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Hereditary spherocytosis (HS) is a genetically and phenotypically heterogeneous hemolytic anemia caused by deficiency in red blood cell (RBC) cytoskeleton proteins leading to disruptions in the vertical association of the cytoskeleton with the RBC lipid bilayer. Monoallelic mutations in the genes encoding ankyrin (ANK1), beta-spectrin (SPTB) and band 3 (SLC4A1) or biallelic mutations in the genes encoding alpha-spectrin (SPTA1), ankyrin, and protein 4.2 (EPB42) result in HS. Autosomal recessive HS due to compound heterozygous defects in SPTA1 is typically severe and diagnosis based on phenotypic assays like RBC morphology, osmotic fragility or ektacytometry is complicated by transfusion dependence resulting in most of the circulating RBCs to be of donor origin. We have developed a rapid comprehensive next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. We describe here patients with hemolytic anemia due to SPTA1 mutations, identified utilizing this assay, and their phenotype-genotype correlation. Each of these cases, when possible, has been also evaluated with ektacytometry and immunoblotting of RBC ghosts for alpha-spectrin quantitation. Wichterle et al in 1996 had estimated that alphaLEPRA(Low Expression PRAgue) mutation (c.4339-99C>T) occurs in SPTA1 gene in about 5% of Caucasians. This mutation leads to activation of an alternate acceptor splice site at position -70 of intron 30, causing frame shift and premature termination, thereby leading to decrease in alpha-spectrin production in this allele to about 16% of normal. We have found a cohort of three transfusion-dependent hereditary hemolytic anemia cases where a nonsense mutation in SPTA1 gene has occurred in trans to alphaLEPRA mutation, resulting in premature termination (see Table 1). Transfusion dependence was alleviated in two of these patients after splenectomy; the third one did not have splenectomy yet. RBC phenotype explored after splenectomy revealed an ektacytometry curve indicating spherocytosis (Figure 1A) and severely decreased alpha-spectrin on immunoblotting along with significant decrease of the associated beta-spectrin (Figure 1B). A patient with moderately severe form of HS, maintaining a hemoglobin value greater than 7 g/dL and requiring only occasional transfusions during periods of illness or stress, was found to have alphaLEPRA occurring in trans to an intronic splicing mutation c.1351-1G>TG where there is substitution at nucleotide-1 of intron positioned between nucleotides 1350 and 1351 of the SPTA1 mRNA. This splicing mutation may allow for some expression of functional alpha-spectrin protein from this allele in contrast to no protein expression in the previous cases of premature termination. Alternatively, other gene mutations, not identified by the next-generation sequencing panel we used, may contribute to this patient's milder phenotype. A couple with history of two fetal losses associated with hydrops fetalis seeked genetic counseling and gave consent to have diagnostic evaluation of genes associated with non-immune hemolytic anemia using targeted next-generation sequencing. Results of the panel revealed a heterozygous frameshift SPTA1 mutation in each of the parents (c.4206delG in the father and c.4180delT in the mother). These mutations in compound heterozygous state in the offspring likely caused total absence of alpha spectrin and fatal hemolytic anemia by the time of birth. Hereditary Spherocytosis is characterized by wide phenotypic variability that will be better understood with studies of genotype-phenotype association. While complete absence of alpha-spectrin expression due to null mutations of both SPTA1 alleles is incompatible with life, a nonsense or splicing SPTA1 mutation in trans to an alphaLEPRA low expression allele causes severe or moderately severe recessive HS, respectively. Targeted next-generation sequencing can be an effective diagnostic tool particularly for patients requiring frequent transfusions that preclude meaningful phenotypical testing of their red blood cells. Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 1. SPTA1 null mutations occurring in trans to alpha-LEPRA causing severe HS Figure 2. Figure 2. Disclosures Begtrup: GeneDx: Employment.

Transcranial Doppler (TCD) screening in children with sickle cell anemia (SCA) identifies abnormally elevated cerebral artery flow velocities that confer an elevated risk for primary stroke. Chronic transfusions offer effective stroke prophylaxis in this setting, but must be continued indefinitely and lead to transfusional iron overload. An alternative treatment strategy that offers similar effective protection against primary stroke, and provides control of iron overload, is needed. TCD With Transfusions Changing to Hydroxyurea (TWiTCH, NCT01425307) was an NHLBI-funded Phase III multicenter randomized clinical trial comparing 24-months of standard treatment (transfusions) to alternative treatment (hydroxyurea) in children with SCA and abnormal TCD velocities. All eligible children had received at least 12 months of transfusions. TWiTCH had a non-inferiority trial design; the primary study endpoint was the 24-month TCD velocity obtained from a linear mixed model, controlling for baseline (enrollment) values, with a non-inferiority margin of 15 cm/sec. The transfusion arm maintained children at HbS 240 cm/sec. Exit brain MRI/MRA exams documented no new parenchymal abnormalities but one child (transfusion arm) developed new vasculopathy. Sickle cell related serious adverse events were more common in the hydroxyurea arm than the transfusion arm (23 to 15), but none was related to study treatment or study procedures. Iron overload improved more in the hydroxyurea arm than in the transfusion arm, with a greater average change in serum ferritin (-1085 compared to -38 ng/mL, p Disclosures Ware: Eli Lilly: Other: DSMB membership; Bayer Pharmaceuticals: Consultancy; Bristol Myers Squibb: Research Funding; Biomedomics: Research Funding. Off Label Use: Hydroxyurea for children with SCA. Owen:Novartis: Speakers Bureau. Rogers:BioRad Labs: Consultancy; Apopharma: Consultancy; Baxter: Consultancy; Glaxo Smith Kline: Consultancy. Kwiatkowski:Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy; Sideris Pharmaceuticals: Consultancy; Novartis: Research Funding; ISIS: Membership on an entity's Board of Directors or advisory committees. Heeney:Sancillio: Consultancy; Eli Lilly: Research Funding. Nottage:Janssen Pharmaceuticals: Employment. Cohen:Novartis: Consultancy; ApoPharma: Other: DSMB member.

