Hemolysis in sickle cell disease

Bensinger, Thomas A.

doi:10.1001/archinte.133.4.624

Cited by 45 publications

(47 citation statements)

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“…45 Therefore, in the context of sickle cell anemia, the effect of the resistance to hepcidin by ferroportin Q248H could be magnified in the delivery of iron from macrophages versus the delivery from enterocytes. Furthermore, sickle cell anemia is characterized by a certain degree of intravascular hemolysis 46 with loss of hemoglobin iron in the urine, 47 which could in part offset increased iron absorption by enterocytes. The net effect of these processes could be reduced iron stores with ferroportin Q248H in the setting of sickle cell anemia, as suggested by the results of this study.…”

Section: Discussionmentioning

confidence: 99%

Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin

Nekhai

Foster

et al. 2012

Haematologica

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ciency or ferroportin Q248H had lower circulating tumor necrosis factor-α concentrations than iron-replete children with WT ferroportin, 25 led us to hypothesize that ferroportin Q248H has reduced sensitivity to hepcidin but that this property would be observed at lower concentrations of hepcidin than those used in previous studies. 23 Here, we analyzed the sensitivity of ferroportin Q248H to hepcidin at various concentrations by determining the levels of ferroportin transiently expressed in cultured cells and in human primary monocytes derived from humans with different ferroportin genotypes. We also measured ferritin concentrations in these cells, levels that are indicative of cellular iron status. Finally, we examined the effect of Q248H on serum iron measures in patients with sickle cell anemia who have markedly increased macrophage iron export because of chronic hemolysis. Design and Methods Predictive analysis and worldwide allele frequenciesMinor allele frequencies for missense mutations in the single nucleotide polymorphism database (dbSNP) were determined using Kaviar and sequence data (http://db.systemsbiology.net/kaviar). 26Mutations were analyzed for effects on protein function using a 571-residue ferroportin protein (UniProtKB/Swiss-Prot Q9NP59) and the predictive analysis tools: SIFT (Sorting Intolerant From Tolerant; http://blocks.fhcrc.org/sift/SIFT.html) 27 and PolyPhen2 (Polymorphism Phenotyping version 2; using HumDiv model; http://genetics.bwh.harvard.edu/pph2/index.shtml). 28 Cells and media293T cells were purchased from ATCC (Manassas, VA, USA) and cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% fetal bovine serum (FBS) (Life Technologies) and 1% glutamine (Invitrogen, Carlsbad, CA, USA) at 37°C in the presence of 5% CO 2 . Ferroportin expression constructsHuman WT ferroportin, ferroportin Q248H and ferroportin C326Y mutants cloned with c-Myc and histidine tags in a pcDNA3.1 expression vector were kindly provided by Dr. Hal Drakesmith. 23 To generate enhanced green fluorescent protein (EGFP)-tagged human ferroportin, the ferroportin coding sequences from WT ferroportin, ferroportin Q248H and ferroportin C326Y mutants were amplified by polymerase chain reaction (PCR) with the following primers that included XhoI and Kpn1 restriction sites (shown in italics): forward primer, GCCTC-GAGATGACCAGGGCGGGAGATCAC and reverse primer, GCGGTACCGTAACAACAGATGTATTTGCTTGATTTTC. The PCR products were purified on agarose gel, digested with XhoI and Kpn1 (BioLabs, Ipswich, MA) and ligated into the pEGFP-N1 vector (Clontech, Mountain View, CA, USA) which was also digested with XhoI and Kpn1 and ligated. The ligation products were transformed into E. coli DH5α cells (Invitrogen) and kanamycin-resistant colonies were selected. WT ferroportin-EGFP, C326Y and Q248H ferroportin -GFP-expressing plasmids were purified using a Qiagen (Valencia, CA, USA) purification kit and sequenced using the Macrogen service (Rockville, MD, USA). Transfection and treatment293T cells were seeded in 6-well ...

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Section: Discussionmentioning

confidence: 99%

Reduced sensitivity of the ferroportin Q248H mutant to physiological concentrations of hepcidin

Nekhai

Foster

et al. 2012

Haematologica

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“…In patients with sickle cell disease, chronic hemolysis leads to the destruction of approximately 10% of the circulating erythrocytes every 24 hours, with 30% of this hemolysis estimated to be intravascular (18,45). The Hb scavenging system in these patients becomes saturated, as indicated by undetectable plasma haptoglobin levels, and cell-free plasma Hb accumulates, as has been demonstrated by increased levels of plasma Hb in patients with sickle cell disease compared with normal patients (18).…”

Section: Figurementioning

confidence: 99%

Hemolysis-associated endothelial dysfunction mediated by accelerated NO inactivation by decompartmentalized oxyhemoglobin

Minneci¹

2005

Journal of Clinical Investigation

286

240

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During intravascular hemolysis in human disease, vasomotor tone and organ perfusion may be impaired by the increased reactivity of cell-free plasma hemoglobin (Hb) with NO. We experimentally produced acute intravascular hemolysis in a canine model in order to test the hypothesis that low levels of decompartmentalized or cell-free plasma Hb will severely reduce NO bioavailability and produce vasomotor instability. Importantly, in this model the total intravascular Hb level is unchanged; only the compartmentalization of Hb within the erythrocyte membrane is disrupted. Using a full-factorial design, we demonstrate that free waterinduced intravascular hemolysis produces dose-dependent systemic vasoconstriction and impairs renal function. We find that these physiologic changes are secondary to the stoichiometric oxidation of endogenous NO by cell-free plasma oxyhemoglobin. In this model, 80 ppm of inhaled NO gas oxidized 85-90% of plasma oxyhemoglobin to methemoglobin, thereby inhibiting endogenous NO scavenging by cell-free Hb. As a result, the vasoconstriction caused by acute hemolysis was attenuated and the responsiveness to systemically infused NO donors was restored. These observations confirm that the acute toxicity of intravascular hemolysis occurs secondarily to the accelerated dioxygenation reaction of plasma oxyhemoglobin with endothelium-derived NO to form bioinactive nitrate. These biochemical and physiological studies demonstrate a major role for the intact erythrocyte in NO homeostasis and provide mechanistic support for the existence of a human syndrome of hemolysis-associated NO dysregulation, which may contribute to the vasculopathy of hereditary, acquired, and iatrogenic hemolytic states. Introduction NO is continuously synthesized from endothelium and regulates homeostatic vascular functions such as vasodilation, inhibition of platelet activation and thrombosis, inhibition of endothelial adhesion molecule and endothelin-1 expression, and modulation of intimal and smooth muscle proliferation (1-5). A paradox and controversy in vascular biology surrounds the near diffusion-limited dioxygenation reaction of NO with oxyhemoglobin that oxidizes NO to nitrate, an inactive metabolite (Reaction 1).

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“…The deoxygenated Hb S molecules form long fibers within the red blood cells, making them rigid and distorting them into the shape of a sickle. This leads to the blocking of capillary vessels and the destabilization of the red blood cell membranes and premature destruction of erythrocytes (5).…”

Section: Hb Smentioning

confidence: 99%