Sickle Cell Disease Pathophysiology

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3 Sickle cell disease pathophysiology
C O N S T A N C E T. N O G U C H I A L A N N. S C H E C H T E R G R I F F I N P. R O D G E R S

Sickle cell anaemia, the genetic disease which led to the concept of 'molecular disease', has provided scientists for decades with the opportunity to use a vast array of research techniques to examine its pathophysiologic mechanisms and to design rational approaches to therapy based upon these findings (Dean and Schechter, 1978; Schechter et al, 1987). The genetic defect arises from a single nucleotide change in the gene for the 13-chain of adult haemoglobin (HbA) (Marotta et al, 1977). The resultant haemoglobin (HbS or sickle haemoglobin) aggregates or polymerizes when the erythrocyte is deoxygenated during its normal transit in the circulation, causing impaired cell flexibility, premature erythrocyte destruction and injury to the tissues by microvascular occlusion (Serjeant, 1992; Hebbel, 1991). In recent years, studies of haemoglobin S solutions using a variety of biophysical techniques have paved the way for studies of the intact erythrocyte containing the abnormal haemoglobin, the sickle cell (Noguchi and Schechter, 1985; Eaton and Hofrichter, 1990). These molecular and cellular studies have clarified pathophysiotogical processes in the microcirculation of the patient. Genetic studies have elucidated some of the factors that determine the remarkably heterogeneous nature of sickle cell disease severity and have allowed the establishment of sensitive and specific prenatal diagnostic methods (Bunn and Forget, 1986; Schechter et al, 1987). Further, molecular genetic studies are beginning to clarify several new approaches to the therapy of this disease targeted at altering globin gene expression-approaches which are now being evaluated in patients (Rodgers et al, 1990, 1993; Charache et al, 1992). We present in this chapter a summary of our knowledge of deoxyhaemoglobin S polymer formation and its effect on sickle erythrocyte properties. The extent of polymer formation is modified by genetic factors which vary haemoglobin composition and concentration within the intact sickle erythrocyte and these factors will be reviewed. Rational approaches to therapy, in addition to the genetic interventions to change haemoglobin composition, based on the biophysical analysis of haemoglobin polymerization, which includes strategies to increase deoxyhaemoglobin S solubility or decrease corpuscular haemoglobin concentration, are also
Bailli~re"s Clinical Haematology--

Vol. 6, No. 1, March 1993 ISBN 0-7020-1692-6

Copyright © 1993, by Bailli~re Tindall All rights of reproduction in any form reserved

57

58

C . T . NOGUCHI ET AL

discussed. Further clinical aspects of sickle cell disease are covered in more detail in Chapter 4 in this volume by Serjeant.
GENETICS

Sickle cell disease is due to the substitution of a T for an A nucleotide in the codon for glutamic acid in the sixth position of the gene for the 13-chain of human haemoglobin (Marotta et al, 1977). The abnormal 13-chains ([3s), which have a substituted hydrophobic valine residue (Ingrain, 1956), combine with the normal or-chains to give HbS rather than HbA in the erythrocytes of sickle cell patients (Figure 1). The reduced solubility of deoxy-HbS, as compared to deoxy-HbA (Hofrichter et al, 1974; Moffat and Gibson, 1974), leads to intracellular polymerization (and in the extreme

HbA
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Figure 1. Overview of the basic molecular processes in the pathophysiology of sickle cell disease. The nucleotide mutation (A--~T) in the sixth codon in the I~-globin chain gives rise to the substitution (Glu--~Val) to form the 13 s chain. Combination of the mutant 13S-globinchains with a-globin chains results in haemoglobin S (FIBS) formation. Upon deoxygenation (filled circles) HbS molecules aggregate into polymers, unlike normal adult haemoglobin (HbA) molecules which remain free in solution in the deoxy as well as the oxygenated form (open circles). The open and dosed circles represent oxyhaemoglobin and deoxyhaemoglobin respectively. Note that the gel that forms in deoxyhaemoglobin S solution or sickle cells is reversible upon the addition of oxygen and is composed of polymer in equilibrium with the surrounding haemoglobin molecules. The extent of haemoglobin polymerization and the nature of the polymer domains within the intact partially deoxygenated sickle erythrocyte determines its morphology and can give rise to the classic sickle, holly-leaf or other appearances. Adapted from Schechter et al (1987).

SICKLE CELL DISEASE PATHOPHYSIOLOGY

59

state, cell sickling) and disease pathophysiology (Noguchi and Schechter, 1985; Eaton and Hofrichter, 1987). Homozygous sickle cell disease (or anaemia) (the SS genotype) is among the most prevalent genetic diseases in the world and is the most frequent in persons of equatorial African origin (Serjeant, 1982). The term sickle cell syndromes (or disorders) is used to connote that spectrum of diseases ranging from the almost symptomless sickle cell trait (the AS genotype) to the most severe homozygous SS genotype found in Africa (Brittenham et al, 1985) (Table 1). This spectrum also includes a variety of genotypes, among which are double heterozygosity for the S gene and for 13-thalassaemia or another haemoglobin variant, such as HbC; and genetic abnormalities co-existing with the S gene, such as that for hereditary persistence of fetal haemoglobin (HbF) (HPFH) or ct-thalassaemia. Table 1 provides haematological data on some of these sickle cell syndromes. As will be explained later, much of the variation in disease severity among these syndromes can now be understood in terms of their primary effects on haemoglobin S polymerization through changing intracellular haemoglobin composition or concentration. The frequency of heterozygotes for the S gene may be 25% or greater in parts of Africa (Allison, 1954a; Lehmann, 1954). The disease is most prevalent in central and western African countries but has a distribution with respect to temperature and rainfall that led to the now generally accepted hypothesis that sickle trait individuals have a selective resistance to malaria (caused by Plasmodium falciparum), accounting for the high prevalence of this very deleterious mutation in equatorial regions (Allison, 1954b, 1957a). Epidemiological studies have supported this hypothesis in that there is a strong geographic correlation of S gene frequencies with the incidence of malaria. In addition, a variety of possible cellular mechanisms-including changes in intracellular pH and potassium in malaria-parasitized erythrocytes---have been proposed to account for the epidemiological and clinical observations (Friedman, 1978; Pasvol et al, 1978). It is expected that in regions where malaria is controlled, the frequency of the S gene will decrease. The recent world-wide upsurge in malaria (World Malaria, 1992) makes the anticipation that this haemoglobinopathy (and perhaps other haemoglobin disorders also linked to malaria) will disappear in the decades ahead less likely than was thought a few years ago. Pioneering genetic studies using restriction enzyme cleavage of i3-globin DNA provided evidence for at least two geographic sites of origin of the sickle mutation, one in West Africa and one in East Africa (Kan and Dozy, 1980), but evidence for other sites within Africa has also been presented (Pagnier et al, 1984). These studies have also been used to define various linked-groupings of DNA restriction cleavage polymorphisms within the 13-globin gene cluster, called haplotypes. Although more than a dozen such 13-globin haplotypes have been identified, most S genes occur on four such haplotypes, known as the Senegal, Benin, Central African Region (or Bantu) and Cameroon types depending on the area with greatest frequency of the cluster of polymorphisms (Powars, 1991a). Another haplotype has been described in Saudi Arabian and Indian sickle cell patients (Labie et al,

Table 1. Haematological data and haemoglobin analysis in some sickling disorders.* Severity Asymptomatic Condition 1. 2. Mild 3. 4. 5. 6. 7. 8. Severe 9. 10. 11. 12.
" +ISD.

n 34 9 3 3 22 15 39 15 44 41 44 88

Hb (g/dl) 14.3 ( + 1.2)" 14.0 11.4 (+ 0.7) 11.1 10.9 (_+ 1.6) 10,5 (_+1.3) 10.3 ( + 1.7) 9.3 (+_0.8) 8.8 (_+ 1.3) 8.6 (_+ 1.0) 8.1 ( + 1.0) 7.8 (+-1.1)

Reticulocytes (%) 2.0 -2.9 -5.2 (+ 3.4) 4.8 (+3.1) 4.0 5,9 (_+2.4) 6.4 7.3 (+ 2.7) 9.3 11.9

MCV (fl) 87.0 86.0 74.0 84.0 72.2 85.0 71.0 67,9 71.2 68.9 84.4 90.1

MCHC (g/d 0 33.9 b 33.3 c 32.7 b 33.0 b 33.4 b 32.00 31.2 ¢ 32.2 c 32.8 a 31.1 a 34.3 d 34.8 d

MC[HbS]C (g/dl) 13.7 20.6 22.6 23.5 23.2 24.4 20.9 25.5 30.3 27.5 31.6 32.0

HbA2 (%)
2.4

HbF (%)
0.8 ~

HbS (%)
40.5

HbAS (African) HbS-°'yA',/-I3°-HPFH (African) HbS-G2t-~'~t3°-thalassaemia (African) HbS-C~/-13°-HPFH (African) HbSS (Saudi Arab) HbSS (Indian) HbS-13+-thalassaemia (African) HbS-13°-thalassaemia (Saudi Arabian) HbSS-et-thalassaemia (African) (a-/a-genotype) HbS-13°-thalassaemia (African) HbSS-a-thalassaemia (African) (ot-/etot genotype) HbSS (African)

2,1 2.2 2.6 2,1 d 2,3 4.8 3.5 3.9 5.0 3.1 2,8

35.9 f 28.8 t 26.1 f 28.5 g 20.0 f 5.0 e 17.3e 3.8 ~ 6.5~ 4.8 ~ 5.3 e

62.0 69.0 71,8 69.4c 77.0 67.0 79.2 92.3 88.5 92.1 91.9
Z © q~

b MCHC by Coulter Counter. c Method unspecified. a MCHC calculated from haemoglobin concentration and packed cell volume. e HbF by alkalai denaturation. t HbF by column chromatography. g HbF by densitometry. * Adapted from Brittenham et al (1985).

t"

SICKLE CELL DISEASE PATHOPHYSIOLOGY

61

1989; Padmos et al, 1991). Evidence has been presented, which will be noted later, that the severity of sickle cell disease may be affected by the haplotype on which the S gene appears (Nagel et al, 1991; Powars, 1991a,b). The existence of individuals affected by sickle cell disease in parts of the world other than Africa can generally be attributed to the movements of individuals or groups--voluntarily or by force--to these other regions and the resultant diffusion of the gene (Serjeant, 1992; Adekile, 1992). SICKLE HAEMOGLOBIN
Solution structure and function

The oxygenated form of HbS appears to be nearly identical to the oxygenated (or other liganded) form of H b A (Baldwin and Chothia, 1979) with respect to its structure and function (Perutz, 1987; Schechter et al, 1987; Eaton and Hofrichter, 1990). This should not be surprising since the mutation in the 13S-chain is on the surface of the molecule (Wishner et al, 1975) and not near the intersubunit contact residues which are involved in determining many of the subtle properties of haemoglobin, such as cooperative oxygenation. As a result, the oxygen-binding properties of dilute HbS and H b A are nearly identical. There are some small differences, however, in the efficiency of interaction of a-chains with 13s. as compared to 13A-chains to form the stable et13(or a13s) dimer that is the structural unit of the haemoglobin tetramer (ot2132 or o~213s2) (Shaeffer et al, 1978). This difference probably accounts for the fact that AS heterozygotes have, in general, a 60 to 40 ratio of H b A to HbS. The stronger interaction of 13-with a-chains, as compared to 13 s and a, leads to selective destruction of the excess 13S-chains, even if the synthetic rates of 13-and 13S-chains are identical. The classical difference usually noted between dilute H b A and HbS is the alteration of charge which causes the electrophoretic abnormality, first noted by Pauling et al (1949), which is the basis of the most common clinical methods to diagnose this haemoglobinopathy. It is in very concentrated solutions, such as the interior of the sickle erythrocyte at a haemoglobin concentration of about 34 g/dl, in which the major difference between HbS and HbA manifests itself. Deoxy-HbS has a markedly reduced solubility compared with oxy-HbS (or as compared with deoxy-HbA or oxy-HbA) (Allison, 1957b; Bertles et al, 1970; Hofrichter et al, 1974). The result of this reduction in solubility is the formation of polymer fibres (also called aggregates, tactoids or a gel) within the sickle erythrocyte upon partial or total deoxygenation (Bertles et al, 1970; Cottam et al, 1974; Moffat and Gibson, 1974; Hofrichter et al, 1976). The allosteric structural change of haemoglobin from the oxygenated R-state to the deoxygenated T-state, with shift of the ~13 subunits with respect to each other (Baldwin and Chothia, 1979; Perutz, 1987), presumably allows for intertetramer contacts that favour the aggregated or polymerized form of HbS over the solution form (Wishner et al, 1975; Padlan and Love, 1985). Such changes in conformation also occur in H b A but the energetics of