Hereditary elliptocytosis (HE) results from defects in the erythrocyte membrane proteins α-spectrin, β-spectrin or protein 4.1R that weaken the ÒhorizontalÓ cytoskeletal associations causing decreased mechanical stability and deformability of the red blood cell (RBC). The hallmark of HE is the presence of elliptical RBCs on peripheral blood smears. Diseases within the spectrum of HE include classic HE and hereditary pyropoikilocytosis (HPP). The traditional diagnosis of HE relies on visualization of elliptocytes on the blood smear, while HPP is characterized by significant poikilocytosis and abnormally shaped RBCs. In vitro assessment of RBC membrane biomechanical properties using ektacytometry is characteristic. However, this evaluation requires a pure RBC population, which may not be available in patients requiring frequent transfusions and does not predict disease course or severity. Using a Next-Generation sequencing panel for 12 genes associated with RBC membrane disorders, we identified the causative genetic mutations in 8 patients with clinically diagnosed or suspected HE or HPP (3 with HE and 5 with HPP). We correlated the identified genetic mutations with clinical presentation, blood smear findings, and ektacytometry results. HE patients: 2 patients with HE had heterozygous mutations in SPTA1 (p.L154_L155insL) resulting in spectrin self-association defect, while the third one had a novel nonsense heterozygous mutation in EPB41 (p.R262*) resulting in defective horizontal interaction between spectrin and actin in the RBC cytoskeleton due to decreased band 4.1R. The same novel mutation was identified in the patient's mother. Both, the patient and the mother, had smear findings and ektacytometry profiles consistent with the diagnosis of HE (Fig. 1A). HPP patients: 3 out of 5 HPP patients were found to be compound heterozygous for a SPTA1 mutation at the self-association site (p.R28H or p.L154_L155insL) and a low-expression SPTA1 allele (c.6531-12C>T known as α-LELY or Low Expression LYon) which allows for the expanded expression of the HE-causing mutation located in trans, resulting in a more severe phenotype. Of note, two siblings carried the same SPTA1 mutation (p.L154_L155insL); one was heterozygous and had mild HE while the other sibling carried α-LELY in trans to the HE-mutation causing a more severe anemia with poikilocytosis and worsened deformability consistent with HPP (Fig. 1B). The ektacytometry profile of these two siblingsÕ RBCs demonstrates the role of α-LELY in modifying the phenotype of elliptocytic disorders. A fourth patient with HPP was found to have compound heterozygosity for mutations in SPTA1 (p.L154_L155insL) and SPTB (p.W2024R) both in the self association sites between α- and β-spectrin. The fifth patient was a 3 y.o. boy with transfusion-dependent hemolytic anemia since infancy and therefore his erythrocyte phenotype could not be evaluated. He was homozygous for a novel mutation in SPTB (p.F2014V); both of his parents were heterozygous for the same mutation. The parents were asymptomatic but their ektacytometry profiles and blood smears were consistent with HE (Fig. 1C), indicating that this mutation in the b-spectrin dimerization domain alters the α- and β-spectrin interaction causing HE in heterozygous state and HPP in the homozygous state. Next-Generation sequencing for genetic diagnosis in HE and HPP can confirm or identify the diagnosis and may be helpful to predict the phenotype and clinical severity in these heterogeneous disorders. Combining clinical data, ektacytometry and family studies is important to understand the relevance of new genetic variants to pathology of RBC cytoskeletal disorders, provide insight into the genotype-phenotype correlation, and improve the genetic counseling and clinical care for these disorders. Figure1. Ektacytometry profiles of : A) a patient and his mother heterozygous for a novel nonsense mutation in EPB41 (p.R262*) causing HE, B) a patient with SPTA1 mutation (p.L154_L155insL) and α-LELY causing HPP compared to a sibling with the same heterozygous mutation in SPTA1 without α-LELY causing HE, C) novel mutation in SPTB (p.F2014V) causing mild HE to heterozygous parents but severe transfusion-dependent anemia in the homozygous child. Ektacytometry was not performed in the child since he was chronically transfused. Figure 1. Figure 1. Disclosures No relevant conflicts of interest to declare.

Red blood cell pyruvate kinase deficiency (PKD) is the most frequent enzyme abnormality of the glycolytic pathway and the most common cause of hereditary nonspherocytic hemolytic anemia. Its incidence has been estimated to be 1 in 20,000, especially in regions where consanguinity is common. It is inherited in autosomal recessive manner occurring as result of homozygous or compound heterozygous mutations affecting both alleles in PKLR gene. Over 200 mutations have been described in patients with PKD causing various severity of hemolysis. Most cases are due to missense mutations, but small deletions, insertions, splice site alterations, frame shifts, disruption of erythroid specific promoters, and nonsense mutations have been reported. Alu elements are the most abundant mobile DNA sequences in the human genome, contributing to almost 11% of its mass. These mobile elements have contributed significantly to evolution and human diversity by causing gene rearrangements. Alu insertions particularly have been associated with a number of human diseases either by disrupting a coding region or a splice signal. In this abstract, we report an Alu-element insertion in the coding region of PKLR gene as a novel cause of PKD causing severe hereditary nonspherocytic hemolytic anemia. The proband was a 16-month-old middle eastern male born to consanguineous parents. He was diagnosed with severe chronic hemolytic anemia and neonatal jaundice requiring regular blood transfusion since birth. There were no known family members with anemia requiring transfusions. The patient's RBC phenotypic analysis was challenging since most of the circulating RBCs were donor cells. Using a Next-Generation sequencing panel for 27 hemolytic anemia associated genes, we identified and characterized a novel homozygous insertional mutation in exon 6 of PKLR gene along with heterozygosity for ANK1 c.1404+15C>T variant. The mutation in exon 6 was identified as Alu element insertion. The inserted Alu element (~370 bp in size), belonging to the youngest Yb8 subfamily, disrupts the reading frame at isoleucine 314 in exon 6 of PKLR gene, leading to a premature stop codon within the inserted sequence. Quantitative reverse transcription-PCR (qRT-PCR) in the patient's reticulocyte RNA using primers for the sequence before the Alu insertion revealed a transcript present but decreased by 80% compared to the average of controls' reticulocyte RNA. This truncated transcript even if translated is expected to cause severe enzyme deficiency. Both parents had a normal hemoglobin level with mild reticulocytosis. The mother had a PK enzyme activity of 4.5 units/g Hb (normal range 6.7-14.3 units/g Hb) indicating that she is heterozygous for PKD. Paternal PK activity level was not determined due to insufficient sample. Carrier testing for mutation in PKLR gene in both parents is currently in process at the time of this submission. The variant in ANK1 gene was predicted to be damaging by affecting the splice site in ankyrin gene. However the father was also heterozygous for this variant and his ektacytometry was normal suggesting a benign nature of ANK1 c.1404+15C>T. This case represents, to our knowledge, the first report of a pathogenic PKLR mutation due to an Alu element insertion and demonstrates a novel mechanism causing severe hereditary nonspherocytic hemolytic anemia. This report also illustrates the challenge of diagnosing congenital hemolytic anemias in chronically transfused patients. The patients often present with a complex clinical picture and ambiguous laboratory findings and may have several potentially damaging genetics variants identified by next generation sequencing. A critical evaluation of the clinical symptoms, laboratory findings and genetic data from other family members and bioinformatics analysis are required to clarify the contribution of these variants and to arrive at an accurate diagnosis, which will guide the institution of targeted interventions and appropriate genetic counseling. Disclosures No relevant conflicts of interest to declare.

