Pathologically stiff erythrocytes impede contraction of blood clots.

BACKGROUND: Blood clot contraction, volume shrinkage of the clot, is driven by platelet contraction and accompanied by compaction of the erythrocytes and their gradual shape change from biconcave to polyhedral, with the resulting cells named polyhedrocytes. OBJECTIVES: Here, we examined the role of erythrocyte rigidity on clot contraction and erythrocyte shape transformation. METHODS: We used an optical tracking methodology that allowed us to quantify changes in contracting clot size over time. RESULTS AND CONCLUSIONS: Erythrocyte rigidity has been shown to be increased in sickle cell disease (SCD), and in our experiments erythrocytes from SCD patients were 4-fold stiffer than those from healthy subjects. On average, the final extent of clot contraction was reduced by 53% in the clots from the blood of patients with SCD compared to healthy individuals, and there was significantly less polyhedrocyte formation. To test if this reduction in clot contraction was due to the increase in erythrocyte rigidity, we used stiffening of erythrocytes via chemical cross-linking (glutaraldehyde), rigidifying Wrightb antibodies (Wrb ), and naturally more rigid llama ovalocytes. Results revealed that stiffening erythrocytes result in impaired clot contraction and fewer polyhedrocytes. These results demonstrate the role of erythrocyte rigidity in the contraction of blood clots and suggest that the impaired clot contraction/shrinkage in SCD is due to the reduced erythrocyte deformability, which may be an underappreciated mechanism that aggravates obstructiveness of erythrocyte-rich (micro)thrombi in SCD.

Erythropoietic protoporphyria in an adult with sequential liver and hematopoietic stem cell transplantation: A case report.

Erythropoietic protoporphyria (EPP) is a rare inherited disorder of the heme biosynthesis pathway resulting in the accumulation of protoporphyrins in the blood, erythrocytes, and other tissues. Because of a gene mutation in the FECH gene, ferrochelatase, the enzyme involved in the final step of heme synthesis, is deficient in these patients. Although the major symptom of this disorder is photosensitivity, rarely, it can cause progressive liver disease requiring liver transplantation (LT). However, LT is not curative and only bone marrow transplantation (BMT) can correct the underlying enzymatic defect. Because liver disease results from accumulation of protoporphyrin in the liver, LT without hematopoietic stem cell transplantation leaves the new liver at risk for similar EPP-related damage. A handful of pediatric patients undergoing sequential LT and stem cell transplantation have been described in the literature; however, to date none has been described in detail in adults. We report a case of an adult male with EPP and liver failure who successfully underwent a sequential liver and hematopoietic stem cell transplantation (HSCT).

Structural basis for the antipolymer activity of Hb ζ2ßs2 trapped in a tense conformation.

The phenotypical severity of sickle-cell disease (SCD) can be mitigated by modifying mutant hemoglobin S (Hb S, Hb α2ßs2) to contain embryonic ζ-globin in place of adult α-globin subunits (Hb ζ2ßs2). Crystallographical analyses of liganded Hb ζζ2ßs2, though, demonstrate a tense (T-state) quaternary structure that paradoxically predicts its participation in--rather than its exclusion from--pathological deoxyHb S polymers. We resolved this structure-function conundrum by examining the effects of α→ζ exchange on the characteristics of specific amino acids that mediate sickle polymer assembly. Superposition analyses of the ßs subunits of T-state deoxyHb α2ßs2 and T-state CO-liganded Hb ζ2ßs2 reveal significant displacements of both mutant ßsVal6 and conserved ß-chain contact residues, predicting weakening of corresponding polymer-stabilizing interactions. Similar comparisons of the α- and ζ-globin subunits implicate four amino acids that are either repositioned or undergo non-conservative substitution, abrogating critical polymer contacts. CO-Hb ζ2ßs2 additionally exhibits a unique trimer-of-heterotetramers crystal packing that is sustained by novel intermolecular interactions involving the pathological ßsVal6, contrasting sharply with the classical double-stranded packing of deoxyHb S. Finally, the unusually large buried solvent-accessible surface area for CO-Hb ζ2ßs2 suggests that it does not co-assemble with deoxyHb S in vivo. In sum, the antipolymer activities of Hb ζ2ßs2 appear to arise from both repositioning and replacement of specific α- and ßs-chain residues, favoring an alternate T-state solution structure that is excluded from pathological deoxyHb S polymers. These data account for the antipolymer activity of Hb ζ2ßs2, and recommend the utility of SCD therapeutics that capitalize on α-globin exchange strategies.