62

C. T. NOGUCHI ET AL

86

B
_ 5

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I'A

Figure 2. Schematic structure of the deoxyhaemoglobin S crystal. (a) The haemoglobin tetramers are arranged in a double-stranded array. The residues identified from crystallographic analysis as making major intermolecular contacts are denoted, including the 13 s6 valine on each HbS tetramer which makes contact with the 13-chain of a neighbouring tetramer. (b) Artistic interpretation of the 13 s6 valine contact region at higher resolution illustrating possible orientation of amino acid side-chains at this intermolecular contact site. (c) IUustration of the normal 13 s6 glutamic acid which suggests that the bulk and charge of this amino acid side chain would not allow the same contact geometry. From Schechter et al (1987), based on Wishner et al (1975) and Dickerson and Geis (1983).

stabilization of the deoxy form are not sufficient without the valine in the 13-chain to make the polymer predominate under the range of conditions found in cells in the body. Crystal and polymer structure During the last decade, X-ray diffraction techniques have been used to determine the structure of crystals of human deoxy-HbS, formed in special crystallizing solvents, at moderately high resolution (Wishner et al, 1975; Padlan and Love, 1985). This structure consists of double strands of haemoglobin tetramers arranged as shown in Figure 2a. There are extensive contacts among tetramers within a strand and on adjacent strands. However, only one 13s6 residue of each HbS tetramer actually makes an intermolecular contact in the crystal to give the stabilization energy unique to HbS. That hydrophobic 13s6valine side-chain fits into a pocket found in a 13-chain on an adjacent HbS tetramer which has a hydrophobic pocket composed, in part, of 1388leucine and 1385phenylalanine residues (Figure

SICKLE CELL DISEASE PATHOPHYSIOLOGY

63

2b). Most of the other atoms in the pocket, however, are also uncharged or poorly polarizable. The glutamic acid residue at this position in the normal 13-chain does not fit into this hydrophobic pocket because of its larger size and its strongly charged nature (Figure 2c). It is likely, but not proven, that these double-stranded structures are the basis of the higher ordered structures that are seen in electron micrographs of deoxygenated sickle cells and concentrated HbS solutions (Dykes et al, 1979; Crepeau et al, 1981). Evidence for this comes from the fact that many likely intermolecular contacts in the sickle polymer structure, that had been deduced from the study of the effects of other mutations in the or- or 13-chain on the solubility of the HbS molecule, are quite consistent with the intermolecular contacts in the double-stranded molecule (Edelstein, 1981). If this is the case, then information on contacts among atoms within the double strand can be used to design compounds which might inhibit polymerization and thus act as therapeutic agents (Dean and Schechter, 1978; Schechter et al, 1987). In concentrated HbS solutions and cells, deoxygenation leads to formation of birefringent gels (Harris, 1950) made up of multiple strands of HbS molecules. The next higher order structure of such gels, which has been clearly defined by electron microscopic and image reconstruction techniques, consists of seven double-strands intertwined in a complex, twisting pattern (Dykes et al, 1979). Higher ordered structures are presumably formed from these 14-strand structures by ordering in parallel arrays into the bundles of fibres seen in electron micrographs (Briehl et al, 1990). Such bundles presumably are capable of distorting the SS cell, by virtue of the cumulative energetics of their formation within the normally biconcave erythrocyte, into sickled and other abnormal shapes. Nothing is known of the detailed molecular contacts within these polymer bundles which also could be targets for drug action. There is some suggestion that small molecular rearrangements may be necessary to allow the double-strand to assemble into the 'macro'-fibres or transform into true crystals. Other fibre patterns have been seen but their physiological relevance is unclear (Josephs et al, 1976). Upon rapid deoxygenation in the SS cell or in concentrated HbS solutions, many small polymer domains form and with time these may rearrange to form one or a few large, cyclindrical domains in the fully 'sickled' cell or in gelled HbS solution (Horiuchi et al, 1990). It is likely that in SS cells, especially in the body, many other polymer forms--such as very short bundles due to shearing forces---may exist but these are not well characterized (Briehl, 1983). Nor are the relative contributions of the various polymer structures to impaired cell rheology understood.

Formation of deoxy-HbS polymer
Kinetics and mechanism

Detailed biophysical studies have clarified the mechanism by which a polymer of deoxy-HbS molecules forms in concentrated HbS solutions or in

64

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C. T. NOGUCHI ET AL

HETEROGENEOUS~ DOMAI1 NON NUCIO; FORMATI
LE<~_AT ,,, <--'-'--

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Figure 3. Model for the polymerization kinetics of deoxyhaemoglobin S solutions. The polymerization of a polymer-free solution involves a nucleation-dependent process which gives rise to a characteristic delay time which varies greatly with total haemoglobin concentration. During this time, deoxyhaemoglobin S molecules are in rapid, reversible equilibria with an intermediate structure or aggregate making up the critical nucleus (homogeneous nucleation). Formation of the critical nucleus is the rate-limiting step. Once formed, growth of long polymers proceeds rapidly and additional nuclei may form on the pre-existing polymer fibres (heterogeneous nucleation), leading to formation of multiple strands of polymer. Further growth and alignment results in domains of ordered haemoglobin polymer. In the cell with pre-existing polymer, growth and alignment process (not nucleation) probably will determine overall kinetics. From Noguchi et al (1985), based on Ferrone et al (1985).

sickle (SS) (or other S genotype) cells upon deoxygenation (Eaton and Hofrichter, 1990). These kinetic studies have defined a nucleationcontrolled process by which deoxy-HbS tetramers assemble to an intermediate structure (thought to be about 15 molecules) which then may grow rapidly by addition of tetramers to this critical nucleus (Figure 3). This process, known as homogeneous nucleation, may be complemented by the formation of other polymer structures upon previously formed polymers--a process known as heterogeneous nucleation (Ferrone et al, 1985). These two proposed nucleation mechanisms account for many aspects of the complex kinetics of HbS polymer formation when HbS polymerization is initiated in concentrated solutions by a temperature jump or other physical perturbation (Hofrichter et al, 1976; Ferrone et al, 1985; Mozzarelli et al, 1987). These kinetic curves exhibit a prolonged delay time before the onset of detectable polymerizationwpresumably the time for the formation of the critical nuclei. The very steep 'progress' curves of polymerization after this reflect the combination of homogeneous and heterogeneous nucleation processes. Very recently evidence has been presented for complex branching and alignment processes as these lower order polymer structures form the 'macro'-structures visible to various microscopic techniques (Samuel et al, 1990). The relevance of these mechanisms to the processes that occur in sickle cells in the patient's body has been somewhat controversial since, in the body, confounding factors may change significantly the relevant mechanisms (Schechter et al, 1987). These factors include: (a) the possibility that many or most cells in the body have pre-existing nuclei (Horiuchi et al, 1990); (b) the difference in concentration and temperature conditions from those in which many of the in vitro studies have been done (Noguchi and

SICKLE CELL DISEASE PATHOPHYSIOLOGY

65

Schechter, 1981); (c) the gradual nature of deoxygenation processes in the body compared with in vitro experimental perturbations; and (d) the effect of shear inside sickle cells during their passage through the circulation (Briehl, 1983). Despite this uncertainty as to the molecular mechanisms of polymerization within cells in the body, the kinetic analysis has provided a framework to understanding HbS polymer formation, and remains the basis for understanding many aspects of the behaviour of HbS molecules (Eaton and Hofrichter, 1987, 1990).

Equilibrium (thermodynamic) aspects
Equilibrium solubility measurements of deoxy-HbS during the last 20 years have also contributed greatly to our understanding of the pathophysiology of the sickle diseases and various approaches to therapy (Bertles et al, 1970; Hofrichter et al, 1976; Magdoff-Fairchild et al, 1976). Early work was done with variants of a gelling assay to give a 'minimum gelling concentration' (or MGC) (Bookchin et al, 1976) but the advent of true solubility measurements allowed recognition of the complex interaction of various haemoglobin chains in polymerization, the role of protein and solvent non-ideality, and many other factors (Minton, 1976; Sunshine et al, 1979; Gill et al, 1980; Noguchi, 1984). When concentrated HbS solutions are deoxygenated and centrifuged at high speed, they may be separated into two phases: the soluble supernatant and the packed pellet (Bertles et al, 1970; Hofrichter et al, 1976; MagdoffFairchild et al, 1976). The pellet consists of the polymer structures that were formed upon deoxygenation with surrounding sedimented-HbS tetramers. Measurement of the concentration of the supernatant provides information about the thermodynamic solubility of HbS under the specified conditions of temperature, buffer, pH, etc. Under these experimental conditions, HbS polymer acts thermodynamically as a two-phase system of gel and free tetramer (Minton, 1974). The solubility of deoxy-HbS under conditions that simulate intracellular conditions is between 16 and 18 g/dl (Poillon and Kim, 1990) (less than the concentration of Hb in the red cell, which averages about 34g/dl as the mean corpuscular haemoglobin concentration (MCHC)). As mentioned earlier, HbS and HbA, in both the oxy and deoxy forms, have solubility values that are much higher than this. In this sense, sickle cell anaemia may be thought of as a disease due to the insolubility of deoxy-HbS inside the erythrocyte. Solubility experiments of HbS with other haemoglobins, such as HbA, Hb A2, HbF and HbC, have clarified how these interact in the deoxygenated state to affect polymerization (Cheetam et al, 1979; Sunshine et al, 1979; Benesch et al, 1980; Bunn et al, 1982). The data are complex but are consistent with a model in which the HbS molecule (a213s2) has the lowest solubility (expressed as the relative probability of entering the polymer of one); all the other homotetramers are much more soluble, even in the deoxy state, and have close to zero probability of entering the polymer (Sunshine et al, 1979; Bunn et al, 1982). It is the mixed hybrids which determine the sparing effects of non-S haemoglobins on polymerization. HbA and C mixed

66

C. T. NOGUCHI ET AL

hybrids with HbS (a213[3s and o~213c13 s) enter the polymer with about 0.5 probability (Bunn et al, 1982), while HbF and HbAz mixed hybrids with HbS (ot2-y[3 s and a2~13s) do not enter the polymer (Sunshine et al, 1979). This difference accounts for much of the importance of these last two haemoglobins, HbF in particular, as a potential therapy for this disease (see later). These techniques of direct solubility measurements at equilibrium have also been used to demonstrate that 2,3-diphosphoglycerate has a small, but distinct, effect on lowering the solubility of deoxy-HbS that is independent of its effect in shifting the allosteric equilibrium from the R- to the T-state (Poillon and Kim, 1990). A very important concept that has come from these thermodynamic studies was the appreciation of the role of non-ideality in affecting the solubility of deoxy-HbS (Minton, 1976; Ross and Minton, 1977). The large size of Hb molecules and their very high concentration inside the erythrocyte makes their chemical activity about 50 times the measured concentration (Ross et al, 1978). It is the activity, not the concentration, that determines the solubility of species in solutions or in cells (Minton, 1976). Similarly, the activity of the aqueous solvent in the cell is different from the nominal value usually assumed (Gill et al, 1980). In analysing experimental data, both kinetic and equilibrium, these factors become very important. In particular both the rate of polymerization (the inverse of the delay time) and the extent of polymerization are dependent to a very high degree on the total intracellular Hb concentration (Noguchi, 1984; Eaton and Hofrichter, 1987, 1990), which explains why reducing M C H C could be an important therapeutic goal (Rosa et al, 1980).

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Figure 4. Polymer fraction within intact sickle erythrocytes measured by t3C-NMR as a function of oxygen saturation from several patients (different symbols). The results indicate that polymer is detected at very high oxygen saturation values (under conditions with few reversibly sickled cells) and that the amount of polymer increases to an average maximum of about 70% as oxygen saturation decreases to zero. Dense cells shift the curve to the right and cause the appearance of detectable polymer near 100% saturation. From Noguchi et al (1980).

SICKLE CELL DISEASE PATHOPHYSIOLOGY

67

A critical example of the effects of non-ideality are the results of measurements of HbS polymerization, as a function of oxygen saturation, that were made with nuclear magnetic resonance (NMR) spectroscopic techniques (Noguchi et al, 1980) (Figure 4). The surprising result of these studies was that polymer formed in the sickle erythrocyte at very high oxygen saturation values (80% or above for a cell with an MCHC of 34 g/dl and higher for more dense cells) and increased monotonically to a maximum of about 70% of the molecules polymerized at complete deoxygenation. Polymer is detected at much higher oxygen saturation values than that for significant cell sickling (morphological distortion) and at far higher oxygen saturation values than simple solubility considerations would have predicted. The explanation for the latter phenomenon is that protein and solvent non-ideality markedly reduce the effective solubility within the sickle erythrocyte (Noguchi et al, 1983; Noguchi, 1984). As will be discussed later, these results have significant implications for understanding the pathophysiology of sickle cell disease and its syndromes. Comparisons of haemoglobin polymer formation in AS and SC erythrocytes (individuals heterozygous for [3s and 13 c) indicated that although comparable mixtures of haemoglobin S and A and of haemoglobins S and C behave similarly (Bunn et al, 1982), SC erythrocytes exhibited an increase in the potential for intracellular haemoglobin polymerization (Noguchi, 1984). In fact, the increased proportion of haemoglobin S in SC disease (about 50%) compared with sickle trait (about 40%) together with the increased MCHC or intracellular haemoglobin concentration in SC erythrocytes due to the presence of haemoglobin C results in an increased potenti.al for haemoglobin polymerization and provides an explanation for the disease severity associated with SC disease in contrast to benign sickle trait (Bunn et al, 1982).