Tubulointerstitial nephritis (TIN) antigen has been recently identified as a novel basement membrane macromolecule. It consists of a single chain of 58 kDa and exhibits a restricted distribution. The interaction between TIN antigen and laminin or type IV collagen has been studied using solid-phase binding assays and found to be for both macromolecules specific, saturable, and with an affinity in the low micromolar range. In similar assays, TIN antigen did not interact with heparin. In turbidimetry assays, it was found that the presence of TIN antigen did not affect the polymerization of type IV collagen but had a concentration-dependent inhibitory effect on laminin polymerization and on preformed laminin polymers. TIN antigen was able to promote adhesion of epithelial cells derived from kidney tubules and of endothelial cells derived from aorta. The data suggest that TIN antigen may be a macromolecule of importance both for basement membrane ultrastructure and cellular adhesion.

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1–7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.

Respiratory infections are associated with clinically significant illness in patients with sickle cell disease (SCD). The 2009 H1N1 pandemic was perceived as a significant threat to this population.We undertook a chart review of all patients with SCD followed at our institution to identify those with confirmed H1N1 infection. Further chart and laboratory data was collected on affected patients to analyze clinical courses and the factors that correlated with disease severity.Approximately half of the patients with confirmed H1N1 infection were managed successfully on an outpatient basis with oseltamivir therapy. Among the patients admitted, the most common diagnosis was acute chest syndrome (ACS). Most admitted patients had uncomplicated clinical courses, with a median length of admission of 3 days and no mortality or requirement for mechanical ventilation. A past history of ACS or reactive airway disease correlated with a higher rate of admission and of ACS incidence during the acute illness. Chronic transfusion therapy or hydroxyurea therapy with high hemoglobin F levels had a strong inverse correlation with incidence of ACS.Our results indicate that that in general the impact of the H1N1 influenza pandemic on patients with SCD was mild but that past clinical history correlated with the severity of illness. Additionally, effective hydroxyurea therapy and chronic transfusion therapy appeared to be protective against the incidence of ACS. Our results suggest guidelines for the management of patients with SCD during future influenza pandemics as well as during seasonal influenza epidemics.

The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells. We have demonstrated that deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin at Ser-724, (corresponding to Ser-726 in human erythrocytes), a domain target of protein kinase C (PKC). PKC phosphorylates adducin and leads to decreased F-actin capping and dissociation of spectrin from actin, implicating a significant role of such phosphorylation in cytoskeletal remodeling. We evaluated adducin phosphorylation in erythrocytes from patients with sickle cell disease and found it consistently increased at Ser-726. In addition, ROS concentration is elevated in sickle erythrocytes by 150-250% compared to erythrocytes from normal control individuals. Here, we review previous studies demonstrating that altered phosphorylation of erythrocyte cytoskeletal proteins and increased ROS production result in disruption of cytoskeleton stability in healthy and sickle cell erythrocytes. We discuss in particular the known and potential roles of protein kinase C and the Rac GTPases in these two processes.

Background The small Rho GTPases Rac1 and Rac2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated.Design and Methods Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background, in mouse hematopoietic cells in vivo. The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry.Results Erythropoiesis in Rac1−/−;Rac2−/− mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and Rac2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen.Conclusions These data suggest that Rac1 and Rac2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.

Restricting transgene expression to maturing erythroid cells can reduce the risk for activating oncogenes in hematopoietic stem cells (HSCs) and their progeny, yet take advantage of their robust protein synthesis machinery for high-level protein production. This study sought to evaluate the feasibility and efficacy of reprogramming erythroid cells for production of a lysosomal enzyme, α-L-iduronidase (IDUA). An erythroid-specific hybrid promoter provided inducible IDUA expression and release during in vitro erythroid differentiation in murine erythroleukemia cells, resulting in phenotypical cross-correction in an enzyme-deficient lymphoblastoid cell line derived from patients with mucopolysaccharidosis type I (MPS I). Stable and higher than normal plasma IDUA levels were achieved in vivo in primary and secondary MPS I chimeras for at least 9 months after transplantation of HSCs transduced with the erythroid-specific IDUA-containing lentiviral vector (LV). Moreover, long-term metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in heart, liver, and spleen. Notably, neurological function and brain pathology were significantly improved in MPS I mice by erythroid-derived, higher than normal peripheral IDUA protein. These data demonstrate that late-stage erythroid cells, transduced with a tissue-specific LV, can deliver a lysosomal enzyme continuously at supraphysiological levels to the bloodstream and can correct the disease phenotype in both viscera and CNS of MPS I mice. This approach provides a paradigm for the utilization of RBC precursors as a depot for efficient and potentially safer systemic delivery of nonsecreted proteins by ex vivo HSC gene transfer.

Erythrocyte production is regulated by balancing precursor cell apoptosis and survival signaling. Previously, we found that BH3-only proapoptotic factor, Nix, opposed erythroblast-survival signaling by erythropoietin-induced Bcl-xl during normal erythrocyte formation. Since erythropoietin treatment of human anemia has limitations, we explored the therapeutic potential of abrogating Nix-mediated erythroblast apoptosis to enhance erythrocyte production. Nix gene ablation blunted the phenylhydrazine-induced fall in blood count, enhanced hematocrit recovery, and reduced erythroblast apoptosis, despite lower endogenous erythropoietin levels. Similar to erythropoietin, Nix ablation increased early splenic erythroblasts and circulating reticulocytes, while maintaining a pool of mature erythroblasts as erythropoietic reserve. Erythrocytes in Nix-deficient mice showed morphological abnormalities, suggesting that apoptosis during erythropoiesis not only controls red blood cell number, but also serves a "triage" function, preferentially eliminating abnormal erythrocytes. These results support the concept of targeting erythroblast apoptosis to maximize erythrocyte production in acute anemia, which may be of value in erythropoietin resistance.