AUF-1 and YB-1 independently regulate ß-globin mRNA in developing erythroid cells through interactions with poly(A)-binding protein.

The normal expression of ß-globin protein in mature erythrocytes is critically dependent on post-transcriptional events in erythroid progenitors that ensure the high stability of ß-globin mRNA. Previous work has revealed that these regulatory processes require AUF-1 and YB-1, two RNA-binding proteins that assemble an mRNP ß-complex on the ß-globin 3'UTR. Here, we demonstrate that the ß-complex organizes during the erythropoietic interval when both ß-globin mRNA and protein accumulate rapidly, implicating the importance of this regulatory mRNP to normal erythroid differentiation. Subsequent functional analyses link ß-complex assembly to the half-life of ß-globin mRNA in vivo, providing a mechanistic basis for this regulatory activity. AUF-1 and YB-1 appear to serve a redundant post-transcriptional function, as both ß-complex assembly and ß-globin mRNA levels are reduced by coordinate depletion of the two factors, and can be restored by independent rescue with either factor alone. Additional studies demonstrate that the ß-complex assembles more efficiently on polyadenylated transcripts, implicating a model in which the ß-complex enhances the binding of PABPC1 to the poly(A) tail, inhibiting mRNA deadenylation and consequently effecting the high half-life of ß-globin transcripts in erythroid progenitors. These data specify a post-transcriptional mechanism through which AUF1 and YB1 contribute to the normal development of erythropoietic cells, as well as to non-hematopoietic tissues in which AUF1- and YB1-based regulatory mRNPs have been observed to assemble on heterologous mRNAs.

Structural determinants of human ζ-globin mRNA stability.

BACKGROUND: The normal accumulation of adult α and ß globins in definitive erythrocytes is critically dependent upon processes that ensure that the cognate mRNAs are maintained at high levels in transcriptionally silent, but translationally active progenitor cells. The impact of these post-transcriptional regulatory events on the expression of embryonic ζ globin is not known, as its encoding mRNA is not normally transcribed during adult erythropoiesis. Recently, though, ζ globin has been recognized as a potential therapeutic for α thalassemia and sickle-cell disease, raising practical questions about constitutive post-transcriptional processes that may enhance, or possibly prohibit, the expression of exogenous or derepresssed endogenous ζ-globin genes in definitive erythroid progenitors. METHODS: The present study assesses mRNA half-life in intact cells using a pulse-chase approach; identifies cis-acting determinants of ζ-globin mRNA stability using a saturation mutagenesis strategy; establishes critical 3'UTR secondary structures using an in vitro enzymatic mapping method; and identifies trans-acting effector factors using an affinity chromatographical procedure. RESULTS: We specify a tetranucleotide 3'UTR motif that is required for the high-level accumulation of ζ-globin transcripts in cultured cells, and formally demonstrate that it prolongs their cytoplasmic half-lives. Surprisingly, the ζ-globin mRNA stability motif does not function autonomously, predicting an activity that is subject to structural constraints that we subsequently specify. Additional studies demonstrate that the ζ-globin mRNA stability motif is targeted by AUF1, a ubiquitous RNA-binding protein that enhances the half-life of adult ß-globin mRNA, suggesting commonalities in post-transcriptional processes that regulate globin transcripts at all stages of mammalian development. CONCLUSIONS: These data demonstrate a mechanism for ζ-globin mRNA stability that exists in definitive erythropoiesis and is available for therapeutic manipulation in α thalassemia and sickle-cell disease.

The RNA binding protein RBM38 (RNPC1) regulates splicing during late erythroid differentiation.

