sickle cell disease pathophysiology

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Sickle cell disease pathophysiologyC O N S T A N C E T . N O G U C H IA L A N N . S C H E C H T E RG 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 ofmolecular disease , has provided scient ists for decades with the o ppor tunityto use a vast array of research techniques to examine its pathophysiologicmechanism s and to design rat ional approaches to therapy based upo n thesefindings (D ean and Schechter, 1978; Schech ter et al , 1987). The g eneticdefect arises fro m a single nucleotide chang e in the gene fo r th e 13-chain ofadult haem oglob in (HbA ) (Marotta e t a l, 1977). The resul tant haem oglob in(HbS or sickle haemo globin) aggregates or polymerizes when the erythro-cyte is deoxygenated during its normal transit in the circulation, causingimp aired cell flexibility, pre m atu re eryth rocyte destruction and injury to thetissues by microvascular occlusion (Serjeant, 1992; Heb bel, 1991). In r ecen tyears, studies of haemoglobin S solutions using a variety of biophysicaltechniques have p aved the w ay for studies of the intact erythrocyte contain-ing the abnormal haemoglobin, the sickle cell (Noguchi and Schechter,1985; Eaton and Hofrichter, 1990). These molecular and cellular studieshave clarified pathophysiotogical processes in the microcirculation of th epat ient . Genetic studies have elucidated some of the factors that de term inethe remarkably heterog eneou s nature of sickle cell disease severity and haveallowed the establishment of sensitive and specific prenatal diagnosticm eth od s (Bu nn and For get, 1986; Schec hter et al , 1987). Fu rthe r, mo lecular

genetic studies are beginning to clarify several new approaches to thetherapy of this disease targeted at a l tering globin gene expression--appro aches which are now being evalu ated in patients (R odg ers et al , 1990,1993; Ch arac he e t al, 1992).We present in this chapter a summ ary of our knowledge of deoxyhaem o-globin S polym er formation and i ts effect on sickle erythrocyte propert ies.The extent of polym er formation is modified by genet ic factors which varyhaemoglobin composit ion and concentrat ion within the intact sickleerythrocyte and these factors will be reviewed. Rational approaches totherapy, in addit ion to the genet ic interventions to change haemoglobincomposition, based on the biophysical analysis of haemoglobin poly-merizat ion, which includes strategies to increase deoxyha emog lobin Ssolubility or decrease corpuscular haemoglobin concentration, are alsoBailli~re s Clinical Haematology--Vol. 6 No. 1 March 1993ISBN 0-7020-1692-6

57Copyright 1993 by Bailli~re TindallAll rights of reproduction in any form reserved


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58 C T NOGUCHI ET AL

discussed. Fu rth er clinical aspects of sickle cell disease ar e cov ered in m or edetai l in C hap ter 4 in this volume by S er jeant .

G E N E T I C S

Sickle cell disease is due to t he substitution of a T for an A nucl eotid e in theco do n for glutam ic acid in the sixth position of the gene fo r the 13-chain ofhu m an haem ogl ob in M arot ta et al, 1977). The abno rm al 13-chains [3s),which hav e a substituted hy drop hob ic valine residu e Ingrain, 1956),combine with the normal or-chains to give HbS rather than HbA in theerythro cytes of sickle cell patients Figure 1). Th e re duc ed solubili ty ofdeoxy-HbS, as com pared to deoxy -HbA Hofr ich te r e t a l, 1974; Moffa t andGibson, 1974), leads to intracellular polyme rization and in the extre m e

Hb HbSCCT GAG GAG CCT GTG GAGPro G lu G Iu Pro VaI Gtu

6 7 6 7

oo :n iOO0 ~ 02 o o o o o ~o ~ ooo .t o o Oo o . .

Normal Abnormal

Figure 1. Overview of the basic molecular processes in the pathophysiology of s ickle cel ldisease. The nuc leotide m utatio n A--~T) in the sixth codon in the I~-globin chain gives rise tothe substitution Glu--~Val) to form the 13 chain. C om bin atio n of the m utan t 13S-globin chainswith a-globin chains resul ts in haemo globin S FIBS) formation. U pon deoxygenat ion f il ledcircles) Hb S molecules aggregate into polymers, unl ike normal adul t haemoglobin HbA )molecules which remain free in solut ion in the deoxy as well as the oxygen ated form opencircles) . The open and dosed circles represent oxyhaemoglobin and deoxyhaemoglobinrespectively. Note that the gel that forms in deox yhaemog lobin S solut ion or s ickle cel ls isreversible upon the addi t ion of oxygen and is composed of polymer in equi l ibr ium with thesurrounding haemoglobin molecules. The extent of haemoglobin polymerizat ion and thenature of the polymer domains within the intact part ial ly deoxygenated sickle erythrocytedetermines i ts mo rphology and can give r ise to the classic sickle, hol ly-leaf or o ther appear-ances. Ad ap ted from Sche chter et al 1987).