The retinoblastoma tumor suppressor protein (RB) plays important roles in the control of the cell division cycle. It is estimated that RB is dysfunctional/inactivated in up to 40% of human leukemias. The consequences of loss of RB on hematopoietic stem and progenitor cell (HSPC) function in vivo are incompletely understood. Here, we report that mice genetically deficient in Rb in all hematopoietic cells (Vav-Cre Rb knockout [KO] animals) showed altered contribution of distinct hematopoietic cell lineages to peripheral blood, bone marrow, and spleen; significantly increased extramedullary hematopoiesis in the spleen; and a 2-fold increase in the frequency of hematopoietic progenitor cells in peripheral blood. Upon competitive transplantation, HSPCs from Vav-Cre Rb KO mice contributed with an at least 4- to 6-fold less efficiency to hematopoiesis compared with control cells. HSPCs deficient in Rb presented with impaired cell-cycle exit upon stress-induced proliferation, which correlated with impaired function. In summary, Rb is critical for hematopoietic stem and progenitor cell function, localization, and differentiation.

Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. Rac1 and Rac2 GTPases regulate actin structures and have multiple overlapping as well as distinct roles in hematopoietic cells; therefore, we studied their role in red blood cells (RBCs). Conditional gene targeting with a loxP-flanked Rac1 gene allowed Crerecombinase–induced deletion of Rac1 on a Rac2 null genetic background. The Rac1–/–;Rac2–/– mice developed microcytic anemia with a hemoglobin drop of about 20% and significant anisocytosis and poikilocytosis. Reticulocytes increased more than 2-fold. Rac1–/–;Rac2–/– RBCs stained with rhodamine-phalloidin demonstrated F-actin meshwork gaps and aggregates under confocal microscopy. Transmission electron microscopy of the cytoskeleton demonstrated junctional aggregates and pronounced irregularity of the hexagonal spectrin scaffold. Ektacytometry confirmed that these cytoskeletal changes in Rac1–/–;Rac2–/– erythrocytes were associated with significantly decreased cellular deformability. The composition of the cytoskeletal proteins was altered with an increased actin-to-spectrin ratio and increased phosphorylation (Ser724) of adducin, an F-actin capping protein. Actin and phosphorylated adducin of Rac1–/–;Rac2–/– erythrocytes were more easily extractable by Triton X-100, indicating weaker association to the cytoskeleton. Thus, deficiency of Rac1 and Rac2 GTPases in mice alters actin assembly in RBCs and causes microcytic anemia with reticulocytosis, implicating Rac GTPases as dynamic regulators of the erythrocyte cytoskeleton organization.

The regulation of cell volume is important for the maintenance of integrity in all cells and is especially critical for the highly specialized red blood cell (RBC) which must withstand pressure changes within the vasculature and remain deformable to traverse small vessels. Disorders that interfere with volume homeostasis result in the premature destruction of RBCs. One protein that appears to play a prominent role in RBC hydration is the recently described nonselective cation channel PIEZO1 which is involved in mechanotransduction. Mutations of PIEZO1 have been associated with an autosomal dominant form of hereditary hemolytic anemia (HHA) characterized by RBC dehydration known as hereditary xerocytosis (HX) (Zarychanski et al., Blood 2012;120:1908). There is evidence that PIEZO1 may also be responsible for a channel activity that participates in the dehydration of sickle cells which exacerbates sickling and vaso-occlusive events in patients with Sickle Cell Disease (reviewed in Gallagher, Curr Opin Hematol 2013, 20:201). Using a Next-Generation sequencing panel containing 27 hemolytic anemia associated genes, we identified and characterized a novel PIEZO1 mutation p.L2023V which results in delayed channel inactivation and a dehydrated RBC phenotype. This single amino acid substitution at a highly conserved site was detected in the heterozygous state in a 15 year old Caucasian young man (CQ15) with chronic hemolysis well compensated with reticulocytosis, along with heterozygosity for the SLC4A1 p.E40K mutation known as band 3 Montefiore. Hematologic characteristics included macrocytosis, elevated MCHC, and a smear showing occasional stomatocytes. Several family members also had hemolysis and jaundice and were given a diagnosis of hereditary pyropoikilocytosis prior to our evaluation. The L2023V mutation was considered possibly damaging by the PolyPhen-2 algorithm with a score of 0.777. The mother was heterozygous for the PIEZO1 L2023V mutation while the father carried the band 3 Montefiore mutation. Ektacytometry was used to evaluate RBC deformability; the mother's RBCs had a profile of classic xerocytosis with decreased Omin and Ohyp, the father had a very mild spherocytosis profile, while the patient's RBCs had mixed ektacytometry characteristics with decreased Ohyp and an intermediate Omin (Figure 1A). Intracellular cation values determined by flame emission spectroscopy for CQ15 and his mother demonstrated K+ loss. The mutation p.L2023V is located at a site predicted to be at the border between membrane and cytoplasm in the carboxy terminal part of the protein, similar to the previously described p.R2456H mutation in a kindred with HX (Zarychanski et al., 2012). To enable detailed physiologic study of the L2023V mutation, we prepared an HEK293 cell line with a stable, single copy integrant of the variant with an inducible promoter. Whole cell patch clamp studies were performed on HEK293 cells expressing wild type PIEZO1 and PIEZO1 L2023V. Traces of mechanically activated currents were recorded from the cells and were normalized to peak current (Figure 1B). The inactivation time constant tau (in ms) was determined from mono-exponential fits for wild type and mutant PIEZO1 channels. The difference in average inactivation time between wild type PIEZO1 and PIEZO1 L2023V was highly significant (Student's t-test; p Patients often present with a complex clinical picture and laboratory results and may have combinations of potentially damaging genetic variants identified by Next-Generation sequencing. The examination of clinical, laboratory, and genetic data from family members and, in some cases, in vitro studies are required to clarify the relative contribution of these variants and to arrive at an accurate diagnosis. The data presented here further our understanding of the role of PIEZO1 in RBCs and its potential pathological contribution in HHAs with associated cellular dehydration and may facilitate the future development of therapeutic targets for treatment of these conditions. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Wiskott-Aldrich Syndrome (WAS) is a primary immunodeficiency that is associated with microthrombocytopenia. Autoimmunity is one of the defining characteristics of severe WAS, and can contribute to the decision to proceed to hematopoietic cell transplantation. Autoimmune Hemolytic Anemia (AIHA) is one of the most common autoimmune syndromes found in WAS. Even in the absence of AIHA, some patients with WAS have anemia, and this is often of unclear etiology. We studied 29 patients with WAS, 6 of whom had had splenectomy, on 46 occasions. All patients were found to have anisopoikilocytosis. Common red blood cell (RBC) variants included stomatocytes, xerocytes, eccentrocytes, dacryocytes, spherocytes and target cells. Two WAS knockout mice whose blood smears were studied also had poikilocytosis, with dacryocytes predominating. The average hemoglobin in humans with WAS was 13.3 g/dL. In 19/46 measurements, hemoglobin was below the lower limit of normal for age. LDH was elevated in 1/43 measurements, and bilirubin was elevated in 7 of 46 measurements, 4 measurements being from a patient with molecularly diagnosed Gilbert’s syndrome. Average MCHC was 34.7 g/dL, with MCHC being elevated in 3/43 measurements. Despite these unremarkable screening assays, haptoglobin was decreased in 10/45 measurements and undetectable in 6 of these 10. The average plasma free hemoglobin and plasma free oxyhemoglobin were elevated to 21.4 and 14.7 mg/dL respectively. Analysis of osmotic fragility revealed average numbers near the lower end of the normal range, with fragility in 0.6 g/dL NaCl being abnormally low in 29/35 specimens and fragility in 0.65 g/dL NaCl being abnormally low in 9/35 specimens. A standard Coomb’s test was positive in 6/39 specimens. 4 such assays were positive for IgG, 1 for C3 and 1 for neither molecule despite a positive screening assay. Using enhanced methodology, 1/5 specimens was positive only with polyspecific serum. Overall, immune mediated hemolysis was detected by either method in 6/39 specimens. These data were interpreted to be consistent with a subclinical non-immune hemolysis in 33/41 specimens from 28/29 patients. In order to evaluate these findings further, ektacytometry was performed in 5 specimens from 5 patients and intracellular cations were measured in 4 of these specimens. Ektacytometry revealed Omin of 135.8±5.1 mOsm/kg in patients with WAS and 156.4±5.3 mOsm/kg in control specimens (P=0.0002). Sodium and potassium concentrations in patients were 53.3±12.7 mmol/kg hemoglobin and 282.3±13.1 mmol/kg hemoglobin respectively. In control specimens, the concentrations were 4.4±10.5 mmol/kg hemoglobin and 297.5±14.4 mmol/kg hemoglobin (P=0.3196 and 0.1684). These data are consistent with non-immune hemolysis in most patients with Wiskott-Aldrich syndrome. This hemolysis is associated with a xerocytic ekatacytometry curve, although cation concentrations in the small number of specimens tested only trended to statistical significance. This is the first intrinsic red cell defect described in patients with WAS, emphasizing that this disease truly affects all hematopoietic lineages and not only cells of the immune system. Whether the roles of the WAS protein in actin polymerization or signal transduction are involved in these abnormal functions remains unclear and requires additional research. These findings also have implications for clinical management of WAS as they indicate that hemolysis is commonly present without necessarily defining the autoimmune, severe clinical phenotype in these patients. As autoimmunity is often an important consideration in the decision to proceed to hematopoietic cell transplantation, these data should inform the decision-making process toward this and other therapeutic modalities for WAS. Disclosures No relevant conflicts of interest to declare.

