Killing me softly - suicidal erythrocyte death.

This book is a completely revised new edition of the definitive reference on disorders of hemoglobin. Authored by world-renowned experts, the book focuses on basic science aspects and clinical features of hemoglobinopathies, covering diagnosis, treatment, and future applications of current research. While the second edition continues to address the important molecular, cellular, and genetic components, coverage of clinical issues has been significantly expanded, and there is more practical emphasis on diagnosis and management throughout. The book opens with a review of the scientific underpinnings. Pathophysiology of common hemoglobin disorders is discussed next in an entirely new section devoted to vascular biology, the erythrocyte membrane, nitric oxide biology, and hemolysis. Four sections deal with and thalassemia, sickle cell disease, and related conditions, followed by special topics. The second edition concludes with current and developing approaches to treatment, incorporating new agents for iron chelation, methods to induce fetal hemoglobin production, novel treatment approaches, stem cell transplantation, and progress in gene therapy.

Disorders of hemoglobin: Genetics, pathophysiology, and clinical management, second edition

The suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage, membrane blebbing and cell membrane phospholipid scrambling resulting in phosphatidylserine exposure at the cell surface. Eryptosis is stimulated in a wide variety of diseases including sepsis, haemolytic uremic syndrome, malaria, sickle-cell anemia, beta-thalassemia, glucose-6-phosphate dehydrogenase (G6PD)-deficiency, phosphate depletion, iron deficiency and Wilson's disease. Moreover, eryptosis is elicited by osmotic shock, oxidative stress, energy depletion as well as a wide variety of endogenous mediators and xenobiotics. Excessive eryptosis is observed in erythrocytes lacking the cGMP-dependent protein kinase type I (cGKI) or the AMP-activated protein kinase AMPK. Inhibitors of eryptosis include erythropoietin, nitric oxide NO, catecholamines and high concentrations of urea. Eryptosis-triggering diseases and chemicals are partially effective by stimulating the formation of ceramide, which in turn fosters cell membrane scrambling. Accordingly, ceramide-induced eryptosis participates in the pathophysiology of several diseases and contributes to the effects of a large number of xenobiotics. The mechanisms underlying ceramide formation in erythrocytes are, however, still ill defined. In case of osmotic cell shrinkage, ceramide formation is apparently due to activation of phospholipase 2, leading to formation of platelet activating factor PAF and PAF-dependent stimulation of ceramide formation, which possibly involves acid sphingomyelinase. Additional experiments are needed to conclusively define the ceramide-generating enzyme and the ceramide-dependent cellular events eventually leading to suicidal erythrocyte death.

/pdf/ceramide-in-suicidal-death-of-erythrocytes-3elk9enqkt.pdf

Ceramide in suicidal death of erythrocytes.

Background Results from recent, highly debated, retrospective studies raised concerns and prompted considerations about further testing the quality of long stored red blood cells from a biochemical standpoint.Design and Methods We performed an integrated mass spectrometry-based metabolomics and proteomics time-course investigation on SAGM-stored red blood cells. In parallel, structural changes during storage were monitored through scanning electron microscopy.Results We detected increased levels of glycolytic metabolites over the first 2 weeks of storage. From day 14 onwards, we observed a significant consumption of all metabolic species, and diversion towards the oxidative phase of the pentose phosphate pathway. These phenomena coincided with the accumulation of reactive oxygen species and markers of oxidation (protein carbonylation and malondialdehyde accumulation) up to day 28. Proteomics evidenced changes at the membrane protein level from day 14 onwards. Changes included fragmentation of membrane structural proteins (spectrin, band 3, band 4.1), membrane accumulation of hemoglobin, anti-oxidant enzymes (peroxiredoxin-2) and chaperones. While the integrity of red blood cells did not show major deviations at day 14, at day 21 scanning electron microscope images revealed that 50% of the erythrocytes had severely altered shape. We could correlate the scanning electron microscopy observations with the onset of vesiculation, through a proteomics snapshot of the difference in the membrane proteome at day 0 and day 35. We detected proteins involved in vesicle formation and docking to the membrane, such as SNAP alpha.Conclusions Biochemical and structural parameters did not show significant alterations in the first 2 weeks of storage, but then declined constantly from day 14 onwards. We highlighted several parallelisms between red blood cells stored for a long time and the red blood cells of patients with hereditary spherocytosis.