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SICKLE CELL DISE SE P THOPHYSIOLOGY 59state , cell s ickling) and disease pathophysiology (Noguchi and Schech ter ,1985; Eaton and Hofrichter, 1987).Ho mo zygo us sickle cel l disease (or anae mia ) ( the SS genoty pe) is am ongthe most p revalent genetic diseases in the wo rld and is the most f req uen t inpersons of equatorial African origin (Serjeant, 1982). The term sickle cellsyndromes (or disorders) is used to connote that spectrum of diseasesranging f rom th e a lmost symptom less s ickle cel l t ra i t ( the AS genoty pe) tothe m os t severe homozygous SS genotype found in A fr ica (Br i t tenham e t a l ,1985) (Table 1). This spec trum also includes a variety of genotyp es, am ongwhich a re doub le h eteroz ygos ity for the S gene an d for 13-thalassaemia oranother haemoglobin var iant , such as HbC; and genetic abnormali t iesco-exis t ing with the S gene, such as that for here ditary pers is tence of fe ta lhaem oglob in (HbF ) (H PF H) or c t- thalassaemia. Table 1 provides hae ma to-logical data on some of these sickle cell syndromes. As will be explainedlater , m uch o f the var ia t ion in disease severi ty amo ng these syn drom es cannow be unders tood in te rms of the i r p r imary e f fec ts on haemoglobin Spolym erization throu gh changing intracellular haem oglobin comp osit ion orconcentra t ion .The f requency of he te rozygotes for the S gene ma y be 25 or grea te r inparts of Africa (Allison, 1954a; Lehmann, 1954). The disease is mostprevalent in central and western Afr ican countr ies but has a dis tr ibutionwith respect to temperature and ra infal l that led to the now generallyacc epte d hypothes is that sickle trait individuals have a selective resistance tomalar ia (caused by Plasmodium falciparum , accounting for the highprevalen ce of this very deleter ious mutatio n in equator ia l regions (All ison,1954b, 1957a). Epidemiological studies have supported this hypothesis inthat t here is a s trong geographic correla t ion of S gene f requencies with theincidence of malar ia . In addit ion, a var ie ty of possible cel lular m ech an ism s--including changes in intracellular pH and potass ium in m alar ia-paras i t izederythrocytes-- -have been propose d to account for the epidemiological andclinical observatio ns (F ried m an, 1978; Pasvol et al, 1978). I t is exp ecte d thatin regions where malar ia is controlled, the f requency of the S gene wil ldecrease. T he re cen t world-wide upsurge in malar ia (World M alar ia , 1992)makes the an t ic ipa t ion tha t th is haemoglobinopa thy (and perhaps o therhaem oglob in disorders a lso l inked to m alar ia) will d isappear in the deca desahead less l ikely than was thoug ht a few years ago.Pio nee ring g enetic studies using restr iction en zy m e cleavage of i3-globinDNA provided evidence for a t leas t two geographic s i tes of or igin of thes ick le muta t ion , o ne in Wes t Af r ica and one in Eas t Af r ica (Kan an d D ozy,1980), but evidence for oth er s i tes within Afr ica has a lso been p resen ted(Pagn ier et al, 1984). Th ese studies have also bee n used to d efine variouslinked-groupings of DNA restr ic t ion cleavage polymorphisms within the13-globin gen e cluster , called haploty pes. Al tho ugh m ore th an a doze n such13-globin haplo types h ave bee n identif ied, most S genes occur on four suchhaplotypes , known as the Senegal , Benin, Central Afr ican Region (orBantu) and C ame roon types depending on the a rea wi th grea tes t f requencyof the c lus ter of polym orphism s (Powars , 1991a). A no the r haplotype hasbeen descr ibed in Saudi A rabian and Ind ian s ickle cell patients (Labie e t a l,


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T ab le 1 . H aem ato log ica l da ta and haem og lob in ana lys i s in s om e s ickl ing d i s o rders . *H b R e t ic u lo c y te s M C V M C H C M C [ H b S ] C H b A 2 H b F H b S