SICKLE ERYTHROCYTES

The sickling of sickle (SS and other sickle syndrome) cells is due to the reversible intracellular polymerization of HbS within the cell upon deoxygenation (Harris et al, 1956) (Figure 1). At complete deoxygenation, polymerization leads to extensive morphological deformation and formation of the classical holly-leaf or sickled forms (Diggs, 1932; Horiuchi et al, 1990). At partial deoxygenation, small amounts of polymer may exist without detectable morphological abnormalities. The rheological properties of the sickle cell (Bessis, 1977, 1982; Itoh et al, 1992) are primarily determined by the extent of intracellular HbS polymerization (Keidan et al, 1989; Mackie and Hochmuth, 1990). As was discussed earlier, the extent of polymerization is determined by the intracellular haemoglobin concentration (MCHC) and composition (percentage of HbS and non-S haemoglobins) as well as oxygen saturation (Noguchi, 1984; Noguchi and Schechter, 1985; Eaton and Hofrichter, 1987, 1990). Other variables such as temperature (Ross et al, 1977), diphosphoglycerate levels and intracellular pH (Poillon and Kim,

68

C. T. NOGUCHI ET AL

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Figure 5. Density distribution of normal (AA) and sickle (SS) erythrocytes. The histogram represents the fraction of cells at each density value and is the difference between adjacent measurements obtained from the density profile generated by phthalate ester density separation in microcapillary tubes (experimental lines). Sickle cell patients have more light cells (largely reticulocytes) and more dense cells (which presumably exacerbate pathology but whose origin is still uncertain) than normal individuals. From Rodgers et al (1985).

1990) also have effects but these are probably small within the range of physiological, or even pathological, variation. Sickle erythrocytes are very heterogeneous with respect to intracellular haemoglobin concentration (Seakins et al, 1973) and various biochemical properties (Clark et al, 1978) as compared to normal (AA) erythrocytes. These abnormalities include changes in diphosphoglycerate concentrations, pH and oxygen affinity and in the properties of the membrane (Hebbel, 1991). The variations in haemoglobin concentration are reflected in a wide spectrum of cell densities when sickle cells are fractionated on density gradients (Rodgers et al, 1985) (Figure 5). It will be noted that there are many light cells--a fraction markedly enriched with reticulocytes--and many dense cells. The dense cells (with MCHC > 37 g/dl) would be expected to be very deleterious in the circulation of the patient with sickle cell anaemia since polymerization of HbS would be markedly enhanced in these cells (Clark et al, 1980; Evans et al, 1984; Noguchi et al, 1983; Keidan et al, 1989; Schmalzer et al, 1989). The origin of the dense cells remains uncertain. It was originally thought that they represented the end stages of repeated cycles of polymerizationdepolymerization ('sickling'-'unsickling') (Bookchin and Lew, 1984; Nash et al, 1988) in the body, as it has been known for decades that deoxygenation of

SICKLE CELL DISEASE PATHOPHYSIOLOGY PRODUCTION CIRCULATION DESTRUCTION

69

DENSE CELLS
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MICROVASCU LAROBSTRUCTION ACUTE CRISIS CHRONIC PROGRESSIVE ORGAN DAMAGE

Figure 6, Schematic outline for the cellular pathophysiology of sickle cell anaemia. Sickle cells produced by the bone marrow represent a heterogeneous population (e.g. with respect to the percentage HbF and cell density). In addition, repeated cycles of intracellular polymerizationdepolymerization can lead to K + toss and dehydration, giving rise to dense cells resulting in more polymer at any oxygen saturation. Reversible intracellular polymerization also leads to membrane damage and the formation of irreversibly 'sickled' cells (ISCs), which are also largely in the dense cell fraction. Dense cells have reduced flexibility due to high MCHC and membrane rigidity but primarily because of increased tendency to polymer formation of any oxygen saturation. Endothelial adhesion may also impair rheological behaviour. These several processes lead to obstruction and haemolysis and, eventually, to the acute and chronic manifestations of the disease. Adapted from Rodgers (1991).

sickle cells leads to K + and H2O fluxes and consequent cellular dehydration (Brugnara et al, 1986) (Figure 6). It was also postulated that these 'premature ageing' changes in circulating SS cells caused the variety of protein and lipid abnormalities, as well as functional changes, detected in sickle cells, particularly the dense ones (Hebbel, 1991). In particular, those cells with extensive membrane abnormalities that always appear deformed, irrespective of the physical state of the intracellular HbS, and are termed 'irreversibly sickled cells' (ISCs) (Bertles and Milner, 1968), are thought to have been formed by such mechanisms (Hebbel, 1991). Recently, however, data have been presented that many erythrocytes may emerge from the marrow more dense than other cells and already manifesting abnormal membranes, including altered membrane proteins and ion transport mechanisms (Mohandas et al, 1989; Bookchin et al, 1991) (Figure 6). These cells are known to be low in HbF (Bertles and Milner, 1968) but other factors that may contribute to this heterogeneity are not known. It does seem likely, however, that these (predetermined) dense cells have a shortened life-time in the circulation (McCurdy and Sherman, 1978) and may be disproportionately involved in the pathogenesis of the acute painful crises of sickle cell disease, the chronic haemolytic anaemia, and other vaso-occtusive complications of this disease (Billett et al, 1988). The oxygen affinities of sickle cells vary from about 40to 60 mmHgvalues of 1'5o, indicating a marked reduction in affinity as compared to normal cells

70

C. T. N O G U C H I ET A L

(Ps0 = 26 mmHg) (May and Huehns, 1975; Winslow, 1976). This change in affinity is not primarily due to the type of Hb as the basic oxygen affinities of HbS and HbA are comparable (Pennelly and Noble, 1978). Rather, the reduced effective oxygen affinity is due to the energetic linkage between polymerization and oxygen binding (May and Huehns, 1975). Since only the deoxy-HbS molecules enter the polymer (Hofrichter, 1979), oxygen binding may be thought of as competing with the binding energy of haemoglobin tetramers in the polymer (Sunshine et al, 1982). Thus effective reduction in oxygen-binding energy leads to the marked 'right-shift' of oxygen equilibrium measurements (May and Huehns, 1975). The physiological consequences of this phenomenon are less clear as this shift facilitates oxygen delivery to the tissues. It should be noted that problems in oxygen delivery due to the anaemia of sickle cell disease do not appear to be a major limitation of these diseases as even severe decrements in haemoglobin level are often well tolerated (Serjeant, 1992). RHEOLOGY OF SICKLE ERYTHROCYTES It is generally accepted that the impaired flow properties of sickle cell erythrocytes (i.e. their abnormal rheology) are due to the acute and chronic effects of the intracellular polymerization of HbS as sickle erythrocytes transverse the circulation from the lungs to the tissues and back (Mohandas and Evans, 1989). During much of the three-quarters of a century after the original description of sickle cell disease, most characterization of the disease related to the morphology of the erythrocytes of patients, both as the cells appeared in the peripheral blood and after deoxygenation with nitrogen or chemical agents (Stuart and Johnson, 1987). During this period, light microscopy has been supplemented by birefringence measurements and electron microscopy (Harris et al, 1956; Dykes et at, 1979; Edelstein, 1981). These studies identified bundles of polymerized haemoglobin molecules within sickled erythrocytes and led to the realization of the relationship between the physical state of the HbS molecules and the morphological abnormalities of the cells (Briehl et al, 1990; Horiuchi et al, 1990; Kaul and Xue, 1991). However, the likely behaviour of cells in the microcirculation cannot be predicted from such observations. Thus, the advent of direct rheological measurements of sickle cells has been of great importance (Ballas, 1991; Morris et al, 1991; Phillips et al, 1991). Filtration measurements through various micropores show that even oxygenated sickle erythrocytes are slightly more rigid than normal cells (Green et al, 1988; Keidan et al, 1989; Mackie and Hochmuth, 1990). This phenomenon is probably due to the effects of the high intracellular haemoglobin concentration in the dense cell subfractions and to the membrane abnormalities discussed earlier. There has been some speculation that abnormal haemoglobin membrane interactions may also contribute to the relative rigidity of sickle erythrocytes but this is still not certain. Upon deoxygenation there is a marked decrease in the filterability of sickle erythrocytes due to the formation of intracellular HbS

SICKLE CELL DISEASE PATHOPHYSIOLOGY
500

400
0 l<c I..J 0
x m ¢3
Z

Z

300

200

/t
,,,,,,,,

71

100

5

1"5

2"0
(kPa)

2"5

OXYGEN TENSION

Figure 7. Effect of decreasing Po2 on the filtration of SS erythrocytes fractionated by density. The index of filtration through a 5 ~M pore diameter increases as filtration becomes impaired, The unfractionated whole blood sample is represented by (0). Fraction 2 (O) and fraction 3 (A) represent middle density fractions while fraction 4 ( 0 ) represents the dense cell fraction. From Keidan et al (1989).

polymers and this must be considered the major factor affecting the rheology of sickle erythrocytes. Indeed detailed filtration studies show that changes in measured properties occur at very high oxygen saturation values (Figure 7) (Keidan et al, 1989). Again the denser cells have greater impairment of filtration at any oxygen saturation and show detectable changes at the highest oxygen saturation values. Other techniques to measure theological properties include viscometry, ektacytometry, micropipette aspirations and various animal blood vessel preparations (Kaul et al, 1989; Mackie and Hochmuth, 1990; Ballas, 1991; Morris et al, 1991). In general, the results with all of these methods are congruent, with different techniques being more or less sensitive to different aspects of the biophysical properties of the sickle erythrocytes. It is generally agreed, however, that none of these in vitro or even in vivo procedures is a really good model of the behaviour of cells in the human microcirculation. For these reasons, and to simulate the pathophysiology of sickle cell anaemia (Figure 6), there has been great interest in developing a true animal model of sickle cell disease. The advent of transgenic techniques during the last decade in which genes may be inserted into the fertilized ova or, more recently, into embryonic

72 v; wal
(~-like Genes

C. T. N O G U C H I ET A L

Chromosome 16

~,-like Genes

5'

"e'" _10
I

G" A
I 20 I 30

.:/ L'{ m
I

w B
II

=

6""B
50
I

-.
I 60

3'

Chromosome 11

~0

Kilobases

Develoemental Pedo~

Embrvonic Hb Gower 1

Fetal

I
I I I I

(~)

Hemoglobins

Hb Gower 2 Hb Portland

HbF

I HbA2

HbA

(~);(~)

t (%a2)
I I I

(~2)

Figure 8. Schematic of the a-like and B-like globin clusters on chromosomes 16 and 11, respectively. For the a-like globin genes, the embryonic ~-globingene is activelytranscribed early during development, followed by a switch to the adult-like a-globin genes during early gestation, which persists during fetal and adult life. The B-like, embryonic ~-globin gene is transcribed during the first trimester 'in utero' followed by a switch to the two 3,-globingenes which persist during fetal development and another switch at birth to the adult 13-globinand 8-globin. Haemoglobin tetramers resulting from combination of these c~- and B-like globin chains are also indicated. From Berg and Schechter (1992). stem cells in culture and animals produced expressing the test gene has made this goal appear attainable (Grosveld et al, 1987). These studies advanced in parallel to basic studies of globin gene expression (Berg and Schechter, 1992) using various animal cells (and the transgenic mouse) as reporter systems. These studies led to the characterization of the important promoter elements for each of the globin genes, to the existence of various enhancers and silencers and, most importantly, to the function of a region upstream of the 13-globin gene cluster (Figure 8), now known as the locus control region (LCR) (Orkin, 1990). This D N A region, and an analogous one for the ot-globin gene cluster (Jarman et al, 1991), is required for high-level, position-independent expression of transferred globin genes in transgenic models. On the basis of these studies, four or more laboratories have produced transgenic mice expressing the human ~S-gene (or analogues engineered for the protein to be even less soluble in the deoxygenated state) and, in some cases, human a-genes (Greaves et al, 1990; Ryan et al, 1990; Rubin et al, 1991; Trudel et al, 1991). Unfortunately, despite the relatively high expression of the human ~s_ genes in these transgenic mice, none of the animals appears to be a very good model for the human disease. A fundamental problem with all of these studies is the continued presence of the complex of mouse globins which combine with the human proteins to give a variety of haemoglobin species whose solubility and oxygen-binding properties are very different from HbS (Ryan et al, 1990). As discussed earlier, for the disease sickle anaemia to be manifest, one needs cells in which the vast bulk of the haemoglobin is the polymerizing species. In addition it is not clear that the mouse, with its

SICKLE CELL DISEASE PATHOPHYSIOLOGY

73

characteristic microvasculature, oxygen metabolism and erythrocyte properties, is a good model for human physiology. It is possible, however, that further genetic engineering--including the very important 'knockout' of the mouse haemoglobins through homologous recombination techniques (Shesely et al, 1991)--and even the use of other animals as models (Wall, 1989; Wall et al, 1991) may eventually lead to the development of an animal model for sickle cell disease. These animals would be very valuable for the studies of the vascular pathophysiology, in vivo behaviour of the sickle erythrocytes, the effects of various pharmacological approaches to therapy and perhaps gene expression and gene transfer therapies.
SICKLE ERYTHROCYTE MEMBRANE

