic Grant NS10281-3/2009. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Identification, Isolation and Transcriptome Analyses of Mouse, Rat and Man Erythroblastic Island Central Macrophages

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 841-841
Author(s):  
Wei Li ◽  
Yaomei Wang ◽  
Huizhi Zhao ◽  
Huan Zhang ◽  
Yuanlin Xu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Negative Control ◽  
Human Macrophage ◽  
Mouse Bone Marrow ◽  
Erythroid Cells ◽  
Wild Type ◽  
Erythroblastic Island ◽  
Hela Cell Lines

Abstract Erythroblastic island (EBI), composed of a central macrophage and surrounding erythroid cells, is the first hematopoietic niche discovered for erythropoiesis. Yet, the identity of the central macrophage has so far remained elusive. Based on the previous findings that F4/80, VCAM1 and CD169 are potential mouse central macrophage markers, we first calculated the number of F4/80+VCAM1+CD169+ mouse macrophages in the mouse bone marrow and compared it to the number of Ter119+ erythroblasts. We found that the ratio of F4/80+VCAM1+CD169+ macrophage and erythroblasts is about 1:2. Given the fact that one central macrophage is surrounded by multiple erythroblasts, the above finding suggests that it is unlikely that all the F4/80+VCAM1+CD169+ macrophages are central macrophages. Erythropoietin (Epo) is essential for erythropoiesis. It has been reported that the Epo receptor (Epor) is expressed in peritoneal macrophages. These findings promoted us to speculate that EBI central macrophages may express Epor so that Epo acts on both erythroid cells and the central macrophages simultaneously in the niche to ensure efficient and optimal red cell production. To test this notion, we first examined whether mouse bone marrow and fetal liver macrophages express Epor using the Epor-GFPcre knockin mouse model. We found that ~5% of bone marrow F4/80+ macrophages and ~35% of fetal liver F4/80+ macrophages express Epor-GFP. As negative control, no Epor-GFP macrophages are noted in wild type F4/80+ macrophages. Importantly, ImageStream analyses revealed the native EBIs in bone marrow and fetal liver are formed by Epor+ but not Epor- macrophages. Bioinformatics analyses of RNA-seq data on the sorted Epor+ and Epor- macrophage populations revealed that molecules involved in central macrophage-erythroblast association such as VCAM1, CD169, and molecules known to be important for central macrophage function such as Dnase2a, ferroportin, are highly expressed in Epor+ macrophages. In marked contrast, highly expressed pathways in Epor- macrophages are associated with immune responses including antigen process and presentation. Intriguingly, the immune related pathways are dramatically downregulated in the Epor+ macrophages, suggesting that the Epor+ macrophages in bone marrow and fetal liver have evolved a specialized function in supporting erythropoiesis. To examine whether expression of Epor in EBI central macrophages is a conserved feature across species, we generated Epor-GFPcre knockin rat using the CRISP/Cas9 technology. Using CD163 as rat macrophage marker, we found that a subpopulation of rat bone marrow CD163+ macrophages expresses Epor-GFP. As a negative control, no Epor-GFP macrophages are noted in wild type CD163+ macrophages. To examine whether EPOR is expressed in human EBI central macrophages, antibody specificity for human EPOR is critical. To this end, we employed CRISP/Cas9 approach to knock out EPOR in K562 and Hela cell lines and validated the specificity of a commercially available anti-human EPOR antibody. Using CD163, CD169 as human macrophage markers, we found that EPOR is also expressed in a subpopulation of human macrophages. Moreover, in vitro EBI formation assay revealed that human EPOR+ but not EPOR- macrophages form EBIs with erythroid cells and that the EBI formation is enhanced by EPO. In summary, we for the first time, after discovery of the EBIs 60 years ago, have identified Epor+ macrophages in mouse bone marrow and fetal liver as EBI central macrophages. Our findings provide solid foundation for studying the mechanisms by which erythropoieis is supported EBI central macrophages. A better understanding of such mechanisms will provide extensive new knowledge on basic biology of erythropoiesis. It is also important to understand the pathology of erythropoietic disorders as well as to improve ex vivo erythrocyte production. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bone marrow glycophorin‐positive erythroid cells of myelodysplastic patients responding to high‐dose rHuEPO therapy have a different gene expression pattern from those of nonresponders

2008 ◽  
Vol 83 (7) ◽  
pp. 531-539 ◽  
Author(s):  
Agostino Cortelezzi ◽  
Gualtiero Colombo ◽  
Caterina Pellegrini ◽  
Ilaria Silvestris ◽  
Lorenza Moronetti Mazzeo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Expression Pattern ◽  
Gene Expression Pattern ◽  
High Dose ◽  
Erythroid Cells

scholarly journals Modulating the Haematological and Cytogenetic Effects of Mitomycin C by Aqueous Extract of Nut Grass (Cyperus rotundus L.)