Alternative pre-mRNA splicing is a prevalent mechanism in mammals that promotes proteomic diversity, including expression of cell-type specific protein isoforms. We characterized a role for RBM38 (RNPC1) in regulation of alternative splicing during late erythroid differentiation. We used an Affymetrix human exon junction (HJAY) splicing microarray to identify a panel of RBM38-regulated alternatively spliced transcripts. Using microarray databases, we noted high RBM38 expression levels in CD71(+) erythroid cells and thus chose to examine RBM38 expression during erythroid differentiation of human hematopoietic stem cells, detecting enhanced RBM38 expression during late erythroid differentiation. In differentiated erythroid cells, we validated a subset of RBM38-regulated splicing events and determined that RBM38 regulates activation of Protein 4.1R (EPB41) exon 16 during late erythroid differentiation. Using Epb41 minigenes, Rbm38 was found to be a robust activator of exon 16 splicing. To further address the mechanism of RBM38-regulated alternative splicing, a novel mammalian protein expression system, followed by SELEX-Seq, was used to identify a GU-rich RBM38 binding motif. Lastly, using a tethering assay, we determined that RBM38 can directly activate splicing when recruited to a downstream intron. Together, our data support the role of RBM38 in regulating alternative splicing during erythroid differentiation.

Structure of fully liganded Hb ζ2ß2s trapped in a tense conformation.

A variant Hb ζ2ß2(s) that is formed from sickle hemoglobin (Hb S; α2ß2(s)) by exchanging adult α-globin with embryonic ζ-globin subunits shows promise as a therapeutic agent for sickle-cell disease (SCD). Hb ζ2ß2(s) inhibits the polymerization of deoxygenated Hb S in vitro and reverses characteristic features of SCD in vivo in mouse models of the disorder. When compared with either Hb S or with normal human adult Hb A (α2ß2), Hb ζ2ß2(s) exhibits atypical properties that include a high oxygen affinity, reduced cooperativity, a weak Bohr effect and blunted 2,3-diphosphoglycerate allostery. Here, the 1.95 Šresolution crystal structure of human Hb ζ2ß2(s) that was expressed in complex transgenic knockout mice and purified from their erythrocytes is presented. When fully liganded with carbon monoxide, Hb ζ2ß2(s) displays a central water cavity, a ζ1-ß(s)2 (or ζ2-ß(s)1) interface, intersubunit salt-bridge/hydrogen-bond interactions, C-terminal ßHis146 salt-bridge interactions, and a ß-cleft, that are highly unusual for a relaxed hemoglobin structure and are more typical of a tense conformation. These quaternary tense-like features contrast with the tertiary relaxed-like conformations of the ζ1ß(s)1 dimer and the CD and FG corners, as well as the overall structures of the heme cavities. This crystallographic study provides insights into the altered oxygen-transport properties of Hb ζ2ß2(s) and, moreover, decouples tertiary- and quaternary-structural events that are critical to Hb ligand binding and allosteric function.

A reverse time-course method for transcriptional chase analyses of mRNA half-lives in cultured cells.

Standard methods for assessing mRNA stabilities in intact cells are labor-intensive and can generate half-life (t(1/2)) measures that are both imprecise and inaccurate. We describe modifications to a conventional tetracycline-conditional transcriptional chase method for analyzing mRNA stability that significantly simplify its conduct, while generating highly reproducible and accurate t(1/2) values. The revised method-which is conducted as a reverse time course, and which accounts for interval expansion in the number of cultured cells-is validated for the analyses of mRNAs with both short and long half-lives. This approach facilitates accurate assessment of mRNA metabolism, providing a user-friendly tool for detailed investigations into their structures and functions, as well as the processes that contribute to their post-transcriptional regulation.

AUF-1 and YB-1 are critical determinants of ß-globin mRNA expression in erythroid cells.

The normal accumulation of ß-globin protein in terminally differentiating erythroid cells is critically dependent on the high stability of its encoding mRNA. The molecular basis for this property, though, is incompletely understood. Factors that regulate ß-globin mRNA within the nucleus of early erythroid progenitors are unlikely to account for the constitutively high half-life of ß-globin mRNA in the cytoplasm of their anucleate erythroid progeny. We conducted in vitro protein-RNA binding analyses that identified a cytoplasm-restricted ß-globin messenger ribonucleoprotein (mRNP) complex in both cultured K562 cells and erythroid-differentiated human CD34(+) cells. This novel mRNP targets a specific guanine-rich pentanucleotide in a region of the ß-globin 3'untranslated region that has recently been implicated as a determinant of ß-globin mRNA stability. Subsequent affinity-enrichment analyses identified AUF-1 and YB-1, 2 cytoplasmic proteins with well-established roles in RNA biology, as trans-acting components of the mRNP. Factor-depletion studies conducted in vivo demonstrated the importance of the mRNP to normal steady-state levels of ß-globin mRNA in erythroid precursors. These data define a previously unrecognized mechanism for the posttranscriptional regulation of ß-globin mRNA during normal erythropoiesis, providing new therapeutic targets for disorders of ß-globin gene expression.