The erythrocyte membrane plays an important role in regulating the ion and water content of the cell and in maintaining the cell volume and intracellular haemoglobin concentration (Agre, 1992). The acquired membrane defects and their potential pathogenic role in sickle cell disease have been summarized recently (Hebbel, 1991). Some SS erythrocytes sustain membrane damage which renders them poorly deformable (Fortier et al, 1988). These may arise through a variety of mechanisms including oxidative damage to membrane proteins. A number of these erythrocytes suffer impaired transport of K ÷ and Ca 2÷, are permanently deformed even in the presence of complete oxygenation and, as noted above, are termed 'irreversibly sickled cells' (ISCs). ISCs have an increased Ca 2÷ content, an increased intracellular haemoglobin concentration and an increased tendency toward haemolysis (Ortiz et al, t986). It has been proposed that ISCs have a noncovalent rearrangement of the spectrin-actin cytoskeleton, resulting from repeated cycles of sickling, and producing abnormally rigid cells. The irreversible membrane damage may include abnormal membrane protein phosphorylation or some other noncovalent modification, a calcium effect on the cytoskeleton or a direct interaction with haemoglobin S (Fairbanks et al, 1983; Apovo et al, 1989). Except for haemolytic anaemia, a correlation between ISCs and disease severity has not been demonstrated. It is possible that the increased tendency toward haemolysis of the ISC results in its accelerated removal from the circulation and minimizes its role in microvascular obstruction. The role of membranes in the polymerization process remains uncertain (Noguchi and Schechter, 1985; Eaton and Hofrichter, 1987). Measurements of polymer formation in the intact erythrocyte using NMR (discussed above) suggest that the amount of polymer is determined primarily by the haemoglobin concentration and composition and by the oxygen saturation, and that contributions of the membrane to amount of haemoglobin polymer is not significant (Noguchi et al, 1980) although the membrane may affect polymer alignment. One integral membrane protein, Band 3, has been shown to bind haemoglobin in the region of the 2,3-diphosphoglycerate pocket (Walder et al, 1984). The role of this interaction in haemoglobin S polymerization (Liu et

74

C. T. NOGUCHI ET AL

al, 1991) and the role of the membrane or membrane-associated proteins serving as nucleation sites for polymerization (thus affecting kinetics but not thermodynamics) have been suggested, but have been difficult to demonstrate (Gotdberg et al, 1981; Mizukami et al, 1986). HbS is more mechanically unstable than H b A and more readily denatured (Hebbet, 1990). Several membrane changes, particularly oxidative damage to the membrane, have been attributed in part to the increase of deposition of native or denatured haemoglobin or Heinz bodies on or near the inner surface of the membrane. The generation of superoxide free radical resulting from denatured or unstable HbS, including from released haem groups or iron atoms, has been proposed as an important mechanism for membrane damage (Hebbel, 1990). Sickle erythrocytes have been observed to adhere to endothelium in tissue culture and in perfusion studies (Francis and Johnson, 1991). Fibrinogen, fibronectin and possibly other acute-phase reactants can facilitate this adherence which appears to be sensitive to the suspending medium but oxygen independent (Mohandas and Evans, 1985; Smith and LaCelle, 1986; Wick et al, 1987). Since adherence of erythrocytes to endothelium is variable and also dependent on shear forces present in the microcirculation, the significance of these findings in the evolution of vaso-occlusion remains uncertain. Repeated cycles of haemoglobin S polymerization lead to a number of membrane changes (Ohnishi, 1983). These may include rearrangement of phospholipid with an increase of aminophospholipids on the outer surface of the membrane compared to normal erythrocytes, particularly upon deoxygenation (Franck et al, 1985). Decreased phospholipid diffusion in sickle erythrocytes has also been observed (Zachowski et al, 1985). The ISC or SS erythrocytes with damaged membranes which are permanently deformed even in the presence of full oxygenation are found predominantly in the dense cell fraction (Berries and Milner, 1968). The change in ionic fluxes that occurs with erythrocyte deoxygenation (Joiner et al, 1988) and HbS polymerization may be responsible for the dense cell formation and the eventual formation of the ISC. Alternatively, it may be membrane damage that accelerates the cation and water loss which gives rise to the increase in cell density (Horiuchi et al, 1988). Owing to the increase in cell density or intracellular haemoglobin concentration, it is likely that these cells, along with the general dense cell population, will contain HbS polymer even at the high oxygen saturations found on the arteriolar side of the circulation (Noguchi and Schechter, 1981). The ISC and dense cell fractions have a disproportionate effect on filtration of SS erythrocytes and appear to increase the haemolytic anaemia in sickle cell disease (McCurdy and Sherman, 1978; Keidan et al, 1989; Mackie and Hochmuth, 1990).
PATHOPHYSIOLOGY AND THERAPEUTIC STRATEGIES

The following may serve as a brief synopsis of the current pathophysiological model or paradigm for understanding the clinical manifestations of sickle

SICKLE CELL DISEASE PATHOPHYSIOLOGY

75

cell disease. As the red cell traverses the body to deliver oxygen from the lungs to the tissue, the partially deoxygenated HbS can polymerize as a consequence of the reduced solubility of deoxygenated HbS which is only about half that of the haemoglobin concentration within the mature erythrocyte (Schechter et al, 1987). The haemoglobin S aggregation which dramatically increases the intracellular viscosity can distort the morphology of the red cell, but the more important change is the marked decrease in the ability of these ceils to flow through the circulation and microvasculature. Other cellular changes such as decreases in cell water (resulting in dense ceils), alteration of ion balance, decreased membrane deformability and membrane damage (resulting in ISCs) can also occur as consequences of HbS polymerization and a small decrease in HbS stability (Hebbel, 1990, 1991). It is likely that obstruction can occur both in the capillary beds as well as in the precapillary arterioles, resulting in compromised oxygen delivery and blood flow. The decreased deformability of the erythrocyte and obstruction to flow eventually leads to premature cell destruction (resulting in anaemia) and tissue damage. Adherence of sickle cells to the endothelium may exacerbate these processes by allowing deoxygenation to increase. Sickle cell anaemia includes a broad spectrum of severity (Serjeant, 1992; Bunn and Forget, 1986) (Table 2). A number of factors have been identified
Table 2. Possible modifiers of sickle cell disease.

Genetic HbF: hereditary persistence of HbF, HbF levels, F-cells et-Thalassaemia: coexistence of (aod-a) or ( - a / - e t ) genotypes 13-Thalassaemia: heterozygous for 13 °-, 13+-thalassaemia Double heterozygosity: C, D or O-Arab haemoglobinopathy Cellular Intracellular haemoglobin concentration: MCHC, density distribution Other intracellular components: 2,3-disphosphoglycerate, pH, Ca2÷ Erythrocyte membrane abnormalities: ISCs, ionic pumps, lipids, proteins Adhesion: endothelial, leukocyte, platelet Physiological Humoral factors: yon Willebrand factor, integrins, cytokines Microvasculature: arteriovenous shunts, collateral circulation, vessel tone, tissue oxygen levels, ambient PO2, oxygen extraction, nitric oxide (?)
HbF, fetal haemoglobin; MCHC, mean corpuscular haemoglobin concentration; ISC, irreversibly sickled cells. Modified from Schechter et al (1987).

which can modify the course of sickle cell disease. These include a large number of other genetic variables (that can be co-inherited with the sickle cell gene such as other mutant haemoglobins and thalassaemia); cellular factors which affect cell volume, haemoglobin concentration, pH, ion balance and other cellular constituents; and other physiologic factors such as vascular tone. Understanding the biophysical nature of sickle cell anaemia and the factors which affect disease severity has given rise to a number of therapeutic strategies (Dean and Schechter, 1978; Schechter et al, 1987; Stamatoyannopoulos and Nienhuis, 1992).

76

C. T. NOGUCHIET AL
Table 3. Approaches to specifictherapy of sickle cell disease.

Inhibitionof polymerization Haemoglobin modification Non-covalent reagents Non-specificalteration of solvent Stereospecificcompetitors (peptides, modifiedamino acids) } 1' deoxyHbS solubility Covalent reagents Amino-terminalresidues (cyanate, pyridoxal) ~" 02 affinity Side chains(glyceraldehyde,acetylationagents) or ~' deoxy-HbS Diphosphoglycerate-bindingsite solubility Erythrocyte modification Increase cell volume Hyponatraemia(DDAVP) | MCH(S)C Membrane modifiers(Cetiedil)} and Modifyion transport | MCHC Decrease 2,3-diphosphoglycerate] Genetic modification Increase "ygene expression DNA methylationinhibitors (5-azacytidine) S-phase cell cycleinhibitors (hydroxyurea) ~ MCH(S)C Differentiatingagents(butyrate, phenylacetate) Gene therapy Viral-mediatedgene transfer into erythroid stem cells Site-specifichomologousrecombinationinto stem cells Bone marrow transplantation

}

}

Decrease mierovascular entrapment

Vasodilators Cell adhesioninhibitors

Modifiedfrom Schechteret al (1987).

The strong dependence of HbS polymerization on haemoglobin composition, haemoglobin concentration, oxygen saturation and the intrinsic solubility of deoxygenated HbS have provided several rational approaches for therapy in sickle cell anaemia (Table 3; Figures 9 and 10).
Haemoglobin S modification

Early attempts at therapy for sickle cell disease focused on increasing the deoxygenated HbS solubility (Dean and Schechter, 1978). The goal is not to eliminate HbS polymerization completely, but to reduce the polymerization potential to levels observed in the more mild sickle syndromes such as HbS/f3+-thalassaemia (with 70% HbS), or better sickle trait (with 40% HbS), where the solubility of deoxygenated haemoglobin from individuals with sickle trait is about 1.5 times that of pure deoxygenated HbS (Sunshine et al, 1978; Noguchi et al, 1981; Noguchi et al, 1988) (Figure 11). Agents known to disrupt hydrophobic interactions such as urea were initially proposed to minimize interactions with the 13S6valine (Murayama, 1966; Cooperative Urea Trials Group, 1974). Other agents known to alter solvent effects, such as ethanol, aromatic alcohols and acids and alkylureas

SICKLE CELL DISEASE PATHOPHYSIOLOGY

77

SS
HbS:IO0%

I

io, O-lo,o~ Iocto~o,o_olP oo~oOoOoOI 0 lip 000 iio~t~ol ,o, Io~o^o_o,o21 _oo~o~1 I__lO _ 3o~O~ ioo ooo- Iooo IOoORoO~:x:)o ~'
12=oollt%~ I Ii°-°: - 0 0 ' T - i l O llO01 oi oo-o-*
,o. o o . . o o . 0 , oOto_O#oO_Ol P • O _ o o O - o o I loll ~ oO~llol l i o o ! o o n ' l o l l IO~lo ~ - i o l o l l io o O T o o ~ t ~o o 1~3 ..O j300CLO0(

# OOo #o i
o~o~ oYo°_4 OpoQp~

SF
HbF=25% HbS=75%

• , o i S = , R ] E6~.~-° ° o:° el
IuoI ll~Ol O0_llOll~ 0 I

OoOoN% ,
0 0-o0 0 0 ~

AS
HbA=60% HbS=40%

I~ eeo e:o?~l

po?.po o moo q I~ cY,-~Oo ~ o 0 o l

,°o~iOi~ I:Oo_?,oi1 ,o_i5o~ o-o~ 00 O0
J " l l " llll~-I~ll-- I l e o l o ipOeo-e I .'~(:~@O 0 0 u C )"P~O OjOOq 0~0 IIu 0 • (

I~,-w~oouoo 0 I

0%

40% 70% Oxygen saturation

100%

Figure 9. Representation of haemoglobin polymerization at equilibrium for a haemoglobin concentration of 34 g/dl. Oxygen saturation varies (from left to right) from 0%, 40%, 70% and 100%. For illustration, haemoglobin composition includes 100% haemoglobin S (top) to model homozygous SS individuals, 75% haemoglobin S with 25% haemoglobin F (middle) to model a pancellular distribution of a high level haemoglobin F and 40% haemoglobin S with 60% haemoglobin A (bottom) to model sickle trait individuals. Polymer formation is maximal for 100% haemoglobin S, decreases with increasing oxygen saturation and is still present at 70% oxygen saturation. Increasing levels of haemoglobin F reduces polymer formation and at 25 % haemoglobin F begins to approach levels associated with sickle trait with 60% haemoglobin A and 40% haemoglobin S.

were found to increase deoxygenated HbS solubility (Waterman et al, 1974; Ross and Subramanian, 1977; Elbaum et al, 1978), but at levels too low for therapeutic benefit. Inhibitors based on the structure of the HbS polymer were designed as stereospecific inhibitors (Schechter et al, 1987). Among these are the aromatic amino acids and short peptides including the oligopeptide mimicking the 13S6valine region (Kubota and Yang, 1977; Noguchi and Schechter, 1979; Votano et al, 1984). While an increase in solubility was observed in some of these agents, other short peptides decreased deoxygenated HbS solubility, presumably because of the nonideal behaviour due to molecular crowding of the concentrated HbS solution required for polymerization (Noguchi et al, 1985). Further increases in efficacy and perhaps specificity are necessary for these compounds and their chemical analogues to be of potential therapeutic value. In general, the covalent and non-covalent inhibitors of haemoglobin S polymerization must be potent enough to be useful at levels well below their toxicity, be specific enough to affect only deoxy-HbS solubility, and be taken up by the erythrocyte (or be able to be delivered to the erythrocyte using such techniques as lipofusion (Kumpati, 1987) or extracorporeal treatment (Cooperative Urea

78

C. T. NOGUCHI ET AL

oo gOoO°
OoOoO o
000 0
0,-,112

100% HbS


.