Sever i ty Cond it ion n (g/dl) (%) ( f l) (g/d0 (g /dl ) (%) (%) (%)Asymp tomatic 1 . HbA S (Afr ican) 34 14.3 2 .0 87.0 33.9 b 13.7 2 4 0 8 ~ 4 0 5(+ 1.2)2. HbS-'yA',/-I3-HPFH (Afr ican ) 9 14.0 -- 86.0 33.3 c 20.6 2,1 35.9 62.0

3. HbS-G2t-~'~t3-thalassaemia (A fri ca n) 3 11.4 2.9 74.0 32.7 b 22.6 2.2 28.8 69.0(+ 0 .7 )3 11.1 -- 84.0 33.0 b 23.5 2.6 26.1 f 71,822 10.9 5.2 72.2 33.4b 23.2 2,1 d 28.5g 69.4c(_+ 1.6) (+ 3.4)15 10,5 4.8 85.0 32.00 24.4 2,3 20.0 77.0

(_+1.3) (+3.1)39 10.3 4.0 71.0 31.2 20.9 4.8 5.0e 67.0(+ 1.7)15 9.3 5,9 67,9 32.2 c 25.5 3.5 17.3e 79.2(+_0.8) (_+2.4)

44 8.8 6.4 71.2 32.8a 30.3 3.9 3.8~ 92.3(_+ 1 .3 )41 8.6 7.3 68.9 31.1 a 27.5 5.0 6 .5~ 88.5(_+ 1.0) (+ 2.7)44 8.1 9.3 84.4 34.3 d 31.6 3.1 4.8 ~ 92.1(+ 1.0)88 7.8 11.9 90.1 34.8 d 32.0 2,8 5.3 e 91.9(+-1.1)

Mild

Severe

4. HbS-C~/-13-HPFH (Afr ican )5 . H bS S (S aud i A rab )6 . H bS S ( Ind ian )7. HbS-13+-thalassaemia (Afr ican)8. HbS-13-thalassaem ia (SaudiA r a b i a n )9. HbSS-et- thalassaemia (Afr ican)( a - / a -geno type)10. HbS-13-thalassaem ia (Afr ican )

11. H bS S -a- tha las s aem ia (A f r i can )(ot-/etot g eno type )12. HbSS (Afr ican)

+ I S D .b M CH C by Cou l te r Coun te r .c Method unspecif ied.a M CH C ca lcu la ted f rom haem og lob in concen t r a t ion and packed ce l l vo lum e.e H bF by a lka la i dena tu ra t ion .t H bF by co lum n ch rom atog raphy .g H bF by dens i tom et ry .* A dap ted f rom Br i t t enham e t a l ( 1985).

q~

t


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SICKLE CELL DISE SE P THOPHYSIOLOGY 61989; P adm os et a l, 1991). E vidence has been prese nted, which wil l be no tedlater, that the sever i ty of s ickle cell d isease may b e affected by the hap lotypeon which the S gen e appear s Nage l et al , 1991; Pow ars, 1991a,b). Theexistence of individuals affected by sickle cell disease in parts of the worldothe r than Afr ica can general ly be a t t r ibuted to the move me nts of indi-v idua ls o r g roups- -vo lun ta r i ly o r by forc e- - to these o ther reg ions and theresultant diffusion of the gene Serje ant, 1992; Ad ekile , 1992).