2007 ◽  
Vol 1 (1) ◽  
pp. 52-54
Author(s):  
Ali H. Ad'hiah ◽  
Mohammed M. F. Al-Halbosiy ◽  
Rakad M. Al-Jumaily
Keyword(s):  
Bone Marrow ◽  
Mitotic Index ◽  
Mitomycin C ◽  
Aqueous Extract ◽  
Leucocyte Count ◽  
Bone Marrow Cells ◽  
Post Treatment ◽  
Cyperus Rotundus ◽  
Micronucleus Formation ◽  
Pre Treatment

The aqueous extract (5, 10 and 15 mg/kg) of nut grass (Cyperus rotundus L.) rhizomes was evaluated orally in albino male mice using some haematological (total leucocyte count) and cytogenetic (mitotic index, micronucleus formation and chromosomal aberrations of bone marrow cells) parameters. The extract interaction with the mutagen mitomycin C (MMC) was also evaluated through two types of treatments (pre- and post-treatments). The results revealed that the dose 15 mg/kg of the extract significantly increased the total count of leucocytes (7634.4 vs. 6783.3 cells/cu.mm. blood), while the mitotic index showed no significant differences, as compared to negative controls. However, the spontaneous formation of micronuclei in the bone marrow cells was significantly decreased in the three investigated doses of the extract (0.30, 0.32 and 0.29, respectively vs. 0.62%), while the chromosomal assay showed similar frequencies in the negative control and nut grass-treated animals. With respect to the interaction with MMC, the pre-treatment (15 mg/kg) enhanced the leucocyte count (10358.6 vs. 3800.2 cells/cu.mm.blood) and mitotic index (11.9 vs. 6.5%), and a similar picture was drawn when the pos-treatment was considered (8884.2 vs. 4292.7 cells/cu.mm.blood; 14.6 vs. 7.6%). However, the doses 5 and 10 mg/kg of the plant extract were much more effective in reducing the MMC-induced micronucleus formation in both types of treatments especially the dose 5 mg/kg (pre-treatment: 4.24 vs. 16.29%; post-treatment: 3.79 vs. 14.34%). With respect to chromosomal aberration assay, the dose 15 mg/kg of the extract was the most effective dose in reducing the MMC-induced aberrations, but the post-treatment was better than pre-treatment in this respect (0.29 vs. 0.79 aberration/cell).

scholarly journals Regulation of divalent metal transporter 1 (DMT1) non-IRE isoform by the microRNA Let-7d in erythroid cells

Haematologica ◽  
2010 ◽  
Vol 95 (8) ◽  
pp. 1244-1252 ◽  
Author(s):  
I. Andolfo ◽  
L. De Falco ◽  
R. Asci ◽  
R. Russo ◽  
S. Colucci ◽  
...  
Keyword(s):  
Divalent Metal ◽  
Erythroid Cells ◽  
Divalent Metal Transporter 1 ◽  
Divalent Metal Transporter ◽  
Metal Transporter ◽  
In Erythroid Cells

Urinary Steroids in Depressive Illness

1971 ◽  
Vol 16 (4) ◽  
pp. 224-227 ◽  
Author(s):  
A. Balfour Sclare ◽  
J. K. Grant
Keyword(s):  
Depressive Illness ◽  
Post Treatment ◽  
Normal Range ◽  
Mean Values ◽  
Female Patients ◽  
Significant Difference ◽  
The Mean ◽  
Pre Treatment ◽  
Depressive Patients ◽  
Source Of Information

A longitudinal study of urinary 17-OHCS in 16 depressive patients showed mean values within the accepted normal range. There were no significant differences between pre-treatment and post-treatment values; only the female patients showed a tendency to have somewhat lower post-treatment levels. 17-KS output was measured in 18 depressive patients. The mean values were within the normal range. There was no significant difference between pre-treatment and post-treatment values although the female patients displayed a tendency towards somewhat lower levels following treatment. DHA and EA excretion were measured in 9 depressive patients. There was a very wide variance in the findings. There was no significant difference between pre-treatment and post-treatment levels. The significance of the above findings is discussed. It is considered that measurements of urinary excretion of corticosteroids in affective disorders constitute a limited source of information.