/pdf/time-course-investigation-of-sagm-stored-leukocyte-filtered-55mkqwmobw.pdf

Time-course investigation of SAGM-stored leukocyte-filtered red bood cell concentrates: from metabolism to proteomics.

Eryptosis, the suicidal death of erythrocytes, is characterized by erythrocyte shrinkage, blebbing, and phospholipid scrambling of the cell membrane. Eryptosis is triggered by increased cytosolic Ca(2+) activity, which may result from Ca(2+) entry through PGE(2)-activated Ca(2+)-permeable cation channels. The Ca(2+) sensitivity of the scrambling machinery is enhanced by ceramide, which is formed by an acid sphingomyelinase, an enzyme stimulated by platelet-activating factor. Eryptosis is enhanced in a variety of clinical conditions such as sickle-cell anemia, β-thalassemia, glucose-6-phosphate dehydrogenase deficiency, hereditary spherocytosis, paroxysmal nocturnal hemoglobinuria, myelodysplastic syndrome, phosphate depletion, iron deficiency, sepsis, hemolytic uremic syndrome, renal insufficiency, diabetes, malaria, mycoplasma infection, and Wilson's disease. Eryptosis is enhanced in mouse models of sickle cell anemia and thalassemia, as well as in mice lacking functional annexin 7, cGMP-dependent protein kinase type I, AMP-activated protein kinase, Janus kinase 3, anion exchanger 1, adenomatous polyposis coli, or Klotho. Eryptosis is triggered by osmotic shock, oxidative stress, energy depletion, hyperthermia, and a myriad of small molecules. Eryptosis is inhibited by a variety of substances including nitric oxide and catecholamines. Erythropoietin counteracts eryptosis in part by inhibiting the Ca(2+)-permeable cation channels. Excessive erythropoietin concentrations lead, however, to formation of erythrocytes, which are particularly sensitive to eryptotic stimuli. Accelerated eryptosis may be compensated by enhanced erythropoiesis, which is apparent from reticulocytosis. If the compensation is not sufficient, clinically relevant anemia develops. Beyond that, adhesion of eryptotic erythrocytes to the vascular wall may lead to impairment of microcirculation.

/pdf/mechanisms-and-significance-of-eryptosis-the-suicidal-death-4izdntie7w.pdf

Mechanisms and Significance of Eryptosis, the Suicidal Death of Erythrocytes

The present work is aimed to study the mechanism of faster erythrocyte clearance in hereditary spherocytosis (HS), a heterogeneous disorders characterized by alterations in the proteins of the red cell membrane skeleton along with different kinds of thalassemia. The maximum exposure of phosphatidylserine (PS) is found in HS compared to those in both α- and β-thalassemia. Interestingly, in HS more PS exposed cells were found in younger erythrocytes compared to normal and the thalassemics where aged cells showed higher loss of PS asymmetry. Loss of sialic acid and GlcNAc bearing glycoconjugates, presumably the glycophorins, was also found upon aging. The loss of PS asymmetry together with the cell surface glycoproteins mediated by membrane vesiculation, seemed to play key role in early clearance of erythrocytes from circulation following a mechanism similar to HbEβ-thalassemia.

Eryptosis in hereditary spherocytosis and thalassemia: role of glycoconjugates.

Summary This study aimed to investigate any correlation between the extent of phosphatidylserine (PS) asymmetry and sialylated glycoconjugate levels with the faster clearance of circulating erythrocytes in haemoglobin E (HbE) b-thalassaemia. Erythrocytes from peripheral blood samples of different HbEb-thalassaemia patients showed loss of PS asymmetry measured by annexin V binding using flow cytometry. Maximum PS exposure was found when HbE was 50–60% and HbF was <20% indicating a possible correlation with severity of the disease. Separation of erythrocytes into aged and younger cells showed higher loss of PS asymmetry in the younger erythrocytes of HbEb-thalassaemia patients when compared with normal blood, where PS asymmetry was lost only in the older cells. Sialylated glycoconjugate measurement using the lectins wheatgerm agglutinin and pokeweed mitogen showed loss of sialic acid and N-acetyl-d-glucosamine-bearing glycoproteins in the order normal<homozygous E<HbEb-thalassaemic upon ageing. A possible correlation was found between the loss of PS asymmetry with HbE level and the reduction of glycophorins from the cell surface, mediated by membrane vesiculation. A more facilitated vesiculation process in HbEbthalassaemic erythrocytes could lead to faster shedding of glycophorincontaining microvesicles, leaving highly PS-exposed erythrocytes accessible to phagocytes.