S I C K L E H A E M O G L O B I No l ut i o n s t ruc ture a nd func t i o n

The oxygena ted form of HbS appears to be near ly iden t ica l to theoxy gena ted or other l iganded) form of H b A Baldwin and Chothia , 1979)with respec t to its structure and function Perutz, 1987; Schec hter et al ,1987; Eaton and Hofrichter, 1990). This should not be surprising since themu tatio n in the 13S-chain is on the surface of the m olecu le W ishner et al ,1975) and not near the intersubunit contact residues which are involved indetermining man y of the subt le proper t ies of haem oglobin, such as co-opera t ive o xygenat ion. As a result , the oxygen-binding proper t ies of di luteHbS and HbA are near ly ident ical . There are some smal l d i f ferences ,how ever , in the eff ic iency of in teraction of a-chains with 13s. as comp ared to13A-chains to form the stable et13 or a13s) dimer that is the structural unit ofthe hae mo glo bin tetram er ot2132 or o~213s2 Shaeffer et al, 1978). Thisdifference probably accounts for the fact that AS heterozygotes have, ingeneral , a 60 to 40 ra t io of H b A to H bS. The s t ronger in teract ion of 13- wi tha-chains , as com pare d to 13 and a , leads to se lect ive dest ruct ion of theexc ess 13S-chains, ev en if the sy nth eti c r ate s of 13- an d 13S-chains are iden tical.The c lass ical d i f ference usual ly noted between di lute HbA and HbS is theal tera t ion of charge w hich causes the e lectrophoret ic a bnormal i ty , f irstnote d by Paul ing e t a l 1949), which is the basis of the m ost com mo n cl inicalmetho ds to d iagnose th is haemoglob inopa thy .It is in very concentrated solutions, such as the interior of the sickleerythro cyte a t a haemo globin concentra t ion of about 34 g/dl , in which themajor d i f fe rence be tween HbS and H bA mani fes ts i tsel f. D eoxy -Hb S has amarked ly reduced so lub il ity comp ared wi th oxy-Hb S or as comp ared wi thde ox y- Hb A or oxy -H bA ) All ison, 1957b; Bert les e t a l , 1970; Ho fr ichter e tal , 1974). The result of this reduction in solubil i ty is the formation ofpo lym er fibres also called aggregates, tactoids or a gel) within the sickleerythro cyte upo n par tia l or to ta l deoxyg enat ion Bert les e t a l , 1970; Co t tamet al , 1974; Moffat and Gibson, 1974; Hofrichter et al , 1976). The allosterics t ruc tura l change of haemoglob in f rom the oxygena ted R-s ta te to thedeoxygenated T-state, with shift of the ~13 subunits with respect to eachother Baldwin and Chothia , 1979; Perutz , 1987), presu mab ly al lows forin te r te tramer con tac ts tha t favour the aggregated or po lymer ized form o fHb S over the solut ion form Wishner e t a l, 1975; Padlan and Lov e, 1985) .Such changes in conformat ion a lso occur in HbA but the energet ics of


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6 C. T. NOGUCHI ET AL

8

I A

_ 5

-:i?::.

Figure 2. Schemat ic s t ruc ture of the deoxyhae moglobin S crys tal . a ) The haemog lobinte t ramers a re a r ranged in a double-s t randed ar ray . The res idues ident i f ied f rom crys ta l lo-graphic ana lys is as mak ing m ajor in termolecu lar contac ts a re d enote d , inc luding the 13 6 va l ineon each Hb S te t ra me r which makes contac t wi th the 13-chain of a ne ighbour ing te t ramer . b)Art is t ic inter pret at io n of the 13 6 val ine con tact reg ion at h igher resolu t ion i l lustrat ing possibleor ienta t ion of amino ac id s ide-chains a t th i s in termolecular contac t s i te . c ) IUust ra tion of thenorm al 13 6 glutam ic acid which suggests that th e bu lk an d charg e of this amino acid side chainwould not a l low the same contac t geometry . Fro m Sc hechter e t a l 1987) , based on W ishner e tal 1975) and Dick erson and Geis 1983).

s tabil ization o f the d eoxy form are no t suff ic ient wi thout th e val ine in the13-chain to ma ke the po lym er predo mina te under the range of condi t ionsfoun d in cells in the body .Crysta l nd polym er structureDuring the las t decade, X-ray dif f ract ion techniques have been used todeterm ine the s t ructure o f crysta ls of hum an deox y-H bS, form ed in specialcrystall izing solvents, at m od era tel y high resolu tion W ishner et al, 1975;Padlan and Lov e, 1985). This s t ructure consists of dou ble s t rands ofhaem oglobin te t ram ers arranged as shown in Figure 2a. There are extensivecontacts among te t ramers within a s t rand and on adjacent s t rands.Ho we ve r , only on e 13s6 res idue o f each Hb S te t ra mer actually ma kes anintermolecular contact in the crysta l to give the s tabi l izat ion energy u niqueto HbS. That hydrophobic 13s6valine side-chain fi ts into a po ck et fou nd in a13-chain on an ad jacent H bS te t ram er which has a hydrop hob ic pock etco mp ose d, in part , of 1388 leucine and 1385 phen ylalanine residue s Figure