ACCELERATION OF BONE-MARROW RECOVERY BY PRE-TREATMENT WITH CYCLOPHOSPHAMIDE IN PATIENTS RECEIVING HIGH-DOSE MELPHALAN

The Lancet ◽  
1978 ◽  
Vol 312 (8097) ◽  
pp. 966-968 ◽  
Author(s):  
D.W Hedley ◽  
J.L Millar ◽  
T.J Mcelwain ◽  
M.Y Gordon
Keyword(s):  
Bone Marrow ◽  
High Dose ◽  
Pre Treatment ◽  
High Dose Melphalan

An Insertional Mutagenesis Screen for Genes That Cooperate with Mll-AF9 in Leukemogenesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3962-3962
Author(s):  
Rachel Joy Bergerson ◽  
Lara S Collier ◽  
Sanne Lugthart ◽  
Raha Allaei ◽  
Molly J Nixon ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Latency Period ◽  
Wild Type ◽  
Aberrant Expression ◽  
Genetic Pathways ◽  
Elevated Expression ◽  
Insertion Sites ◽  
Murine Leukemia

Abstract Abstract 3962 Poster Board III-898 By altering the activity of specific transcription complexes, the MLL-AF9 fusion oncogene can initiate the process of acute myeloid leukemia (AML) development. However, all the genetic pathways that can cooperate with MLL-AF9 expression to cause full-blown AML are unknown. These pathways will provide therapeutic targets for MLL-AF9-associated AML. Mice with constitutive expression of the Mll-AF9 fusion oncoprotein under the control of the endogenous promoter develop AML but only after a prolonged latency. This model thus provides a system for understanding the evolution of AML initiated by an MLL fusion oncoprotein. We hypothesized that infection with a recombinant Murine Leukemia Virus, abbreviated M4070, could cooperate with MLL-AF9 expression to accelerate the onset of leukemia by causing the secondary mutations required for cancer progression. We bred Mll-AF9 heterozygous males to wild type females, and the offspring were injected at three days of age with M4070 virus (n=211) or were mock infected (n=68). All mice were genotyped and observed for disease progression. Virally infected Mll-AF9/+ mice succumb to disease with a significantly reduced latency period when compared to virally infected wild type (WT) mice (p < .0001) and uninfected Mll-AF9/+ mice (p < .0001), indicating that M4070 infection causes significant leukemia acceleration in these mice. Histopathology, immunohistochemical staining, analysis of the surface immunophenotype by flow cytometry, and Southern blot analysis of T and B cell receptor rearrangement indicated that infected Mll-AF9/+ animals developed primarily myeloid leukemia (myeloperoxidase positive, Mac1 or Mac1/Gr1 positive, CD3 negative) while infected WT animals developed mostly lymphoid leukemia (CD3 positive, CD4 and/or CD8 positive, myeloperoxidase negative). Retroviral insertion sites were cloned from 167 leukemic tissues from the accelerated leukemia mice using two different restriction enzymes in a shotgun-based, linker-mediated, cloning protocol to identify the genes most frequently mutated in Mll-AF9 positive leukemia. More than 4,100 independent insertions were isolated and 101 common insertion sites (CIS), defined as genomic locations with several proviral insertions from at least 3 mice, were identified. The majority of the CIS harbored proviral insertions in both Mll-AF9/+ and wild type mice, but a subset of CIS were found in only one group or the other. Some of the genes closest to the CIS have been identified as target genes in other proviral screens and some are known cancer genes. We studied a subset of the CIS-associated genes for aberrant expression in leukemic tissues. There was elevated expression of Mn1, and a trend towards increased expression of Bcl11a and Fosb, in our Mll-AF9 murine leukemia samples with proviral insertions nearby these genes. Moreover, elevated expression of MN1, FOSB, and BCL11A has been observed in microarray studies of human patients with AML. We have completed a bone marrow transduction/transplantation experiment to seek functional evidence of cooperation with Mll-AF9. Mice transplanted with Mll-AF9/+ bone marrow that had been transduced with a retrovirus encoding the candidate gene MN1 succumb to myeloid malignancy faster than mice transplanted with wild type bone marrow transduced with MN1, or Mll-AF9/+ bone marrow transduced with a retrovirus encoding just the Green Fluorescent Protein gene. This data suggests that MN1 can cooperate with Mll-AF9 to accelerate myeloid leukemia in a mouse model. We are currently using shRNA knockdown strategies in human cell lines to confirm cooperation of more candidate genes with MLL-AF9 in AML development. Thus, CIS-associated genes from leukemias accelerated by M4070 in Mll-AF9/+ mice may help define important genetic pathways that are altered during progression of AML induced by MLL fusion oncogenes. Disclosures: No relevant conflicts of interest to declare.