Introduction : Stroke is a major cause of morbidity and mortality in children with sickle cell anemia (SCA), and current efforts seek to prevent primary stroke in this high-risk population through transcranial Doppler (TCD) screening programs and prophylactic blood transfusions for children with abnormally high intracranial TCD velocities. During the SWITCH trial (ClinicalTrials.gov NCT00122980), a novel MRA vasculopathy grading (VOG) scale was developed, which documented baseline bilateral vessel stenosis and parenchymal injury in children receiving transfusions for secondary stroke prophylaxis, and provided a severity scale suitable for future clinical trials. We now describe the baseline brain magnetic resonance imaging/angiography (MRI/MRA) results and vasculopathy grading scores in children with SCA on primary stroke prophylaxis, and report correlations with intracranial TCD velocities and duration of transfusion. Methods: TCD With Transfusions Changing to Hydroxyurea (TWiTCH) is a Phase three randomized, controlled, multicenter clinical trial (ClinicalTrials.gov NCT01425307), comparing transfusions versus hydroxyurea for children with abnormal TCD velocities. Children were eligible for TWiTCH enrollment if they currently receive monthly transfusions for primary stroke prophylaxis, and had no known history of stroke, transient ischemic attack, or severe vasculopathy. All TWiTCH participants underwent baseline MRI/MRA examinations with central review after study enrollment. Baseline TCD examinations were also centrally reviewed and Time-Averaged Mean Velocities (TAMV) were recorded for the distal internal carotid artery (dICA), internal carotid bifurcation (BIF), first segment middle cerebral artery (MCA) = M1, mid MCA (MCA), and posterior cerebral artery (PCA). MRA vasculopathy grades were recorded bilaterally and compared to the TCD velocities and duration of transfusion. Results: Of 159 enrolled subjects, 143 had complete baseline MRI/MRA data. The mean age at study enrollment was 9.8 years, with a 2:3 male:female ratio, and mean age at the original abnormal (Index) TCD exam was 5.4 years. A total of 62 children had parenchymal abnormalities identified on baseline brain MRI, including 2 with infarcts, 54 with leukoencephalopathy, 5 with encephalomalacia, and 20 with lacunae lesions (not mutually exclusive). A total of 124 children had baseline MRA vasculopathy scores ≤3, while 19 had grades 4-6 (severe vasculopathy) that required removal from the study. There were significant positive associations between the baseline hemisphere vasculopathy grades and the corresponding TAMV velocities in the Left dICA (p=0.02), Left BIF (p˂.0001), Left MCA (p=.0002), Right BIF (p=.0003) and Right MCA (p=.0002). Likewise, when TCD velocities were categorized as low ( Conclusions: Since the TWiTCH study excluded the enrollment of children on chronic transfusion therapy for primary stroke prevention with a known history of stroke, TIA, or severe vasculopathy, the prevalence of baseline brain MRI and MRA abnormalities was relatively higher than expected and 19 participants failed screening due to unrecognized severe vessel stenosis. The recent published SWiTCH vasculopathy grading scale was validated in the TWiTCH population, with more severe stenosis found to be associated with higher ipsilateral TCD velocities. Exit brain MRI/MRA exams will be compared to entry studies, to provide longitudinal data including comparisons between the effects of continued transfusions and hydroxyurea. Disclosures No relevant conflicts of interest to declare.