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SICKLE CELL DISE SE P THOPHYSIOLOGY 62b) . Mo st of the other a toms in the pocke t , how ever , are a lso uncharged orpoorly polarizable. The glutamic acid residue at this posit ion in the normal13-chain doe s n ot f it into this h ydr oph obi c po ck et beca use o f its larger sizeand i ts strongly charged nature (Figure 2c).I t is l ikely , but not prove n, that these do uble-s t rand ed s t ructures are thebasis of the higher o rdere d s t ructures that are seen in e lectron m icrographsof deoxygenated s ickle cel ls and concentra ted HbS solut ions (Dykes e t a l ,1979; Cre pe au et al, 1981). Ev iden ce for this com es from th e fact that m anyl ikely intermolecular contacts in the s ickle poly mer s t ructure , that had bee nde du ce d fr om the study o f the effects of other mu tation s in the or- or 13-chainon the solubi l i ty of the HbS molecule , are qui te consis tent wi th the inter-molecular contacts in the double-s t randed molecule (Edels te in , 1981) . I fth is is the case , then informat ion on contacts among atoms within the dou blest rand can be used to design comp ound s which might inhibi t polymerizat ionand thus act as therapeu t ic agents (De an and S chechter , 1978; Schechter e tal, 1987).In concen tra ted H bS solut ions and cel ls , deoxyg enat ion leads to format ionof birefr ingent gels (Harris , 1950) ma de up of mult iple s t rands of H bSmolecules . The next higher order s t ructure of such gels , which has beenclear ly def ined by e lectron microscopic and image reconstruct ion tech-niques, consists of seven double-strands intertwined in a complex, twistingpat tern (Dykes e t a l , 1979) . Higher ordered s t ructures are presumablyform ed from these 14-st rand s t ructures b y order ing in paralle l arrays into thebun dles o f f ibres seen in electron m icrographs (Briehl et al , 1990). Suchbundles presumably are capable of dis tor t ing the SS cel l , by vir tue of thecumulat ive energet ics of their format ion within the normal ly biconcav eerythrocyte , in to s ickled and other abnormal shapes . Nothing is known ofthe deta i led molecular contacts wi thin these polymer bundles which a lsocould be targets for drug act ion. There is some suggest ion that smal lmolecular rearrangements may be necessary to a l low the double-s t rand toassemble into the macro - f ibres or t ransform into true crysta ls. O ther f ibrepat terns have b een seen bu t their physiological re levance is unclear (Josephset al, 1976).Upon rapid deoxygenat ion in the SS cel l or in concentra ted HbSsolut ions , many smal l polymer domains form and with t ime these mayrearran ge to form one or a few large, cyclindrical domains in the fullysickled cell or in gelled Hb S solution (Ho riuch i et al , 1990). I t is l ikely thatin SS cel ls , especially in the b ody, m any o ther po lym er form s--s uch as veryshort bundles due to shear ing forces---may exis t but these are not wel lcharacterize d (Briehl, 1983). No r are the relative contribution s of thevar ious polymer s t ructures to impaired cel l rheology unders tood.Formation of deoxy HbS polymer

inetics and mechanismDetai led biophysical s tudies have c lar i f ied the mechanism by which apo lym er o f deoxy -HbS molecu les fo rms in concen tra ted Hb S so lu t ions o r in


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o o o HO M O G E NEO USo o o GRO WTHo o o o NUCLEA TION P~b HETEROGENEOUS~OMAINUC IO; FORMATIONE