Erythroid Differentiation Regulator 1 (ERDR1) From Nurse-Like Cells Promotes the Survival of Chronic Lymphocytic Leukemia Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1372-1372
Author(s):  
Hendrik W. Van Deventer ◽  
Robert Mango ◽  
Jonathan Serody
Keyword(s):  
Bone Marrow ◽  
Chronic Lymphocytic Leukemia ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Conflicts Of Interest ◽  
Erythroid Differentiation ◽  
Mesenchymal Cells ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Wild Type

Abstract Abstract 1372 Background: Chemotherapy resistance in chronic lymphocytic leukemia (CLL) can be mediated by anti-apoptotic signals produced by stromal or nurse-like cells. Developing strategies to overcome this resistance is hindered by the lack of suitable “stromal” targets responsible for these signals. We have discovered that erythroid differentiation regulator 1 (ERDR1) may be a candidate target for such a strategy. In this study, we show Erdr1 is generated by several stromal cell types including bone marrow stromal cells, fibrocytes, and nurse-like cells. Furthermore, inhibition of stroma-generated Erdr1 results in increased apoptosis of co-cultured CLL cells. Methods/Results: We initially identified Erdr1 on an Affymetrix array that compared the gene expression of wild type and CCR5-/- mice with pulmonary metastasis. The increased expression of Erdr1 in the wild type mice was particularly pronounced in the pulmonary mesenchymal cells. Therefore, these cells were transfected with one of two shRNAs (shRNA #9 or shRNA#11) and the survival of these cells was compared with mesenchymal cells transfected with a non-targeted control vector. After 15 days in culture, the control cells expanded normally; however, no significant expansion was seen in either the shRNA#9 or shRNA#11 transfected cells. These differences in cellular expansion were associated with differences in apoptosis. 21.4+1.6% of the Erdr1 knockdown cells were annexin V+ compared to 11.2+1.9% of the non-targeted control (p<0.03). Using GFP as a marker for transfection, we were also able to show that knockdown of Erdr1 increased the apoptosis of surrounding non-transfected mesenchymal cells. Thus, Erdr1 is a critical protein for the survival of stromal cells. Further analysis of the mesenchymal cell subpopulations revealed the greatest expression of Erdr1 in the CD45+, thy1.1+/− fibrocytes. When compared to CD45- fibroblasts, the fibrocytes expressed CCR5 and increased Erdr1 expression by 14.2+/−2.9 fold when treated with the CCR5 ligand CCL4. Given the similarities between fibrocytes and nurse-like cells, we went on to measure the effect of Erdr1 inhibition on CLL cells. In these experiments, stable Erdr1 knockdown and control clones were selected after the transfection of the bone marrow stromal cell line M2-10B4. These clones were then co-cultured with primary CLL cells. At 96 hours, leukemia cells co-cultured with the control lines had expanded by 1.33 + 0.9 compared to 0.74 + 0.22 fold in the knock-down lines (p<0.03). As before, the lack of cellular expansion was associated with an increase in apoptosis. To further show the relevance of these findings to CLL, we demonstrated that human fibrocytes and nurse-like cells expressed mRNA and protein for ERDR1 in all patient samples tested. Implications for the treatment of human disease: Our data demonstrate that ERDR1 is a critically important protein for the survival of nurse-like cells. These data suggest that targeting ERDR1 or the upstream pathway through CCR5 might be a novel approach for the treatment of CLL. Disclosures: No relevant conflicts of interest to declare.