Mutation in the factor VII hepatocyte nuclear factor 4α-binding site contributes to factor VII deficiency.

Severe coagulant factor VII (FVII) deficiency in postpubertal dizygotic twin males results from two point mutations in the FVII gene, a promoter region T→C transition at -60 and a His-to-Arg substitution at amino acid 348; both mutations prevent persistence of plasma functional FVII. This report documents longitudinal laboratory measurements from infancy to adulthood of FVII coagulant activity (FVII:C) in the twin FVII-deficient patients; it also details specific biochemical analyses of the -60 T→C mutation. The results revealed FVII:C levels of less than 1% in infancy that remain severely decreased through puberty and into adulthood. In-vitro analyses utilizing hepatocyte nuclear factor 4α (HNF4α) co-transfection and a chromatin immunoprecipitation assay indicate that the -60 T→C mutation severely diminishes functional interaction between the FVII promoter and transcription factor HNF4α. The importance of interaction between the FVII gene and HNF4α in normal FVII expression provides an in-vivo illustration of the regulated expression of an autosomal gene encoding a coagulation protein. The constancy of FVII:C and peripubertal patient symptomatology reported here illustrates androgen-independent expression in contrast to expression with an analogous mutation in the promoter region of the gene encoding coagulation FIX.

Hb Baden: structural and functional characterization.

Hb Baden (ß18Val→Met) is a rare variant hemoglobin that has never been functionally or clinically characterized. We describe a Hb Baden heterozygote who exhibits normal growth and development, as well as age- and gender-appropriate hematological values. Surprisingly, in vitro analyses demonstrate that Hb Baden is relatively unstable and exhibits an abnormally high affinity for O2. These properties are likely to affect the physiologies of individuals who inherit the ß(Baden) mutation in trans to a determinant for either a functionally relevant hemoglobinopathy or a mild thalassemia. The data also provide insights into the function of the A-helix/AB-segment of ß globin, supporting a structural model in which this poorly understood region serves as a scaffold that fixes the positions of other helices that directly impact ß-globin function.

Developmental expression of human hemoglobins mediated by maturation of their subunit interfaces.

Different types of human hemoglobins (Hbs) consisting of various combinations of the embryonic, fetal, and adult Hb subunits are present at certain times during development representing a major paradigm of developmental biology that is still not understood and one which we address here. We show that the subunit interfaces of these Hbs have increasing bonding strengths as demonstrated by their distinct distribution of tetramers, dimers, and monomers during gel filtration at very low-Hb concentration. This maturation is mediated by competition between subunits for more favorable partners with stronger subunit interactions. Thus, the protein products of gene expression can themselves have a role in the developmental process due to their intrinsic properties.

A potential regulatory role for mRNA secondary structures within the prothrombin 3'UTR.

The distal 3'UTR of prothrombin mRNA exhibits significant sequence heterogeneity reflecting an inexact 3'-cleavage/polyadenylation reaction. This same region encompasses a single-nucleotide polymorphism that enhances the normal post-transcriptional processing of nascent prothrombin transcripts. Both observations indicate the importance of 3'UTR structures to physiologically relevant properties of prothrombin mRNA. Using a HepG2-based model system, we mapped both the primary structures of reporter mRNAs containing the prothrombin 3'UTR, as well as the secondary structures of common, informative 3'UTR processing variants. A chromatographic method was subsequently employed to assess the effects of structural heterogeneities on the binding of candidate trans-acting regulatory factors. We observed that prothrombin 3'UTRs are constitutively polyadenylated at seven or more positions, and can fold into at least two distinct stem-loop conformations. These alternate structures expose/sequester a consensus binding site for hnRNP-I/PTB-1, a trans-acting factor with post-transcriptional regulatory properties. hnRNP-I/PTB-1 exhibits different affinities for the alternate 3'UTR secondary structures in vitro, predicting a corresponding regulatory role in vivo. These analyses demonstrate a critical link between the structure of the prothrombin 3'UTR and its normal function, providing a basis for further investigations into the molecular pathophysiology of naturally occurring polymorphisms within this region.