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SICKLE CELL DISE SE P THOPHYSIOLOGY 65Schech ter , 1981); (c) the gradual nat ure o f deoxygena tion processes in thebod y com pared with in vitro exper imenta l per turbations; and (d) the effectof shear inside sickle cells during their passage through the circulation(Briehl , 1983). Despite this uncer ta in ty as to th e m olecular mechanism s ofpolym erization within cells in the body, the kinetic analysis has provid ed af rame work to unders tanding HbS p olym er format ion , and remains the bas isfor unders tanding man y aspec ts o f the behaviour of HbS m olecules (Ea tonand Hofrichter, 1987, 1990).Equilibrium thermodynamic) aspectsEquil ibr ium solubili ty me asure me nts of deoxy-HbS du r ing the las t 20 yearshave also contr ibuted greatly to ou r unde rs tanding of the pathop hysiologyof the sickle diseases and various app roach es to the rap y (Bertles et al, 1970;Ho frich ter et al, 1976; Magdo ff-Fairchild et al, 1976). E arly wo rk was do newith var iants of a gelling assay to give a mi nim um gell ing conce ntrat ion (orM GC ) (Bookc hin e t a l, 1976) but the advent of true solubil i ty me asure m entsallowed recognit ion of the complex interaction of var ious haemoglobinchains in polym erization, the role of protein and solvent non- ideali ty , andmany other factors (Minton, 1976; Sunshine et al, 1979; Gill et al, 1980;Noguchi, 1984).W hen concentra ted HbS so lu t ions a re deoxy gena ted and cen tr ifuged a thigh speed, they ma y be separated into two phases: th e soluble supernatan tand t he p ack ed pellet (Be rtles et al, 1970; Hofri cht er et al, 1976; Ma gdoff-Fairchild et al, 1976). Th e pellet consists of the poly m er structures tha t we reform ed upon deoxygena t ion wi th sur rounding sed imented-HbS te t ramers .Me asurem ent of the concentra t ion of the superna tan t p rovides in format ionabout the therm odyn amic so lubi li ty of HbS unde r the spec i fied condi tions oftem pera ture , buf fe r , pH, e tc. U nd er these exper imenta l condi tions, HbSpoly m er acts therm ody nam ically as a two-phase system of gel and f reete tra m er (Minton, 1974). The solubili ty of deoxy-H bS und er condit ions thatsimu late intracellu lar condition s is betw een 16 and 18 g/dl (Poillon and Kim ,1990) ( less than the concentrat ion of Hb in the red cell , which averagesabout 34g/d l as the mean corpuscula r haemoglobin concentra t ion(MC HC )) . As ment io ned ear l ie r , HbS and Hb A, in bo th the oxy and deoxyforms, have solubili ty values that are mu ch higher than this . In this sense,s ickle cel l anae m ia m ay be th oug ht of as a disease due to th e insolubil i ty ofdeoxy-HbS ins ide the e ry throcyte .Solubil i ty exper im ents of HbS with other haemoglob ins , such as H bA , HbA2, HbF and HbC, have clar if ied how these interact in the deoxygenatedstate to affect polymerization (Cheetam et al, 1979; Sunshine et al, 1979;Benesch et a l , 1980; Bunn et a l , 1982) . The data are complex but areconsisten t with a m od el in which th e H bS mo lecul e (a213s2) has th e lo westsolubil ity (expressed as the re la t ive probabili ty of enter ing the poly m er ofone) ; a l l the o th er hom ote t ram ers a re much more so luble , even in the deoxystate , and have close to zero probabil ity of enter ing the pol ym er (Sunshineet al, 1979; Bun n et al, 1982). I t is the m ixed hybrids which det erm in e th espar ing effects of non-S haemoglo bins on polymerization. Hb A and C m ixed


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66 C. T. NOGUCHI ET ALhyb rid s wit h H bS a213[3 an d o~213c13 ) en te r th e p ol ym er wit h ab ou t 0.5probabil ity Bunn et a l , 1982) , while Hb F and HbAz m ixed hybr ids withHb S ot2-y[3 and a2~13 ) do no t ente r the po lym er Suns hine et al, 1979). Thisdif ference accounts for much of the importance of these las t two haemo-globins, HbF in particular, as a potential therap y for this disease see later) .These techniques of direct solubil i ty measu remen ts a t equil ibr ium have alsobeen used to demonstrate that 2 ,3-diphosphoglycerate has a small , butdistinct, effect on lowering the solubility of deoxy-HbS that is ind ep end en tof its effect in shifting the allosteric equilibrium from the R- to the T-statePoillon and Kim, 1990).A very impo rtant concept that has come from these thermo dyna mic studieswas the appreci ation of the role of non-ideality in affecting the solubility ofdeoxy-H bS Minto n, 1976; Ross and Minton , 1977). T he large size of Hbmolecules and their very high concentrat ion ins ide the erythrocyte m akestheir chemical act ivi ty about 50 t imes the measu red concentrat ion Ross e tal, 1978). I t is the activity, not the concentration, that determines thesolubility of species in solutions or in cells Mi nto n, 1976). Similarly, th eactivity of the aqu eou s solvent in the cell is different from the nom inal valueusually assum ed Gill et al, 1980). In analysing experi men tal data, bo thkinetic and equil ibr ium, these factors becom e very important . In part icularboth the ra te of polymerization the inverse of the delay time) and the extentof polymerization are dependent to a very high degree on the tota l intra-cellular Hb c once ntratio n Noguc hi, 1984; Eat on and Hofrichter, 1987,1990) , which explains why reducing M C H C could be an im portan tthera peut ic goal Rosa et al, 1980).

zot

r ru 0 5r rLU

n

m

D

oqp

~~ogx

Aollbx xAOD XA Q

J j~J0 50 100

O X Y G E N S A T U R A T IO NFigure 4 . Polymer f rac t ion wi th in in tac t s ickle e ry throcytes measured by t3C-NMR as a funct ionof oxygen sa tura t ion f rom severa l pa t ients (d i f ferent symbols) . The resul t s indica te tha tpolymer i s de tec ted a t very h igh oxygen sa tura t ion va lues (un der condi t ions wi th few revers ib lys ickled ce ll s ) and tha t the amou nt of polymer increases to an average maximum of about 70 asoxygen sa tura t ion decreases to zero . Dense ce l l s sh i f t the curve to the r ight and cause theappearance of de tec table polym er near 100 sa tura t ion . From Noguchi e t a l (1980).