Regulation of Gata1 Expression by HS+3.5

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3864-3864
Author(s):  
Julia E Draper ◽  
William G Wood ◽  
Catherine Porcher ◽  
Paresh Vyas
Keyword(s):  
Bone Marrow ◽  
Colony Formation ◽  
Transgene Expression ◽  
Conflicts Of Interest ◽  
Es Cells ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Haematopoietic Stem Cell ◽  
Wild Type ◽  
Foetal Liver

Abstract Abstract 3864 Precise regulation of Gata1 expression is required in order to control the balance between lymphoid/granulomonocytic (GM) and megakaryocytic-erythroid (MegE) specification, as well as to ensure correct differentiation of the MegE lineages. Transcriptional control is conferred in part by cis regulatory elements. An upstream enhancer, HS-3.5, and the erythroid first exon IE of Gata1 are necessary and sufficient to direct transgene expression in primitive but not definitive erythroid cells. Transgene expression in definitive red blood cells is restored by inclusion of an intronic DNaseI hypersensitive site, HS+3.5. Here we report the characterization of the HS+3.5 null embryonic stem cells and the HS+3.5 knockout mouse. In vitro differentiation of HS+3.5 null ES cells resulted in reduced myeloid and megakaryocytic colony formation compared to wild type. The ΔHS+3.5 ES cells retained normal primitive erythroid colony formation. ΔHS+3.5 definitive erythroid colony progenitors displayed a decreased sensitivity to Interleukin 3 (IL3) signalling compared to wild type. ΔHS+3.5 mice were viable and had normal blood counts and films. GM and erythroid progenitors also developed normally. However, there was a mild expansion of the E14.5 foetal liver Megakaryocytic Progenitor (MkP) compartment and an increase in Gata2 expression in both the bone marrow and foetal liver MkPs. Turning to Gata1, a decrease in Gata1 expression was observed in the following compartments: the bone marrow long term haematopoietic stem cell (LT-HSC) and the foetal liver common myeloid progenitor (CMP). The relationship between the effect of the HS+3.5 deletion on Gata1 expression and the haematopoietic phenotype will be discussed. Disclosures: No relevant conflicts of interest to declare.

Desialylated Platelets Are Cleared From the Circulation by the Hepatic Asialoglycoprotein Receptor,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3257-3257
Author(s):  
Renata Grozovsky ◽  
Silvia Giannini ◽  
Karin M. Hoffmeister
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Asialoglycoprotein Receptor ◽  
Regulatory Mechanisms ◽  
Wild Type ◽  
Cell Counts ◽  
Blood Cell Counts ◽  
Terminal Galactose

Abstract Abstract 3257 The regulatory mechanisms of platelet homeostasis remain elusive. We investigated here the role of hepatic asialoglycoprotein receptor (a.k.a. Ashwell-Morell receptor) in platelet clearance. Mice lacking the hepatic asialoglycoprotein receptor Asgpr2 subunit had increased platelet survivals (T1/2 = 49.5±2h) when compared to wild type (WT, T1/2 = 31±4h) mice. Consequently, Asgpr2−/− mice had platelet counts increased by ∼20%, compared to WT, with increased terminal galactose exposure, as demonstrated using the galactose specific lectin RCA1. Bone marrow and spleen megakaryocyte numbers were reduced by ∼15% and ∼20% in Asgpr2−/− mice, compared to WT mice. Sialidase (NA, Clostidium perfringens, 50mU/mice) maximally desialylated circulating platelets when injected intravenously, as evidenced by increased RCA1 binding. Sialidase injection resulted in a ∼60% depletion of circulating platelets after 24h in Asgpr2−/− mice, compared to >90% in WT mice, indicating that desialylated platelets were partially removed by Asgpr1/2. In contrast to platelets, red blood cell counts were unaffected by sialidase treatment. Sialidase injection for 72h resulted in a 2.3-fold and 1.2-fold increase in megakaryocyte numbers in the spleen and bone marrow of WT mice, respectively, but not in Asgpr2−/− mice. In contrast to sialidase treatment, injections of rabbit anti-mouse platelet serum (RAMPS) depleted >95% of circulating platelets and increased by 70% bone marrow, but not spleen MK numbers in both WT and Asgpr2−/− mice. The data shows that removal of desialylated, i.e, senescent, platelets by the hepatic Ashwell-Morell receptor differs to that of antibody-mediated platelet clearance. Disclosures: No relevant conflicts of interest to declare.

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