Loss of phospholipid membrane asymmetry and sialylated glycoconjugates from erythrocyte surface in haemoglobin E

Sickle cell disease (SCD) is a hemolytic disorder caused by a mutation in beta-globin gene and affects millions of people worldwide. Though clinical manifestations of the disease are quite heterogeneous, many of them occur due to erythrocyte sickling at reduced oxygen concentration and vascular occlusion mediated via blood cell adhesion to the vessel wall. We have followed proteomic approach to resolve the differentially regulated proteins of erythrocyte cytosol. The deregulated proteins mainly fall in the group of chaperone proteins such as heat shock protein 70, alpha hemoglobin stabilizing protein, and redox regulators such as aldehyde dehydrogenase and peroxiredoxin-2 proteoforms. Proteasomal subunits are found to be upregulated and phospho-catalase level also got altered. Severe oxidative stress inside erythrocyte is evident from the ROS analysis and Oxyblot(TM) experiments. Peroxiredoxin-2 shows significant dimerization in the SCD patients, a hallmark of oxidative stress inside erythrocytes. One interesting fact is that most of the differentially regulated proteins are also common for hemoglobinopathies such as Eβ thalassemia. These could provide important clues in understanding the pathophysiology of SCD and lead us to better patient management in the future.

2D DIGE based proteomics study of erythrocyte cytosol in sickle cell disease: altered proteostasis and oxidative stress.

This study aimed to investigate any correlation between the extent of phosphatidylserine (PS) asymmetry and sialylated glycoconjugate levels with the faster clearance of circulating erythrocytes in haemoglobin E (HbE) beta-thalassaemia. Erythrocytes from peripheral blood samples of different HbEbeta-thalassaemia patients showed loss of PS asymmetry measured by annexin V binding using flow cytometry. Maximum PS exposure was found when HbE was 50-60% and HbF was <20% indicating a possible correlation with severity of the disease. Separation of erythrocytes into aged and younger cells showed higher loss of PS asymmetry in the younger erythrocytes of HbEbeta-thalassaemia patients when compared with normal blood, where PS asymmetry was lost only in the older cells. Sialylated glycoconjugate measurement using the lectins wheatgerm agglutinin and pokeweed mitogen showed loss of sialic acid and N-acetyl-D-glucosamine-bearing glycoproteins in the order normal<homozygous E<HbEbeta-thalassaemic upon ageing. A possible correlation was found between the loss of PS asymmetry with HbE level and the reduction of glycophorins from the cell surface, mediated by membrane vesiculation. A more facilitated vesiculation process in HbEbeta-thalassaemic erythrocytes could lead to faster shedding of glycophorin-containing microvesicles, leaving highly PS-exposed erythrocytes accessible to phagocytes.

Loss of phospholipid membrane asymmetry and sialylated glycoconjugates from erythrocyte surface in haemoglobin E β‐thalassaemia

In search of genetic alterations responsible for high fetal hemoglobin (Hb F) phenotypes in the population of eastern India, 91 probands were screened for four polymorphisms by sequencing and/or restriction fragment length polymorphism (RFLP) analysis. These are the A>G allele on the rs4895441 locus in the intergenic region between HBS1L and MYB on chromosome 6, the G>A allele on the rs4671393 locus on chromosome 2 (BCL11A gene), the A>C allele on the rs2071348 (HBBP1 gene) and the XmnI polymorphism (rs7482144, -158 position of HBG2) on chromosome 11. We found a significant association (p = 0.002 and 0.0013) of Hb F levels with rs2071348 and rs4895441, respectively. However, the polymorphism rs4671393 gene did not show significant association with Hb F levels (p = 0.0655). As is well known, the XmnI polymorphism (p <0.0001) showed the strongest association.

Debasis Banerjee

Papers

Eryptosis in hereditary spherocytosis and thalassemia: role of glycoconjugates.

Loss of phospholipid membrane asymmetry and sialylated glycoconjugates from erythrocyte surface in haemoglobin E

2D DIGE based proteomics study of erythrocyte cytosol in sickle cell disease: altered proteostasis and oxidative stress.

Loss of phospholipid membrane asymmetry and sialylated glycoconjugates from erythrocyte surface in haemoglobin E β‐thalassaemia

Influence of BCL11A, HBS1L-MYB, HBBP1 single nucleotide polymorphisms and the HBG2 XmnI polymorphism on Hb F levels