I"
• •
•DGNDI • .

75% HbS and 25% HbF

0[]00 0 O 0[3 ~ 0 []

0 [Do
formation
Hybrid

~0~ O~ 0

0 i_.1 oOgO (]]

(~

[~)

,1+02

-02

o o

O%oo
O = HbS ] [] = HbF (]] = HbS/HbFhybrid

•.,







Figure 10. Sparing effect of haemoglobin F (HbF). Deoxygenation of a pure solution of

haemoglobin S near physiologic conditions, or of cells of average haemoglobin content and little HbF results in a polymer fraction of about 0.7. Mixture of HbS and HbF contain haemoglobin tetramers of HbS (a213s2),HbF (ot2~2)and the hybrid haemoglobin (a213s'y).The sparing effect of HbF results from the fact that neither HbF nor the hybrid haemoglobin (u213s~) enters into the polymer phase unlike "HbAin which the hybrid haemoglobin (a213s13) can enter the polymer, but at reduced tendency. Hence, for a mixture of 75% HbS and 25% HbF near physiologic conditions, deoxygenation results in a polymer fraction of 0nly 0.4. Factors which increase HbF tend to cause a reduction in HbS, for reasons that are not entirely clear, and this fact should also be considered as part of the sparing effect. Trials G r o u p , 1974) ) with a sufficiently long resident time in the erythrocyte relative to the erythrocyte life span (Schechter et al, 1987).

Sickle erythrocyte modifications
The strong dependence of H b S polymerization on corpuscular haemoglobin concentration suggests that increasing cell water or decreasing the intracellular H b S concentration would provide some reduction in the polymerization potential of the erythrocyte (Izumo et al, 1987). Preliminary studies using medically induced hyponatraemia by strict regimen of fluid limitation and the administration of desmopressin acetate resulted in the osmotic swelling of the erythrocyte with a reported reduction in the frequency of crises (Rosa et al, 1980). Although limited in scope, these early results illustrate the potential of increasing cell water or decreasing M C H C (mean corpuscular haemoglobin concentration) as a therapeutic strategy. Several other pharmacological agents which affect red cell m e m b r a n e and erythrocyte ion balance have been proposed as ' m e m b r a n e active' treatments (Benjamin et al, 1980; Clark et al, 1982; A s a k u r a et al, 1984; Johnson et al, 1989; Ohnishi et al, 1989; Vitoux et al, 1989; Joiner, 1990).

SICKLE

CELL

DISEASE

PATHOPHYSIOLOGY

79
1.0
J v • i | v i i =

1.0

......... ,

,

,

,

,

.

.

.

.

z

_o
ecer" ILl 0 n
0
0 0.5

a

b

0
1.0 0

OXYGEN SATURATION
z

1.0 0.5 OXYGEN SATURATION
, , i i i ~ r

'c
%

d
~ b 6 ~

(.) e¢-

0

0

0.5 1.0 0 0.5 1.0 OXYGEN SATURATION OXYGEN SATURATION

'

0

J

Figure 11. Predictions for expected polymer formation versus oxygen saturation for several conditions reflecting potential therapies for sickle cell disease. The predictions were calculated from total haemoglobin concentration and haemoglobin composition and oxygen saturation. (a) Polymer fraction predicted for pure haemoglobin S at 26, 30, 34, 38 and 42 g/dl. For a whole cell population of sickle erythrocytes, cell heterogeneity will skew the polymer curve to the right due to the higher polymerization potential of dense cells (see Figure 4). (b) Polymer fraction predicted for homozygous sickle cell disease, without (SS) and with (SS/(-od-a)) homozygous ~t-thalassaemia, and for sickle trait (AS) based on the mean corpuscular haemoglobin concentration and mean percentages of haemoglobins S, F, A2 and A. (c) Polymer fraction predicted for haemoglobin concentration of 34 g/dl for Hb S and F mixtures for increasing amounts of HbF from 0 to 40%. (d) Polymer fraction predicted for haemoglobin concentration of 34 g/dl with increasing solubility of deoxyhaemoglobin S from 16 to 26 g/dl. From Noguchi et al (1989). While s o m e o f these agents m a y increase cell water, their effects o n o t h e r sickle e r y t h r o c y t e properties, cell survival or clinical benefit remain to be tested. T h e increase in o x y g e n affinity p r o p o s e d b y inactivation o f 2,3d i p h o s p h o g l y c e r a t e (Poillon a n d Kim, 1990) o r by chemical agents such as cyanate which m o d i f y h a e m o g l o b i n (Nigen et al, 1974) has the potential to decrease the extent o f p o l y m e r i z a t i o n at any o x y g e n tension by reducing the

80

C. T. NOGUCHI ET AL

proportion of deoxygenated sickle haemoglobin (Beddell et al, 1984; Franklin et al, 1986; Abraham et al, 1991). Preliminary trials with cyanate including extracorporeal administration resulted in a decrease in haemolysis and anaemia associated with sickle cell disease, but no significant reduction in painful crises was observed (Balcerzak et al, 1982; Lee et al, 1982). Because of the uncertainties regarding absolute oxygen delivery to the tissues, the therapeutic value of increasing HbS oxygen affinity remains to be demonstrated.

Genetic approaches
The approach to therapy of the disease aimed at augmenting fetal haemoglobin synthesis was predicated on clinical and epidemiological observations of geographical groups of sickle cell patients demonstrating a milder clinical course when the HbF level exceeded 20% (Noguchi et al, 1988). Though previous studies involving a few hundred patients concluded that HbF levels substantially lower than the 20%, which is commonly found among US sickle cell individuals imparted a minimal effect on disease manifestations (Powars et al, 1984), the more recent analysis of data obtained on several thousand patients in the Cooperative Study of Sickle Cell Disease suggests an ameliorating effect on the pain rate is demonstrable with only modest increases in steady-state HbF (Platt et al, 1991). Biophysical studies have now delineated the quantitative bases for these observations. An increase in the proportions of non-S haemoglobins, particularly HbF, within a red cell is beneficial since, as mentioned above, it effectively reduces the intracellular concentration of HbS and the mixed hybrids of HbS and HbF do not enter the polymer. This 'sparing' effect conferred by HbF on HbS polymerization therefore would tend to normalize the rheological properties of the sickle erythrocyte in which it is contained (Schechter et al, 1987) (Figures 9 and 10). The initial attempts to 'reactivate' the synthesis of HbF, were extensions of observations made in cell cultures that eukaryotic gene expression was related (at least in part) to the extent of methylation within and around the gene of inquiry (Fesel, 1985). Thus, 5-azacytidine (5-Aza C), a potent inhibitor of DNA methylation, was administered to anaemic primate models (DeSimone, 1982), and subsequently to patients with B-thalassaemia (Ley et al, 1982) and sickle cell disease (Charache et al, 1983; Ley et al, 1983) and was found to increase dramatically the synthesis of HbF. Indeed, baboons who were maintained chronically anaemic through regular phlebotomy, after receiving subcutaneous courses 5-Aza C, were able to increase consistently the synthesis of HbF (up to 70-80%), while reciprocally decreasing [3-globin synthesis (DeSimone, 1982). Phenotypically, these changes effectively recapitulated a reversal in the fetal-to-adult haemoglobin switch and, therefore, provided the rationale for its experimental application to patients with [3-thalassaemia and sickle cell anaemia. Following short courses of parenteral 5-Aza C, sickle cell patients have invariably shown an increase in percentage HbF and in F-cell numbers, generally within 2-3 days from the initiation of therapy and reaching maximal values of four to seven times baseline values (Charache et al, 1983;

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Ley et al, 1983). Such enhancement in HbF production has been associated when measured, with a decrease in the proportion of dense red cells and in the rate of haemolysis (Ley et al, 1983). Other S-phase specific drugs such as arabinosylcytosine (Ara C) (Veith et al, 1985a), vinblastine (Veith et al, 1985b) and myleran (Liu et al, 1990) have been also found to be effective in stimulating HbF production, but because of their perceived carcinogenic potential, especially given the requisite chronicity of administration to maintain elevated HbF levels in these patients, their use has been curtailed except in individuals with end-stage ~-thalassaemia (Lowery and Nienhuis, 1991). Hydroxyurea (HU) is a cytostatic agent, useful for the treatment of myeloproliferative disorders (Alter and Gilbert, 1985) and head and neck neoplasms (Donehower, 1992), by virtue of its inhibitory effect on the enzyme ribonucleotide reductase which results in the perturbation of DNA synthesis in rapidly dividing cells. Alter and Gilbert (1985) first reported that patients with chronic myelogenous leukaemia treated palliatively with HU showed substantial elevations in HbF levels above baseline, with those on daily therapy attaining greater levels of HbF than those on intermittent treatment. Letvin et al (1984) demonstrated that HU could augment HbF levels in a primate model which led to its successful application to sickle cell patients in small pilot studies (Platt et al, 1984; Charache et al, 1987; Dover and Charache, 1989). To date, nearly 100 SS patients have been entered into clinical trials with HU (Goldberg et al, 1990; Rodgers et al, 1990; Charache et al, 1992), which has substantiated this agent to be a potent inducer of HbF production. Several general and relevant conclusions can be drawn from these trials which have included treatment periods from 3 months (Rodgers et al, 1990) to several years (Charache et al, 1992). First, under close medical supervision most patients, but not all, will respond to HU with at least doubling from baseline in the HbF and F-reticulocyte numbers. The 20-25% of non-responders (or poor responders) cannot be distinguished at the present time by current haematological, biochemical or molecular analyses. Second, in contrast to the response to 5-Aza C, increases in HbF production on HU occur more gradually (over weeks) with some patients not attaining a plateau on a stable dose of HU for several months. Third, the HbF response to HU occurs near myelotoxic dosages and the magnitude of this response can be quite variable, with the best responders achieving maximal HbF levels of 15-20%, invariably associated with a striking macrocytosis. Anecdotal experience accumulated during these trials suggests that a substantial number of these responders will experience fewer, less severe crisis and an increased sense of well-being. The subjective nature of these symptoms, however, dictates that HU should be viewed as an experimental agent pending the outcome of controlled clinical trials. In view of the variability of the response to HU, together with the myelotoxicity observed at the optimal dose of HU, other agents, including cytokines which exert an independent effect on HbF production have been studied for their potential synergistic effects. The observations that recombinant human erythropoietin (Epo) alone (A1-Khatti et al, 1988) or in

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combination with HU (McDonagh et al, 1989) may result in substantial increases in both F-cell number and HbF levels in a primate model raised the possibility that Epo may exert an additive or synergistic effect to HbF stimulation. An initial report involving a small number of sickle cell patients showed no additive effect of Epo when administered to patients receiving daily HU (Goldberg et al, 1990). More recently, it has been found that the timing of the recombinant Epo with HU, the absolute dose of Epo, and/or the concomitant administration of oral iron with Epo therapy are important variables that determine the response to this form of combination therapy (Rodgers et al, 1993). Figure 12 demonstrates the HbF and F-reticulocyte response to HU alone and HU alternating with Epo (given with supplemental iron).

Patient I HU->HU/EPO
50 r HU . . %F-retics . . ~ 20 40 30
IJ-

1


....... ~'-"~-

-'0-'- %HbF

1
LL

o~

f 20

;
o~0< --200

i ;:



0 --300

-t00 Treatment Day

0

100

Figure 12. Response of patient with sickle cell anaemia to combination therapy of hydroxyurea and erythropoietin. Treatment with hydroxyurea (HU, as indicated) alone caused an increase in percentage reticulocytes containing haemoglobin F (F-retics) followed by an increase in percentage HbF levels. Supplementation of hydroxyurea with erythropoietin (Epo, as indicated) resulted in further increases in percentage F-retics and percentage HbF. When erythropoietin was stopped and the patient continued on hydroxyurea therapy alone, percentage F-retics and percentage HbF levels gradually decreased to levels obtained with hydroxyurea alone before erythropoietin therapy. From Rodgers et al (1993).