To delineate the role of specific members of β1 integrins in stress erythropoiesis in the adult, we compared the response to phenylhydrazine stress in 3 genetically deficient models. The survival of β1-conditionally deficient mice after phenylhydrazine is severely compromised because of their inability to mount a successful life saving splenic erythroid response, a phenotype reproduced in β1Δ/Δ reconstituted animals. The response of bone marrow to phenylhydrazine-induced stress was, unlike that of spleen, appropriate in terms of progenitor cell expansion and mobilization to peripheral blood although late differentiation defects qualitatively similar to those in spleen were present in bone marrow. In contrast to β1-deficient mice, α4Δ/Δ mice showed only a kinetic delay in recovery and similar to β1Δ/Δ, terminal maturation defects in both bone marrow and spleen, which were not present in VCAM-1Δ/Δ mice. Convergence of information from these comparative studies lends new insight to the distinct in vivo roles of α4 and α5 integrins in erythroid stress, suggesting that the presence of mainly α5β1 integrin in all hematopoietic progenitor cells interacting with splenic microenvironmental ligands/cells is instrumental for their survival and accumulation during hemolytic stress, whereas presence of α4, or of both α5 and α4, is important for completion of terminal maturation steps.

Hereditary hemolytic anemia is caused by defects in hemoglobin, in the red blood cell (RBC) cytoskeleton proteins, or by deficiencies in RBC enzymes. RBC cytoskeletal disorders include hereditary spherocytosis (HS), elliptocytosis (HE), pyropoikilocytosis (HPP), and stomatocytosis (HSt), which are typically inherited as autosomal dominant disorders but can also present as recessive forms, frequently severe. The RBC enzymopathies, such as glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) deficiency, are X-linked or recessively inherited disorders causing hemolytic anemia. Although rare, congenital dyserythropoietic anemias (CDA) are inherited red cell lineage disorders which occasionally, especially CDA-II, can be misdiagnosed as HS. Diagnosis of an inherited hemolytic anemia not due to a hemoglobin disorder is based upon morphology of the RBCs, functional analysis of the RBCs through ektacytometry or osmotic fragility, and enzymatic assays. The diagnosis of severely affected patients is complicated by transfusion dependence. Diagnosis based on genetic mutations is attractive, especially in transfused patients, and provides additional insight into the mechanisms of disease. Despite these advantages, genetic diagnoses have been limited by expense and long turn-around times for clinical results. To address these issues, we have developed a rapid comprehensive clinical next-generation sequencing-based assay that evaluates 27 genes with published disease-causing mutations for RBC cytoskeletal disorders, enzymopathies, and CDAs. The protein-coding exons plus 25 bases of exon/intron junction as well as promoter sequences with known relationship to clinical phenotypes were included in the design. Genomic DNA was digested with a panel of 8 restriction enzymes and oligonucleotide probes were used to enrich the target regions. Enriched samples were then sequenced on an Illumina MiSeq benchtop sequencer with 150 base pair, paired-end reads. Enrichment and sequencing were completed within 48 hours. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using NextGENe software. Initial validation included 5 affected probands, 1 affected sibling of a proband, 4 parental samples, and 2 unrelated control individuals with no history of hemolytic anemia. Overall, > 99% of all nucleotides in the regions of interest had at least 20X sequencing coverage. Our assay confirmed a previously identified maternally inherited nonsense mutation, Y1089X, and a paternally inherited A970D missense variant in SPTA1 in a patient (SPCA) with severe HS. The A970D SPTA1 missense change was also seen in two unaffected parents and an unaffected control, further validating that this missense variant alone does not cause dominant HS. The common SPTA1 allele, αLELY was seen in the clinically unaffected mother of SPCA, but was not found in the affected child, suggesting that this allele must be inherited in trans with a pathologic SPTA1 mutation to have clinical effect. Analysis of additional probands revealed both dominant and recessive forms of HS due to mutations in ANK1. Patient AAHS2 presented with typical dominant HS, and was found to have a novel ANK1 frameshift mutation, c.3464delG. Patient HEEM presented with severe transfusion-dependent anemia and was found to have one reported ANK1 missense mutation, T1075I (ankyrin Tubarao) in the spectrin-binding domain, and one novel nonsense mutation, Y735X. In addition, we identified novel amino acid changes in the newly identified dehydrated HSt gene PIEZO1. HEGR, a patient with hemolytic anemia, splenomegaly and portal vein thrombosis in infancy was found to have a novel 6 base pair insertion at R1462 in PIEZO1. Thal1 was previously diagnosed with dominant β-thalassemia, however had a more severe presentation than expected. We found a PIEZO1missense mutation R2302H in Thal1 that likely explains the severe phenotype. The initial validation of this comprehensive sequencing assay has demonstrated that this is a robust and rapid diagnostic tool for patients with severe hereditary hemolytic anemia. Simultaneous investigation of the key protein-coding genes involved in proper RBC function and survival, provides new insight into the variable phenotypes of patients with hemolytic anemia and ultimately will improve the management of patients with severe disease. Disclosures: No relevant conflicts of interest to declare.

RhoA GTPase is known to regulate cell adhesion, actomyosin cytoskeleton, and cytokinesis. Specifically, it contributes to cytokinesis in sea urchin embryos, Xenopus and HeLa cells by associating with the microtubule ends at the abscission area marking the position for the daughter cell separation and by activating myosin through phosphorylation of the myosin regulatory light chain (MRLC) (Piekny et al, Trends in Cell Biology 2005). Erythroblast enucleation, the process through which orthochromatic erythroblasts expel their nucleus during the final stage of mammalian erythropoiesis, is a multi-step process resembling asymmetric cytokinesis (reviewed by Li, Developmental Cell 2013). It requires establishment of cell polarity through microtubule function, followed by formation of a contractile actomyosin ring between reticulocyte and pyrenocyte (Konstantinidis et al, Blood 2012). To define the mechanistic contribution of RhoA signaling in terminal erythroid maturation, we generated mice with erythroid specific RhoA deletion (EpoR-GFPcre-driven). Erythroid-specific deletion of RhoA, confirmed in the CD71-positive cells by PCR and immunoblotting, caused severe anemia, fatal in utero by E15.5. RhoA-deficient peripheral primitive blood cells were large with significant poikilocytosis and anisocytosis, frequently binuclear or multinuclear with polyploidy of the genetic material as demonstrated by flow cytometry with propidium iodide, and with incomplete clearance of the Golgi network and mitochondria. RhoAΔ/Δ fetal livers had progressively decreased cellularity. The erythroblast populations as determined by CD71-Ter119-FSC flow cytometry as well as by multispectral high-speed cell imaging in flow, demonstrated progressive decline and significantly decreased reticulocyte production (n=3, p Thus, RhoA GTPase regulates erythroblast differentiation and enucleation by dynamic coordination of the microtubule-actomyosin machineries to achieve successful abscission. In addition, RhoA may also regulate erythroblast adhesive interactions with the central macrophage in erythroblastic islands and intracellular vesicular transport. Our results reveal novel molecular components and cellular processes in erythropoiesis that may be important for improving the efficiency of red blood cell production in vitro. Disclosures: No relevant conflicts of interest to declare.