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SICKLE CELL DISE SE P THOPHYSIOLOGY 67A critical exam ple of the effects of non-ideality are the results of measure -me nts of HbS polymerization, as a function of oxygen saturat ion, that weremade with nuclear magnetic resonance (NMR) spectroscopic techniques

(Noguc hi et al, 1980) (Figure 4). Th e surprising result of these studies wasthat poly mer fo rme d in the s ickle erythrocyte a t very high oxygen saturat ionvalues (80 or above for a cell with an MC HC of 34 g/dl and higher for mo redense cells) and increased monotonically to a max imum of about 70 of themolecules polymerized at com plete deoxygenation. Polym er is detected a tmu ch hig her ox ygen saturatio n values than that for significant cell sickling(morphological distortion) and at far higher oxygen saturation values thansimple solubili ty considerat ions would have predicted. T he explanation forthe la t ter phenomenon is that protein and solvent non- ideali ty markedlyreduc e t he effective solubility within the sickle erythrocyte (Noguchi et al,1983; N ogu chi, 1984). As will be discussed later, these results have signifi-cant implications for understanding the pathophysiology of sickle celldisease and its syndromes.Compar isons of haemoglobin po lymer format ion in AS and SC ery thro-cytes (individuals hete roz ygo us for [3 and 13 ) ind icate d that alt hou ghcom parabl e mixtures of haemoglo bin S and A and of haemoglobins S and Cbeha ve similarly (Bunn et al, 1982), SC erythrocy tes exhibited an increase inthe poten tial for intracellular haemo glob in polym erization (Noguchi, 1984).In fact , the increased propor t ion of haemoglobin S in SC disease (about50 ) com pared with s ickle trai t (about 40 ) together with the increasedMC H C or intracellular haemo globin concentrat ion in SC erythrocytes dueto the presence of haemoglobin C results in an increased potenti.al forhaemoglobin polymerization and provides an explanation for the diseaseseverity associated with SC disease in contrast to benign sickle trait (B unn etal, 1982).

SI KLE ERYTHRO YTESThe sickling of sickle (SS and other sickle syndrome) cells is due to thereversible intracellular polymerization of HbS within the cell upon deoxy-genation (Harris et al, 1956) (Figure 1). At complete deoxygenation, poly-merization leads to extensive morphological deformation and formation ofth e classical holly-leaf or sickled forms (Diggs, 1932; H ori uch i et al, 1990).At par t ia l deoxygenation, small amounts of polymer may exis t withoutdetectable morpholog ical abnormali t ies . The rheological proper t ies of thesickle cell (Bessis, 1977, 1982; It oh et al, 1992) are prim arily de te rm in ed bythe exte nt of intracellular HbS poly meriza tion (Keid an et al, 1989; Mack ieand Hochmuth, 1990). As was discussed earlier , the extent of polymeriza-t ion is dete rmi ned by the intracellular haemoglobin concentrat ion (M CH C)and com posit ion (percentage of HbS and non-S haemoglobins) as well asoxygen saturatio n (N oguchi, 1984; Noguch i and Schechter, 1985; Ea ton andHofrichter, 1987, 1990). Other variables such as temperature (Ross et al,1977), diphosphoglycerate levels and intracellular pH (Poillon and Kim,


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_o 0 5I-U

uJoz

z_oE

O1

0 5

01 o56

S S

1 i

1 . 0 9 6 1 . 1 3 6DENSITY NUMBER

Figure 5. Density dis tribution of normal AA ) an d sickle SS) erythrocytes. The histogramrepresents the fraction of cells at each density value and is the difference between adjacentmeasurements obtained from the density profile generated by phthalate ester densityseparatio n in microcapillary ubes exper imen tal lines). Sickle cell patients have mo re light cellslargely reticulocytes) and m ore den se cells which presum ably exacerbate pathology butwhose origin is still unc ertai n) than no rm al individuals. Fro m Ro dgers et al 1985).