The butyrate analogues (sodium butyrate, butyric acid, a-amino butyrate) have long been known to be potent inducers of differentiation in haematopoietic cells. These compounds have been shown to retard the normal fetal-to-adult haemoglobin switch when infused in utero into the umbilical veins of developing sheep fetuses (Perrine et al, 1988). Subsequently, sodium butyrate has been used clinically in a small pilot (Phase II) study in which children with sickle cell anaemia and t3-thalassaemia have received several day infusions, with promising results (Perrine et al, 1993). More recently, a report has suggested that phenylbutyrate, which has previously been used as

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treatment for patients with urea cycle defects, results in an augmentation in HbF production suggesting a potential role for this agent in sickle cell disease (Dover et al, 1992). Unlike the myelosuppressive effects noted with the cytotoxic agents, administration of the butyrate analogues has been largely free of this side-effect making them attractive candidates for trials in paediatric and adolescent patients, or in combination with the others. Finally, molecular therapy directed at substituting for the defective 13S-gene in the sickle cell syndromes is theoretically attractive but at the present time not feasible. Formidable, though not insurmountable obstacles to overcome include the development of better delivery systems to allow for more efficient gene insertion, as well as improvement on the current physical means to isolate, purify and amplify pluripotent haemopoietic stem cells. Nonetheless, since many of the principles of eukaryotic molecular genetics were derived from the analysis of primary and cultured erythroid cells (Berg and Schechter, 1992), it is likely that genetic engineering will be applied to the therapy of the severe [3-globin disorders. In the meanwhile, it is likely that therapies based on modulation of (existing) gene expression, as discussed above, offer the most promise in favourably altering the underlying pathogenic mechanism in sickle cell anaemia and its genetic variants.
SUMMARY

The primary pathophysiological event in the erythrocytes of individuals with the various sickle syndromes is the intracellular aggregation or polymerization of sickle haemoglobin (HbS). The extent of polymerization is determined by the intracellular haemoglobin composition (% HbS and % HbS A, A2 and F), concentration (MCHC and % of dense cells) and oxygen saturation, as well as minor factors such as intracellular pH and DPG concentration. Intracellular HbS polymerization leads to a marked decrease in the flexibility or rheological properties of the sickle erythrocytes and obstruction in various microcirculatory beds, as well as chronic anaemia. Other abnormalities in the properties of the sickle erythrocytes, including membrane abnormalities, changes in ion fluxes and volume and endothelial adhesion, result from acute and chronic oxygen-linked polymerization events and may, in turn, modify polymerization. However, within a good approximation, many aspects of sickle cell disease pathophysiology--for example variations in anaemia among the different sickle syndromes---can be explained in terms of differences in polymerization tendency. Thus, the effects of ot-thalassaemia can be explained with reference to changes in MCHC and syndromes with high HbF are understandable in terms of the sparing effect of HbF on polymerization. Recent therapeutic approaches to sickle cell disease focus on attempts to reduce intracellular HbS polymerization by altering the haemoglobin molecules, erythrocyte properties, or the distribution of intracellular haemoglobin species. The last, through pharmacological elevation of HbF, has become the central focus of much laboratory and clinical research in recent years. Agents such as hydroxyurea (with or without recombinant

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erythropoeitin) and butyrate compounds elevate HbF (and reduce HbS) in a majority of sickle erythrocytes, thus decreasing intracellular polymerization. Current prospective protocols are designed to see if these changes cause clinical improvement at acceptable doses. Other treatment strategies, including bone marrow transplantation and possible gene replacement therapies, are also under active clinical or laboratory investigation. REFERENCES
Adekile AD (1992) Anthropology of the beta S gene-flow from West Africa to north Africa the Mediterranean, and southern Europe. Hemoglobin 16: 105-121. Agre P (1992) Clinical relevance of basic research on red cell membranes. Clinical Research 40: 176-186. A1-Khatti A, Papayannopoulou T, Knitter G, Fritsch EF & Stamatoyannopoulos G (1988) Cooperative enhancement of F-cell formation in baboons treated with erythropoietin and hydroxyurea. Blood 72: 817-819. Allison AC (1954a) The distribution of the sickle-cell trait in East Africa and elsewhere, and its apparent relationship to the incidence of subtertian malaria. Transactions of the Royal Society of Tropical Medicine and Hygiene 48: 312-318. Allison AC (1954b) Protection afforded by sickle-cell trait against subtertian malarian infection. British Medical Journal 1: 290-294. Allison AC (1954c) Notes on sickle-cell polymorphism. Annals of Human Genetics 19: 39-51. Allison AC (1957a) Parasitological Reviews. Malaria in carriers of the sickle-cell trait and in newborn children. Experimental Parasitology 6: 418-447. Allison AC (1957b) Properties of sickle-ceU hemoglobin. Biochemical Journal 65: 212-219. Alter BP & Gilbert HS (1985) The effect of hydroxyurea on hemoglobin F in patients with myeloproliferative syndromes. Blood 66: 373-379. Apovo M, Gascard P, Rhoda MD, Beuzard Y & Giraud F (1989) Alteration in protein kinase C activity and subcellular distribution in sickle erythrocytes. Biochimica et Biophysica Acta 984: 26-32. Asakura T, Shibutani Y, ReiUy MP & DeMeio RH (1984) Antisickling effect of tetlurite: a potent membrane-acting agent in vitro. Blood 64: 305-307. Balcerzak SP, Grever MR, Sing DE, Bishop JN & Segal ML (1982) Preliminary studies of continuous extracorporeal carbamylation in the treatment of sickle cell anemia. Journal of Laboratory and Clinical Medicine 100: 345-355. Baldwin J & Chothia C (1979) Hemoglobin: the structural changes related to ligand binding and its allosteric mechanism. Journal of Molecular Biology 129: 175-220. Ballas SK (1991) Sickle cell anemia with few painful crises is characterized by decreased red cell deformability and increased number of dense cells. American Journal of Hematology 36: 122-130. Beddell CR, Goodford P J, Kneen G, White RD, Wilkinson S & Wootton R (1984) Substituted benzaldehydes designed to increase the oxygen affinity of human haemoglobin and inhibit the sickling of sickle erythrocytes. British Journal of Pharmacology 82: 397-407. Benesch RE, Edalji R, Benesch R & Kwong S (1980) Solubilization of hemoglobin S by other hemoglobins. Proceedings of the National Academy of Sciences, USA 77: 5130-5134. Benjamin LJ, Kokkini G & Peterson CM (1980) Cetiedil: its potential usefulness in sickle cell disease. Blood 55: 265--270. Berg P & Schechter AN (1992) The impact of molecular biology on the diagnosis and treatment of hemoglobin disorders. In Friedmann T (ed.) Molecular Genetic Medicine, Vol. 2, pp 1-38. San Diego, CA: Academic Press. Bertles JF & Milner PFA (1968) Irreversibly sickled erythrocytes: a consequence of the heterogeneous distribution of hemoglobin types in sickle-cell anemia. Journal of Clinical Investigation 47: 1731-1741. Bertles JF, Rabinowitz R & DObler J (1970) Hemoglobin interaction: modification of solid phase composition in the sickling phenomenon. Science 169: 375-377.

SICKLE CELL DISEASE PATHOPHYSIOLOGY

85

Bessis M (ed.) (1977) Red cell rheology. Blood Cells 3(1): 229. Bessis M (ed.) (1982) Sickle cells. Blood Cells 8(9): 201. Billett HH, Fabry ME & Nagel RL (1988) Hemoglobin distribution width: a rapid assessment of dense red cells in the steady state and during painful crisis in sickle cell anemia. Journal of Laboratory and Clinical Medicine 112: 339-344. Bookchin RM & Lew VL (1984) Red cell membrane abnormalities in sickle cell anemia. In Brown E (ed.) Progress in Hematology, pp 1-24. New York: Grune & Stratton. Bookchin RM, Balazs T & Landau LC (1976) Determinants of red cell sickling. Effects of varying pH and of increasing intracellular hemoglobin concentration by osmotic shrinkage. Journal of Laboratory and Clinical Medicine 87: 597-616. Bookchin RM, Ortiz OE & Lew VL (1991) Evidence for a direct reticulocyte origin of dense red cells in sickle cell anemia. Journal of Clinical Investigation 87: 113-124. Briehl RW (1983) Rheology of hemoglobin S gels: possible correlation with impaired microvascular circulation. American Journal of Pediatric HematoIogy/Oncology 5: 390-398. Briehl RW, Mann ES & Josephs R (1990) Length distribution of hemoglobin S fibers. Journal of Molecular Biology 211: 693-698. Brittenham GM, Schechter AN & Noguchi CT (1985) Hemoglobin S polymerization: primary determinant of the hemolytic and clinical severity of the sickling syndromes. Blood 65: 183--189. Brugnara C, Bunn HF & Tosteson DC (1986) Regulation of erythrocyte cation and water content in sickle cell anemia. Science 232: 388-390. Bunn HF & Forget BG (1986) Hemoglobin: Molecular, Genetic and Clinical Aspects. Philadelphia: WB Saunders. Bunn HF, Noguchi C'T, Hofrichter HJ, Schechter GP, Schechter AN & Eaton WA (1982) Molecular and cellular pathogenesis of hemoglobin SC disease. Proceedings of the National Academy of Sciences, USA 79: 7527-7531. Charache S, Dover G, Smith K, Talbot CC, Moyer M & Boyer S (1983) Treatment of sickle cell anemia with 5-azacytidine results in increased fetal hemoglobin production and is associated with non hypomethylation of DNA around the gamma-delta-beta-globin gene complex. Proceedings of the National Academy of Sciences, USA 8t1: 4842-4846. Charache S, Dover G, Moyer M & Moore J (1987) Hydroxyurea-induced augmentation of fetal hemoglobin production in patients with sickle cell anemia. Blood 69: 109-116. Charache S, Dover GJ, Moore RD et al (1992) Hydroxyurea: effects on hemoglobin F production in patients with sickle cell anemia. Blood 79: 2555--2565. Cheetam RC, Huehns ER & Rosemeyer MA (1979) Participation of hemoglobins A, F, A2 and C in polymerisation of hemoglobin S. Journal of Molecular Biology 129: 45-61. Clark MR, Unger RC & Shohet SB (1978) Monovalent cation composition and ATP and ligand content of irreversibly sickled cells. Blood 51: 1169-1178. Clark MR, Mohandas N & Shohet SB (1980) Deformability of oxygenated irreversibly sickled cells. Journal of Clinical Investigation 65: 189-196. Clark MR, Mohandas N & Shohet SB (1982) Hydration of sickle cells using the sodiumionophore Monensin. A model for therapy. Journal of Clinical Investigation 70: 1074--1080. Cooperative Urea Trials Group (1974) Treatment of sickle cell crisis with urea in invert sugar. A controlled trial. Cooperative Urea Trials Group. Journal of the American Medical Association 228:1125-1128. Cottam GL, Valentine KM, Yamaoka & Waterman MR (1974) The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relaxation measurements. Archives of Biochemistry and Biophysics 162: 487--492. Crepeau RH, Edelstein SJ, Szalay M et al (1981) Sickle cell hemoglobin fiber structure altered by a-chain mutation. Proceedings of the National Academy of Sciences, USA 78: 14061410. Dean J & Schechter AN (1978) Sickle cell anemia: molecular and cellular basis of therapeutic approaches. New England Journal of Medicine 299: 752-763, 804-811,863-870. DeSimone J, Heller P, Hall L & Zwiers D (1982) 5-Azacytidine stimulates fetal hemoglobin synthesis in anemic baboons. Proceedings of the National Academy of Sciences, USA 79: 4428--4431. Dickerson RE & Geis I (1983) Hemoglobin. Menlo Park, CA: Benjamin/Cummings. Diggs LW (1932) The blood picture in sickle cell anemia. Southern Medical Journal 25: 615-620.