Abstract 3198 Background: Red blood cell (RBC) extravasation contributes to the growth of atherosclerotic plaque, and is associated with plaque rupture. High Fat Diet (HFD) fuels systemic inflammation by promoting monocyte activation and their recruitment/transendothelial migration. RBC bind chemokines such as monocyte chemotactic protein-1 (MCP-1) via Duffy Antigen Receptor for Chemokines (DARC). While it was previously reported that HFD may impact RBC cholesterol content, very little is known about the impact – if any – of HFD on RBC function(s). Hence, we studied whether HFD affects biochemical and functional properties of RBC in ways potentially relevant to the progression of atherosclerosis. Methods and Results: Wild type (WT) C56BL6/J mice were fed either chow (10% total fat) or HFD (60% total fat) for 312 weeks. MCP-1 levels were measured in plasma before and after the release of DARC-bound MCP-1 by heparin. Released MCP-1 was 1.5 fold higher in HFD RBC (12 weeks on diet, n=3) compared to chow RBC. Levels of intracellular reactive oxygen species (ROS) were measured by DCFH fluorescence using flow cytometry, and were increased in HFD RBC (17 weeks on diet, n=3) by ∼1.2 fold compared to chow RBC (p Conclusions: We here show that HFD elicits an increase in surface levels of MCP-1 in RBC, which is associated with an enhanced macrophage adhesion to the intact vascular endothelium and increased transendothelial migration in vitro. Further, HFD increases ROS levels and PS externalization in RBC, and HFD RBC exhibit an enhanced splenic uptake in vivo. These effects of HFD on circulating RBC are likely to promote chronic inflammation, rendering RBC a heretofore unknown contributor to local as well as systemic changes that lead to atherosclerosis. Disclosures: No relevant conflicts of interest to declare.

Abstract 976 Introduction: Erythrocyte cytoskeleton disorders, a common cause of hereditary hemolytic anemia, consist of a genetically and phenotypically variable group of diseases that include hereditary spherocytosis (HS), elliptocytosis (HE), pyropoikilocytosis (HPP), and stomatocytosis (HSt) syndromes. The diagnosis is most commonly based on the morphology of the red blood cells (RBCs) on the blood smear and on ektacytometry and osmotic fragility studies. However, in infants and children with transfusion-dependent hemolytic anemia these studies are challenging to obtain since the patient has mostly transfused RBCs. Molecular diagnosis has not been easily attainable so far due to the number and large size of the genes involved in pathogenesis and due to the fact that each kindred has frequently a private mutation in the responsible gene(s) (Gallagher, Hematology, 2005). Methods and Results: We have developed a high-throughput assay for the diagnosis of known and discovery of new genetic mutations causing erythrocyte cytoskeleton disorders. The complete exon sequences of 24 genes encoding cytoskeleton proteins (spectrin a-chain (SPTA1), spectrin b-chain (SPTB), ankyrin 1 (ANK1), band 3 (SLC4A1), protein 4.1 (EPB41), protein 4.2 (EPB42), adducins (ADD), dematin (EPB49), tropomyosins (TPM), tropomodulins (TMOD), Rh-associated glycoprotein (RhAG), erythrocyte protein p55 (MPP1), stomatin (EPB72), Glut1-glucose transporter (SLC2A1), and K-Cl-cotransporter (SLC12A4, SLC12A6, SLC12A7) were determined using a Next Generation Sequencing technology. Analysis was performed on eleven patients diagnosed with erythrocyte cytoskeleton disorder along with a negative control. Informed consent was obtained from all subjects under an Institutional Review Board-approved protocol. The RainDance Technologies RDT1000 instrument was used for target enrichment covering the exons, 20 bases of exon/intron junctions, and 500 bases up and downstream of the 24 genes of interest. The products were then sequenced on an Illumina HiSeq2000 system. Bioinformatic analysis was performed in a blinded fashion as to the disease characteristics and inheritance mode of the patient using the GATK software package from the Broad Institute (DePristo et al, Nature Genetics, 2011). Mutations predicted to have significant impact to the corresponding protein structure and therefore likely to cause disease were identified in 9 out of the 11 patients ( Table 1 ). Shared pathogenic mutation(s) were identified blindly in family members, e.g. siblings HS-S1 and HS-S2, or parents sHS-M, sHS-F with child sHS. Two EPB49 mutations predicted to impair gene function were identified in an infant with clinical picture of HPP, implicating dematin in HPP pathogenesis. Immunoblotting of RBC membranes from this patient demonstrated decreased dematin. Conclusions: Next generation sequencing for the genetically variable erythrocyte cytoskeleton disorders can provide a cost-effective and faster patient diagnosis compared with a gene-by-gene approach and it is a feasible diagnostic method in a transfusion-dependent child. Moreover, a precise genetic diagnosis can facilitate natural history studies to understand genotype-phenotype correlations in the erythrocyte cytoskeleton disorders and offer valuable insights into the structure-function relationship of the erythrocyte cytoskeleton proteins. Table 1 . Characteristics of patients with erythrocyte cytoskeleton disorder tested and corresponding gene mutation(s) identified. # Subject Phenotype Gene Mutation Comment 1 HS-S1 Mild HS SPTB Q1946* Sibling of HS-S2 2 HS-S2 Mild HS SPTB Q1946* 3 sHS Severe HS SPTA1 SPTB I2262T R1972Q 4 sHS-M Asymptomatic SPTA1 I2262T Mother of sHS 5 s-HS-F Asymptomatic SPTB R1972Q Father of sHS 6 TDHA Transfusion-dependent hemolytic anemia SPTA1 SPTA1 L1432* A970D No family history 7 HS-3 Mild HS SLC4A1 E473Gfs*38 Father with HS 8 HS-4 Moderate HS SPTA1 c.1351-1G>T Father with HS 9 HPP Neonate with HPP, transfusion requirement resolved after 4 months of age EPB49 R19Q and K170R No family history 10 HS-5 Moderate chronic hemolysis None found No family history 11 HE-1 Severe hemolytic anemia, compensated after 18 months of age None found Mother with mild HE Disclosures: No relevant conflicts of interest to declare.