Cytokine-mediated increases in fetal hemoglobin are associated with globin gene histone modification and transcription factor reprogramming.

Therapeutic regulation of globin genes is a primary goal of translational research aimed toward hemoglobinopathies. Signal transduction was used to identify chromatin modifications and transcription factor expression patterns that are associated with globin gene regulation. Histone modification and transcriptome profiling were performed using adult primary CD34(+) cells cultured with cytokine combinations that produced low versus high levels of gamma-globin mRNA and fetal hemoglobin (HbF). Embryonic, fetal, and adult globin transcript and protein expression patterns were determined for comparison. Chromatin immunoprecipitation assays revealed RNA polymerase II occupancy and histone tail modifications consistent with transcriptional activation only in the high-HbF culture condition. Transcriptome profiling studies demonstrated reproducible changes in expression of nuclear transcription factors associated with high HbF. Among the 13 genes that demonstrated differential transcript levels, 8 demonstrated nuclear protein expression levels that were significantly changed by cytokine signal transduction. Five of the 8 genes are recognized regulators of erythropoiesis or globin genes (MAFF, ID2, HHEX, SOX6, and EGR1). Thus, cytokine-mediated signal transduction in adult erythroid cells causes significant changes in the pattern of globin gene and protein expression that are associated with distinct histone modifications as well as nuclear reprogramming of erythroid transcription factors.

Energetic differences at the subunit interfaces of normal human hemoglobins correlate with their developmental profile.

A previously unrecognized function of normal human hemoglobins occurring during protein assembly is described, i.e. self-regulation of subunit pairings and their durations arising from the variable strengths of their subunit interactions. Although many mutant human hemoglobins are known to have altered subunit interface strengths, those of the normal embryonic, fetal, and adult human hemoglobins have not been considered to differ significantly. However, in a comprehensive study of both types of subunit interfaces of seven of the eight normal oxy human hemoglobins, we found that the strengths, i.e., the free energies of the tetramer-dimer interfaces, contrary to previous reports, differ by 3 orders of magnitude and display an undulating profile similar to the transitions ("switches") of various globin subunit types over time. The dimer interface strengths are also variable and correlate linearly with their developmental profile. Embryonic hemoglobins are the weakest; fetal hemoglobin is of intermediate strength, and adult hemoglobins are the strongest. The pattern also correlates generally with their different O(2) affinities and responses to allosteric regulatory molecules. Acetylation of fetal hemoglobin weakens its unusually strong subunit interactions and occurs progressively as its level of expression diminishes and adult hemoglobin A formation begins; a causal relationship is suggested. The relative contributions of globin gene order and competition among subunits due to differences in their interface strengths were found to be complementary and establish a connection among genetics, thermodynamics, and development.

A post-transcriptional process contributes to efficient gamma-globin gene silencing in definitive erythroid cells.

OBJECTIVES: The expression of human gamma globin is developmentally regulated through mechanisms that affect the transcriptional activity of its encoding gene. The current manuscript investigates whether the efficiency of this process might be enhanced though an unrecognized post-transcriptional event that defines the stability of gamma-globin mRNA. METHODS: Experiments were conducted in vivo in transgenic mice expressing human gamma globin in their adult erythroid cells. The expression of gamma-globin protein was manipulated by breeding the transgene into animals producing different levels of endogenous mouse beta-globin. Changes in the expression of gamma globin were then correlated to measures of gamma-globin mRNA stability in vivo. RESULTS: Human gamma globin was expressed at higher levels in thalassemic than in than non-thalassemic control transgenics, paralleling a highly significant increase in the stability of gamma-globin mRNA. Other molecular events-including possible transcriptional induction of the transgene, or an increase in the stability of the gamma-globin protein-did not appear to contribute to the observed increase in transgene expression. As anticipated, the stability of gamma-globin mRNA also fell in bitransgenic animals that co-expressed human beta-globin mRNA. CONCLUSIONS: Our results are consistent with a model for dynamic post-transcriptional control of gamma-globin gene expression, through modulation of the stability of its encoding mRNA. Moreover, the stability of gamma-globin mRNA appears to be inversely related to ambient levels of co-expressed beta-globin mRNA. This data suggests that therapeutic gene-reactivation and/or gene-replacement therapies may be particularly effective in individuals with severe forms of beta-thalassemia.