86

C, T. NOGUCHI ET AL

Donehower RS (1992) An overview of the clinical experience with hydroxyurea. Seminars in Oncology 19 (suppl 9): 11-19. Dover G & Charache S (1989) Stimulation of fetal hemoglobin production by hydroxyurea in sickle cell anemia. In Nienhuis A & Stamatoyannopoulos G (eds) Hemoglobin Switching, Part B: Cellular and Molecular Mechanisms, pp 295--306. Baltimore: Johns Hopkins University Press. Dover GJ, Brusilow S & Samid D (1992) Increased fetal hemoglobin in patients receiving sodium 4-phenylbutyrate. New England Journal of Medicine 327:569-570 (letter). Dykes GW, Crepeau RH & Edelstein SJ (1979) Three-dimensional reconstruction of the 14-filament fibers of hemoglobin S. Journal of Molecular Biology 130: 451--472. Eaton WA & Hofrichter J (1987) Hemoglobin S gelation and sickle cell disease. Blood 70: 1245-1266. Eaton WA & Hofrichter J (1990) Sickle cell hemoglobin polymerization. Advances in Protein Chemistry 40: 263-279. Edelstein SJ (1981) Molecular topology in crystals and fibers of hemoglobin S. Journal of Molecular Biology 150: 557-575. Elbaum D, Harrington JP, Bookchin RM & Nagel RL (1978) Kinetics of HB S gelation. Effect of alkylureas, ionic strength and other hemoglobins. Biochimica et Biophysica Acta $34: 228-238. Evans E, Mohandas N & Leung A (1984) Static and dynamic rigidities of normal and sickle erythrocytes. Major influences of cell hemoglobin concentration. Journal of Clinical Investigation 73: 477-488. Fairbanks G, Palek J, Dino JE & Liu PA (1983) Protein kinases and membrane protein phosphorylation in normal and abnormal human erythrocytes" variation related to mean cell age. Blood 61: 850-857. Ferrone FA, Hofrichter J & Eaton WA (1985) Kinetics of sickle hemoglobin polymerization II. A dual nucleation mechanism. Journal of Molecular Biology 183: 611-631. Ferrone FA, Basak S, Martino AJ & Zhou HX (1987) Polymer domains, gelation models and sickle cell crises. Progress in Clinical and Biological Research 240: 47-58. Fesel YJ (1985) Mode of action and effects of 5-azacytidine and of its derivatives in eukaryotic cells. Pharmacology and Therapeutics 28: 17-27. Fortier N, Snyder LM, Garver F, Kiefer C, McKenney J & Mohandas N (1988) The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity. Blood 71: 1427-1431. Francis RB Jr & Johnson CS (1991) Vascular occlusion in sickle cell disease: current concepts and unanswered questions. Blood 77: 1405-1414. Franck PF. Bevers EM, Lubin BH et al (1985) Uncoupling of the membrane skeleton from the lipid bilayer. The cause of accelerated phospholipid flip-flop leading to an enhanced procoagulant activity of sickled cells. Journal of Clinical Investigation 75: 183-190. Franklin JM, Huehns ER, Rosemeyer MA (1986) Increasing haemoglobin affinity to prevent sickling: abnormal haemoglobin variants as models. British Journal of Haematology 64: 319-329. Friedman MJ (1978) Erythrocyte mechanism of sickle cell resistance to malaria. Proceedings of the National Academy of Sciences, USA 75: 1994-1997. Gill SJ, Spokane R, Benedict RC, Fall L & Wymann JH (1980) Ligand-linked phase equilibria of sickle cell hemoglobin. Journal of Molecular Biology 140: 299-312. Goldberg MA, Lalos AT & Bunn HF (1981) The effect of erythrocyte membrane preparations on the polymerization of sickle hemoglobin. Journal of Biological Chemistry 256: 193-197. Goldberg MA, Brugnara C, Dover GJ et al (1990) Treatment of sickle cell anemia with hydroxyurea and erythropoietin. New England Journal of Medicine 323: 366-372. Greaves DR, Fraser P, Vidal MA et al (1990) A transgenic mouse model of sickle cell disorder. Nature 343: 183-185. Green MA, Noguchi CT, Keidan A J, Marwah SS & Stuart J (1988) Polymerization of sickle cell hemoglobin at arterial oxygen saturation impairs erythrocyte deformability. Journal of Clinical Investigation 81: 1669-1674. Grosveld F, van Assendelft GB, Greaves DR & Kollias G (1987) Position-independent, high-level expression of the human beta-globin gene in transgenic mice. Cell 51: 975-985. Harris JW (1950) Studies on the destruction of red blood cells. VIII. Molecular orientation in

SICKLE CELL DISEASE PATHOPHYSIOLOGY

87

sickle cell hemoglobin solutions. Proceedings of the Society of Experimental Biology 75: 197-201. Harris JW, Brewster HH, Ham TH & Castle WB (1956) Studies on the destruction of red blood cells. X. The biophysics and biology of sickle cell disease. Archives oflnternal Medicine 97: 145-168. Hebbel RP (1990) The sickle erythrocyte in double jeopardy: autoxidation and iron decompartmentalization. Seminars in Hematology 27: 51-69. Hebbel RP (1991) Beyond hemoglobin polymerization: the red blood cell membrane and sickle disease pathophysiology. Blood 77: 214-237. Hofrichter J (1979) Ligand binding and gelation of sickle cell hemoglobin. Journal of Molecular Biology 128: 335-369. Hofrichter J, Ross PD & Eaton WA (1974) Kinetics and mechanism of deoxyhemoglobin S gelation: a new approach to understanding sickle cell disease. Proceedings of the National Academy of Sciences, USA 71: 4864-4868. Hofrichter J, Ross PD & Eaton W A (1976) Supersaturation in sickle cell hemoglobin solutions. Proceedings of the National Academy of Sciences, USA 73: 3035-3039. Horiuchi K, Ballas SK & Asakura T (1988) The effect of deoxygenation rate on the formation of irreversibly sickled cells. Blood 71: 46-51. Horiuchi K, Ohata J, Hirano Y & Asakura T (1990) Morphologic studies of sickle erythrocyte by image analysis. Journal of Laboratory and Clinical Medicine 115: 613--620. Ingram VM (1956) A specific chemical difference between the globins of normal human and sickle-cell hemoglobin. Nature 178: 792-794. Itoh T, Chien S & Usami S (1992) Deformability measurements on individual sickle cells using a new system with pO2 and temperature control. Blood 79: 2141-2147. Izumo H, Lear S, Williams M, Rosa R & Epstein FH (1987) Sodium-potassium pump, ion fluxes, and cellular dehydration in sickle cell anemia. Journal of Clinical Investigation 79: 1621-1628. Jarman AP, Wood WG, Sharpe JA, Gourdon G, Ayyub H & Higgs DR (1991) Characterization of the major regulatory element upstream of the human alpha-globin gene cluster. Molecular and Cellular Biology 11: 4679-4689. Johnston MN, Ellory JC & Stuart J (1989) Bepridil protects sickle cells against the adverse rheological effects of cyclical deoxygenation. British Journal of Haematology 73: 522-526. Joiner CH (1990) Deoxygenation-induced cation fluxes in sickle cells. II. Inhibition by stilbene disulfonates. Blood 76: 212-220. Joiner CH, Dew A & Ge DL (1988) Deoxygenation-induced cation fluxes in sickle ceils: relationship between net potassium efflux and net sodium influx. Blood Cells 13" 339-358. Josephs R, Jarosh HS & Edelstein SJ (1976) Polymorphism of sickle cell hemoglobin fibers. Journal of Molecular Biology 102: 409-426. Kan YW & Dozy AM (1989) Evolution of the hemoglobin S and C genes in world populations. Science 209: 388-391. Kaul DK & Xue H (1991) Rate of deoxygenation and rheologic behavior of blood in sickle cell anemia. Blood 77: 1353--1361. Kaul DK, Fabry ME & Nagel RL (1989) Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications. Proceedings of the National Academy of Sciences, USA 86: 3356--3360. Keidan A J, Noguchi CT, Player M, Chalder SM & Stuart J (1989) Erythrocyte heterogeneity in sickle cell disease: effect of deoxygenation on intracellular polymer formation and rheology of sub-populations. British Journal of Haematology 72: 254-259. Kubota S & Yang JT (1977) Oligopeptides as potential antiaggregation agents for deoxyhemoglobin S. Proceedings of the National Academy of Sciences, USA 74: 5431-5434. Kumpati J (1987) Liposome-loaded phenylalanine or tryptophan as sickling inhibitor: a possible therapy for sickle cell disease. Biochemical Medicine and Metabolic Biology 38: 170-181. Labie D, Srinivas R, Dunda O et al (1989) Haplotypes in tribal Indians bearing the sickle gene: evidence for the unicentric origin of the beta S mutation and the unicentric origin of the tribal populations of India. Human Biology 61: 479--491. Lee MY, Uvelli DA, Agodoa LC, Scribner BH, Finch CA & Babb AL (1982) Chnical studies of a continuous extracorporeal cyanate treatment system for patients with sickle cell disease. Journal of Laboratory and Clinical Medicine 100: 334-344.

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C. T. NOGUCHI ET AL

Lehmann H (1954) Distribution of the sickle cell gene. A new light on the origin of the East Africans. Eugenics Review 46: 101-121. Letvin N, Linch D, Beardsley P, Mclntyre K & Nathan D (1984) Augmentation of fetal hemoglobin production in anemic monkeys by hydroxyurea. New England Journal of Medicine 310: 869-873. Ley T J, DeSimone ,i, Anagnou Net al (1982) 5-Azacytidine selectively increases gamma-globin synthesis in a patient with t3+-thalassemia~ New England Journal of Medicine 307: 14691475. Ley TJ, DeSimone J, Noguchi CT et al (1983) 5-Azacytidine increases gamma-globin synthesis and reduces the proportion of dense cells in patients with sickle cell anemia. Blood 62: 370-380. Liu DP, Liang CC, Ao ZH et al (1990) Treatment of severe beta-thalassemia (patients) with myleran. American Journal of Hematology 33: 50-55. Liu SC, Derick LH, Zhai S & Palek J (1991) Uncoupling of the spectrin-based skeleton from the lipid bilayer in sickled red cells. Science 252: 574-576. Lowrey C & Nienhuis AN (1991) Pharmacological manipulation of globin gene expression with 5-azacytidine has produced long-term therapeutic benefits in two patients with betathalassemia. Blood 78 (suppl 1): 368a. Mackie LH & Hochmuth RM (1990) The influence of oxygen tension, temperature, and hemoglobin concentration on the rheologic properties of sickle erythrocytes. Blood 76: 1256-1261. Magdoff-Fairchild B, Poillon WN, Li T-I & Berries .IF (1976) Thermodynamic studies of polymerization of deoxygenated sickle cell hemoglobin. Proceedings of the National Academy of Sciences, USA 73: 990-994. Marotta CA, Wilson JT, Forget BG & Weissman SM (1977) Human 13-globinmessenger RNA, Journal of Biological Chemistry 252:5040-5051. May A & Huehns ER (1975) The concentration dependence of the oxygen affinity of hemoglobin S. British Journal of Haematology 30: 3t7-335. McCurdy PR & Sherman AS (1978) Irreversibly sickled cells and red cell survival in sickle cell anemia: a study with both DF32p and 5~Cr. American Journal of Medicine 64: 253-258. McDonagh KT, Dover GJ, Donahue R et al (1989) Manipulation of the HbF production with hematopoietic growth factor. In Stamatoyannopoulos G & Nienhuis AW (eds) Hemoglobin Switching, Part B. Cellular and Molecular Mechanisms, pp 295-306. New York: Liss. Minton AP (1974) A thermodynamic model for gelation of sickle-cell hemoglobin. Journal of Molecular Biology 82: 483-498. Minton AP (1976) Relations between oxygen saturation and aggregation of sickle-cell hemoglobin. Journal of Molecular Biology 100: 519-542. Mizukami H, Bartnicki DE, Burke S, Brewer GJ & Mizukami IF (1986) The effect of erythrocyte membrane on the bire fringence formation of sickle cell hemoglobin. American Journal of Hematology 21: 233-241. Moffat K & Gibson AH (1974) The rates of polymerization and depolymerization of sickle celt hemoglobin. Biochemical and Biophysical Research Communkations 61: 237-242. Mohandas N & Evans E (1985) Sickle erythrocyte adherence to vascular endothelium, Morphologic correlates and the requirement for divalent cations and collagen-binding plasma proteins, Journal of Clinical Investigation 76: 1605-1612. Mohandas N & Evans E (1989) Rheological and adherence properties of sickle cells. Potential contribution to hematologic manifestations of the disease. Annals of the New York Academy of Science 565: 327-337. Mohandas N, Johnson A, Wyatt ,iet al (1989) Automated quantitation of cell density distribution and hyperdense cell fraction in RBC disorders. Blood 74: 442-447. Morris CL, Gruppo RA, Shukla R & Rucknaget DL (1991) Influence of plasma and red cell factors on the rheologic properties of oxygenated sickle blood during clinical steady state. Journal of Laboratory and Clinical Medicine 118: 332-342. Mozzarelli A, Hofrichter J & Eaton WA (1987) Delay time of hemoglobin S polymerization prevents most cells from sickling in vivo. Science 237: 500-506. Murayama M (1966) Molecular mechanism of red cell 'sickling'. Science 153: 145-149. Nagel RL, Erlingsson S, Fabry ME et al (1991) The Senegal DNA haplotype is associated with the amelioration of anemia in African-American sickle cell anemia patients. Blood 77: 1371-1375.