Diabetic retinopathy is characterized by microaneyrisms derived from vessels which have selectively lost their pericytes and demonstrate focal proliferation of endothelial cells. The proliferation of retinal endothelial cells and the pericytic degeneration followed by replacement with pericytic “ghosts” is an almost invariable and early feature of diabetic retinopathy 1.

Abstract 1634 Chronic inflammation has emerged as an important pathogenic mechanism in sickle cell disease. One component of this inflammatory response is oxidant stress mediated by reactive oxygen species (ROS) generated by leukocytes, endothelial cells, plasma enzymes, and sickle erythrocytes. Sickle RBC ROS generation has been attributed to sickle hemoglobin auto-oxidation and Fenton chemistry reactions catalyzed by denatured heme moieties bound to the RBC membrane. The potential role of enzymatic mechanisms in ROS production in RBCs has not been fully explored. One candidate for enzyme-mediated ROS production in SS RBCs is NADPH oxidase, which is activated by the small Rho GTPases Rac1 and Rac2 in a variety of cell types (Hordijk P.L. Circ. Res. 2006; 98:453-462). Using flow cytometry with 5-(and 6-)-chloromethyl-2`,7`-dichlorodihydrofluorescein diacetate, a peroxide-sensitive probe, we determined that ROS generation is elevated in HbSS RBCs by 150–250% relative to that in HbAA RBCs. The NADPH oxidase NOX subunit homologs NOX1 and NOX5 were expressed in erythrocytes, and treatment of SS RBCs with the NADPH oxidase inhibitor diphenyleneiodonium (DPI) reduced ROS generation in a dose-dependent manner. Inhibition of PKC or Rac activity by the small molecule-inhibitors calphostin and NSC23766, respectively, also resulted in decreased ROS production in HbSS-RBCs in a dose-dependent fashion, while PKC activation by phorbol 12-myristate 13-acetate (PMA) increased ROS production. This effect of PMA could be inhibited by parallel treatment with NSC23766, implicating a PKC-Rac axis in erythrocyte ROS production. Enzymatic ROS production in sickle RBCs was dependent on the availability of free intracellular calcium, since it was inhibited by BAPTA-AM, a cell-permeable calcium chelator. Moreover, we found that ROS generation in SS RBCs was modulated by humoral factors. Incubation of AA RBCs in blood type-matched SS patient plasma resulted in increased ROS generation, while the incubation of SS RBCs in AA plasma decreased ROS production. Immunoblotting of AA and SS RBC membranes with specific antibodies revealed receptors for the inflammatory signaling molecules TGFβ1 and Endothelin-1 (ET-1), both of which are present in elevated levels in the plasma of patients with SCD. Incubation of AA RBCs with TGFβ1 or ET-1 resulted in increased Rac activation and increased ROS production in these treated cells. Our results suggest that ROS production in sickle RBCs is mediated by NADPH oxidase through Ca2+-regulated PKC and Rac signals, which in turn are modulated by plasma TGFβ1 and ET-1 via their receptors. ROS-mediated damage to RBC membrane components is known to contribute to erythrocyte deformation and fragility in sickle cell disease. Erythrocyte ROS generation, RBC lysis, vaso-occlusion, and the inflammatory response to tissue damage may therefore act in a positive feedback loop to drive the pathophysiology of sickle cell disease. These data may offer new therapeutic targets to counteract inflammation and RBC fragility and deformation in sickle cell disease. Disclosures: No relevant conflicts of interest to declare.

Abstract 901 Although sickle hemoglobin (HbSS) polymerization in hypoxic conditions is the trigger for the sickling deformation of red blood cells (RBC) containing HbSS, subsequent changes in the cytoskeleton and the mechanisms by which reversibly sickled cells become irreversibly malformed have not been fully elucidated. Actin oligomers are a significant structural component of the erythrocyte cytoskeleton. The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells (Schwartz.M., J.Cell Science 2004). Deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin (an F-actin capping protein) at Ser-726, a domain-target of protein kinase C (PKC) (Kalfa et al, Blood 2006). We evaluated the cytoskeleton phosphorylation changes in RBCs from patients with HbSS and from control subjects with hemoglobin AA (HbAA) by immunoblotting with phospho-specific antibodies. We found that phosphorylation of adducin at Ser-726 and of band 4.1 at Ser-331 were significantly increased in the erythrocyte cytoskeleton of HbSS-RBCs. This was associated with elevated PKC activity in these cells, compared with normal RBCs. ROS concentration was also elevated in HbSS-RBCs by 150-250% compared to that in HbAA-RBC, as determined by flow cytometry using 5-(and 6-)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, a peroxide-sensitive probe. Inhibition of PKC or Rac activity in HbSS-RBCs by small molecule-inhibitors, calphostin and NSC23766 respectively, resulted in decreased ROS production in a dose-dependent fashion, while PKC activation by phorbol 12-myristate 13-acetate (PMA) increased ROS production, implicating a PKC-Rac axis in erythrocyte ROS production. Additionally, Rac inhibition resulted in dose-dependent increase of adducin phosphorylation while PKC inhibition had the opposite effect. Preincubation of HbSS RBCs with calphostin appeared to decrease the subsequent susceptibility of these cells to sickling under hypoxic conditions, suggesting a role for PKC activity in the transition from normal to sickle morphology, likely through a combination of effects on volume regulation and membrane stability of the cells. Our results imply that aberrant cytoskeleton protein phosphorylation and ROS production in HbSS-RBCs, mediated by signaling pathways involving PKC and Rac GTPases, may contribute to membrane changes induced by cell sickling. Elucidation of these pathways may identify new therapeutic targets in sickle cell disease. Disclosures: No relevant conflicts of interest to declare.