SICKLE CELL DISEASE PATHOPHYSIOLOGY

89

Nash GB, Johnson CS & Meiselman HJ (1988) Rheologic impairment of sickle RBCs induced by repetitive cycles of deoxygenation-reoxygenation. Blood 72: 539-545. Nigen AM, Njikam N, Lee CK & Manning JM (1974) Studies on the mechanism of action of cyanate in sickle cell disease. Oxygen affinity and gelling properties of hemoglobin S carbamylated on specific chains. Journal of Biological Chemistry 249: 6611-66t6. Noguchi CT (1984) Polymerization in erythrocytes containing S and non-S hemoglobins. Biophysical Journal 45: 1154-1158. Noguchi CT & Schechter AN (1978) Inhibition of sickle hemoglobin gelation by amino acids and related compounds. Biochemistry 17: 5455-5459. Noguchi CT & Schechter AN (1980) The intracellular polymerization of sickle haemoglobin and its relevance to sickle ceil disease. Blood 58: 1057-1068. Noguchi CT & Schechter AN (1985) Sickle haemoglobinopolymerization in solution and in cells. Annual Review of Biophysics and Biophysical Chemistry 14: 239-263. Noguchi CT, Torchia DA & Schechter AN (1980) Determination of deoxyhemoglobin S polymer in sickle erythrocytes upon deoxygenation. Proceedings of the NationalAcademy of Sciences, USA 77: 5487-5491. Noguchi CT, Torchia DA & Schechter AN (1981) Polymerization of hemoglobin in sickle trait erythrocytes and lysates. Journal of Biological Chemistry 256: 4168--4171. Noguchi CT, Torchia DA & Schechter AN (1983) IntraceUular polymerization of sickle hemoglobin: effects of cell heterogeneity. Journal of Clinical Investigation 72: 846-852. Noguchi CT, Luskey KL & Pavone V (1985) Dipeptides as inhibitors of the gelation of sickle hemoglobin, Molecular Pharmacology 28: 4G-44. Noguchi CT, Rodgers GP, Serjeant G & Schechter AN (1988) Levels of fetal hemoglobin necessary for treatment of sickle cell disease. New England Journal of Medicine 318: 96-99. Noguchi CT, Rodgers GP & Schechter AN (1989) Intracellular polymerization: disease severity and therapeutic predictions. Annals of the New York Academy of Sciences 565: 75-82. Ohnishi ST (1983) Inhibition of the in vitro formation of irreversibly sickled cells by cepharanthine. British Journal of Haematology 55: 665--671. Ohnishi ST, Katagi H & Katagi C (1989) Inhibitionof the in vitro formation of dense cells and of irreversibly sickled cells by charybdotoxin, a specific inhibitor of calcium-activated potassium efflux. Biochimica et Biophysica Acta I010: 199-203. Orkin SH (1990) Globin gene regulation and switching: circa 1990. Cell 63: 665-672. Ortiz OE, Lew VL & Bookchin RM (1986) Calcium accumulated by sickle cell anemia red cells does not affect their potassium (86Rb+) flux components. Blood 67: 710-715. Padlan EA & Love WE (1985) Refined crystal structure of deoxyhemoglobin S. Journal of Biological Chemistry 2611:8272-8279. Padmos MA, Roberts GT, Sackey K et al (1991) Two different forms of homozygous sickle cell disease occur in Saudi Arabia. British Journal of Haematology 79: 93-98. Pagnier J, Meats JG, Dunda-Belkhokja Oet al (1984) Evidence for the multicentric origin of the sickle hemoglobin gene in Africa. Proceedings of the National Academy of Sciences, USA 81: 1771-1773. Pasvol G, Weatherall DJ & Wilson RJM (1978) Cellular mechanism for the protective effect of hemoglobin S against P. falciparum malaria. Nature 274: 701-703. Pauling L, Itano HA, Singer SJ & Wells IC (1949) Sickle cell anemia, a molecular disease. Science 110: 543-548. PennellyRR & Noble RW (1978) Functional identity of hemoglobins S and A in the absence of polymerization. In Caughey W (ed.) Biochemical and Clinical Aspects of Hemoglobin Abnormalities, pp 401-412. New York: Academic Press. Perrine SP, Rudolph A, Faller DV et al (1988) Butyrate infusions in the ovine fetus delay the biologic clock for globin gene switching. Proceedings of the Nationat Academy of Sciences, USA 85: 8540-8542. Perrine SP, Ginder GD, Failer DV et al (1993) A short-term trial of butyrate to stimulate fetal globin gene expression in the 13gtobin gene disorders. New England Journal of Medicine in press. Perutz MF (1987) Molecular anatomy, physiology and pathology of hemoglobin. In Stamatoyannopoulos G, Nienhuis AW, Leder P & Majures PW (eds) The Molecular Basis of Blood Diseases, pp t27-177. Philadelphia: W.B. Saunders.

90

C. T. NOGUCHI ET AL

Phillips G Jr, Coffey B, Tran-Son-Tay R, Kinney TR, Orringer EP & Hochmuth RM (1991) Relationships of clinical severity to packed cell rheology in sickle cell anemia. Blood 78: 2735-2739. Platt O, Orkin S, Dover G e t al (1984) Hydroxyurea enhances fetal hemoglobin production in patients with sickle cell anemia. Blood 69: 109-116. Platt OS, Falcone JF & Lux SE (1985) Molecular defect in the sickle erythrocyte skeleton. Abnormal spectrin binding to sickle inside-out vesicles. Journal of Clinical Investigation 75: 266-271. Platt O, Thorington B, Branbilla D et al (1991) Pain in sickle cell disease. New England Journal of Medicine 325: 11-16. Poillon WN & Kim BC (1990) 2,3-Diphosphoglycerate and intracellular pH as interdependent determinants of the physiologic solubility of deoxyhemoglobin S. Blood 76: 1028-1036. Powars DR (1991a) Beta s-gene-cluster haplotypes in sickle cell anemia. Clinical and hematologic features. Hematology/Oncology Clinics of North America. 5: 475--493. Powars DR (1991b) Sickle cell anemia: beta s-gene-cluster haplotypes as prognostic indicators of vital organ failure. Seminars in Hematology 28: 202-208. Powars DR, Weiss JN, Chan LS & Schroeder WA (1984) ls there a threshold of fetal hemoglobin that ameliorates morbidity in sickle cell anemia. Blood 63: 921-926. Rodgers GP (1991) Recent approaches to the treatment of sickle cell anemia. Journal of the American Medical Association 265: 2097-2101. Rodgers GP, Dover GJ, Noguchi CT, Schechter AN & Nienhuis AW (1990) Hematologic responses of patients with sickle cell disease to treatment with hydroxyurea. New England Journal of Medicine 322: 1037-1045. Rodgers GP, Dover GJ, Uyesaka N, Noguchi CT, Schechter AN & Nienhuis AW (1993) Erythropoietin augments the fetal hemoglobin response to hydroxyurea in sickle cell patients. New England Journal of Medicine 328: 73-80. Rodgers GP, Schechter AN & Noguchi CT (1985) Cell heterogeneity in sickle cell disease. Quantitation of the erythrocyte density profile. Journal of Laboratory and Clinical Medicine 106: 30--37. Rosa RM, Bierer BE, Thomas R et al (1980) A study of induced hyponatremia in the prevention and treatment of sickle cell crisis. New England Journal of Medicine 303: 1138-1143. Ross PD & Minton AP (1977) Analysis of non-ideal behavior in concentrated hemoglobin solutions. Journal of Molecular Biology 112: 437-452. Ross PD & Subramanian S (1977) Inhibition of sickle cell hemoglobin gelation by some aromatic compounds. Biochemical and Biophysical Research Communications 77: 12171223. Ross PD, Hofrichter J & Eaton WA (1977) Thermodynamics of gelation of sickle cell deoxyhemoglobin. Journal of Molecular Biology 115: 111-134. Ross PD, Briehl RW & Minton AP (1978) Temperature dependence of non-ideality in concentrated solutions of hemoglobin. Biopolymers 17: 2285-2288. Rubin EM, Witkowska HE, Spangler E et al (1991) Hypoxia-induced in vivo sickling of transgenic mouse red cells. Journal of Clinical Investigation 87: 639-647. Ryan TM, Townes TM, Reilly MP et al (1990) Human sickle hemoglobin in transgenic mice. Science 247: 566-568. Samuel RE, Salmon ED & Briehl RW (1990) Nucleation and growth of fibres and gel formation in sickle cell haemoglobin. Nature 345: 833-835. Schechter AN, Noguchi CT & Rodgers (1987) Sickle cell disease. In Stamatoyannopoulos G, Nienhuis AW, Leder P & Majerus PW (eds) The Molecular Basis of Blood Diseases, pp 179--218. Philadelphia: Saunders. Schmalzer EA, Manning RS & Chien S (1989) Filtration of sickle cells: recruitment into a rigid fraction as a function of density and oxygen tension. Journal of Laboratory and Clinical Medicine 113: 727-734. Seakins M, Gibbs WN, Milner PF & Bertles JF (1973) Erythrocyte HbS concentration: an important factor in the low oxygen affinity of blood in sickle cell anemia. Journal of Clinical Investigation 52: 422-432. Serjeant GR (1970) Irreversibly sickled cells and splenomegaly in sickle-cell anemia. British Journal of Haematology 19: 635-641. Serjeant GR (1992) Sickle Cell Disease, 2nd edn. Oxford: Oxford University Press.

SICKLE CELL DISEASE PATHOPHYSIOLOGY

91

Serjeant GR, Serjeant BE & Milner PF (1969) The irreversibly sickled cell: a determinant of haemolysis in sickle cell anaemia. British Journal of Haematology 17: 527-533. Shaeffer JR, Kingston RE, McDonald MJ & Bunn HF (1978) Competition of normal beta chains and sickle hemoglobin beta chains for alpha chains as a post-translational control mechanism. Nature 276: 631-633. Shesely EG, Kim HS, Shehee WR, Papayannopoulou T, Smithies O & Popovich BW (1991) Correction of a human beta S-globin gene by gene targeting. Proceedings of the National Academy of Sciences, USA 88: 4294-4298. Smith BD & La Celle PL (1986) Erythrocyte-endothelial cell adherence in sickle cell disorders. Blood 68: 1050-1054. Stamatoyannopoulos JA & Nienhuis AW (1992) Therapeutic approaches to hemoglobin switching in treatment of hemoglobinopathies. Annual Review of Medicine 43: 497-521. Stuart J & Johnson CS (1987) Rheology of the sickle cell disorders. Baillibre's Ch;nical Haematology 1: 747-775. Sunshine HR, Hofrichter J & Eaton WA (1978) Requirement for therapeutic inhibition of sickle haemoglobin gelation. Nature 275: 238-240. Sunshine HR, Hofrichter J & Eaton WA (1979) Gelation of sickle cell hemoglobin in mixtures with normal adult and fetal hemoglobins. Journal of Molecular Biology 133: 435--467. Sunshine HR, Hofrichter J, Ferrone FA & Eaton WA (1982) Oxygen binding by sickle celt hemoglobin polymers. Journal of Molecular Biology 158:251-273. Trudel M, Saadane N, Garel MC et al (1991) Towards a transgenic mouse model of sickle cell disease: hemoglobin SAD. EMBO Journal 10: 3157-3165. Veith R, Galanello R, Papayannopoulou T & Stamatoyannopoulos G (1985a) Stimulation of F-cell production in patients with sickle cell anemia treated with cytarabine or hydroxyurea. New England Journal of Medicine 313: 1571-1575. Veith R, Papayannopoulou T, Kuracho S & Stamatoyannopoulos G (1985b) Treatment of baboon with vinblastine: insights into the mechanism of pharmacologic stimulation of HbF in the adult. Blood 66: 456-459. Vitoux D, Olivieri O, Garay RP, Cragoe EJ Jr, Galacteros F & Beuzard Y (1989) Inhibition of K + efflux and dehydration of sickle cells by [(dihydroindenyl)oxy]alkanoic acid: an inhibitor of the K + C l - cotransport system. Proceedings of the National Academy of Sciences, USA 86: 4273--4276. Votano JR, Altman J, Wilchek M, Gorecki M & Rich A (1984) Potential use of biaromatic L-phenylalanyl derivatives as therapeutic agents in the treatment of sickle cell disease. Proceedings of the National Academy of Sciences, USA 81: 3190-3194. Walder JA, Chatterjee R, Steck TL et al (1984) The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane. Journal of Biophysical Chemistry 259: 10238-10246. Wall ILl (1989) Use of transgenic animals in livestock improvement. Animal Genetics 20: 325-327. Wall RJ, Pursel VG, Shamay A, McKnight RA, Pittius CW & Henninghausen L (1991) High-level synthesis of a heterologous milk protein in the mammary glands of transgenic swine. Proceedings of the National Academy of Sciences, USA 88(5): 1696-1700. Waterman MR, Yamaoka K, Dahm L, Taylor J & Cottam GL (1974) Noncovalent modification of deoxyhemoglobin S solubility and erythrocyte sickling. Proceedings of the National Academy of Sciences, USA 71: 2222-2225. Wick TM, Moake JL, Udden MM, Eskin SG, Sears DA & Mclntire LV (1987) Unusually large yon Willebrand factor multimers increase adhesion of sickle erythrocytes to human endothelial cells under controlled flow. Journal of Clinical Investigation 80: 905-910. Winslow RM (1976) Blood oxygen equilibrium studies in sickle cell anemia. In Hercules JI et al (eds) Proceedings of the Symposium on Molecular and Cellular Aspects of Sickle Cell Disease, pp 235-255. Bethesda: NIH. Wishner BL, Ward KB, Lattam EE & Love WE (1975) Crystal structure of sickle-cell deoxyhemoglobin at 5A resolution. Journal of Molecular Biology 98: 179--194. World malaria situation in 1990. Part I. Weekly Epidemiology Record (1992) 67: 161-167. Zachowski A, Craescu CT, Galacteros F & Devaux PF (1985) Abnormality of phospholipid transverse diffusion in sickle erythrocytes. Journal of Clinical Investigation 75: 1713-1717.

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