1990) also have effects but these are probably small within the range ofphysiological, or even pathological, variation.Sickle erythro cytes are ve ry heterog eneo us with respect to intracellularhaem oglob in concentra t ion (Seakins e t a l , 1973) and var ious biochemicalproper t ies (Clark e t a l , 1978) as compared to normal (AA) erythrocytes .The se abn ormali t ies include changes in diphosph oglycerate concentrat ion s ,pH and oxygen a ff in i ty and in the proper t ies of the m em bran e (Hebb el ,1991). Th e var ia tions in haemog lobin conc entrat ion are ref lected in a widespectrum of cel l densit ies when s ickle cel ls are f ract ionated on densitygradients (Rodgers et al, 1985) (Figure 5). I t will be noted that there aremany l igh t ce l ls - -a f rac t ion markedly enr iched wi th re t icu locytes - -andm any den se cells . The den se cells (with MC HC > 37 g/dl) would be exp ectedto be very deleter ious in the c irculat ion of the patient with s ickle cel lanaemia s ince po lymer iza t ion of HbS would be m arkedly enhanc ed in thesecells (Clark et al, 1980; Ev ans et al, 1984; No guch i et al, 1983; Kei dan et al,1989; Sch m alze r et al, 1989).The or igin of the dense cells remains uncer ta in . I t was or iginally thoug httha t th ey represen ted the en d s tages of repea ted cyc les of po lym er iza t ion-depoly meriza tion ( s ickling - unsickling ) (Bookchin and Lew , 1984; Nash eta l , 1988) in the body , as it has bee n kno wn for decades that d eoxy genatio n of


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SICKLE CELL DISEASE PATHOPHYSIOLOGY 6 9PRODUCTION CIRCUL TION DESTRUCTION

DENSECELLSo.~ O~ ~

INTRACELLULAR bS ~ ' ~ ~ =~ ' ~ ~ ~ -O2 POLYMERIZATIONOoO~--~ ~ ~ ~' DECREASiD ILTERABILITY

fF-cells MEMBRANEDAMAGE ~-'~. HEMOLYSIS SEQUESTRATIONBONEMARROW ~.. . . .

MICROVASCUAROBSTRUCTIONACUTE CHRONICCRISIS PROGRESSIVEORGANDAMAGE

Figure 6, Sche matic outl ine for the cel lular pathophy siology of sickle cel l anaem ia. Sickle cel lsproduced by the bone marro w represe nt a he terogeneous popula t ion (e .g . wi th respect to thepercentage Hb F and ce l l dens i ty). In addi t ion , repea ted cycles of in t race l lu lar polym er iza t ion-depoly meriza t ion ca n lead to K + toss and d ehyd ration , giving r ise to dense cel ls result ing inmore polyme r a t any oxygen sa tura t ion . Revers ib le in t race l lu lar polymer iza t ion a lso leads tomembrane damage and the format ion of i r revers ib ly s ickled ce l l s ( ISCs) , which are a l solargely in the de nse cel l fract ion. Den se cel ls have reduced f lexibil i ty due to high MC HC andme mb rane r igid ity but pr imar i ly because of increased tendency to polymer format ion of anyoxygen sa tura t ion . Endothe l ia l adhes ion may a lso impai r rheologica l behaviour . These severa lprocesses lead to obs t ruc t ion and haemolys is and, eventua l ly , to the acute and chronicmanifes ta t ions of the d isease . Ada pted f rom R odgers (1991) .

sickle cells leads to K + an d H O f luxes and conseq uent cel lular dehyd rat ion(Br ugn ara et al , 1986) (Figure 6). I t was also postulated that thes e pre m atu reageing chang es in circulating SS cells caused th e variety of protein and l ipidabno rmali t ies, as well as functiona l changes, d etec ted in sickle cells , part icu-larly the den se ones (He bbe l, 1991). In part icular, those cells with extensivem em bra ne abnormal i t ies that a lways appear d eform ed, i r respect ive of thephysical state of the intracellular HbS , and are ter m ed irreversibly sickledcells ( ISCs) (Ber t les and Milner , 1968), are thought to have been fo rm ed bysuch mechanisms (Hebbel, 1991).Recent ly , however , da ta have been presented tha t many e ry throcytesmay emerge f rom the mar row more dense than o ther ce l l s and a l readymani fes t ing abnormal membranes , inc luding a l t e red membrane pro te insand ion transp ort m echan isms (Moh and as et al, 1989; Bo okch in et al , 1991)(Figure 6) . Th ese cells are k nown to be low in H bF (Ber t les and Milner ,1968) but oth er factors that m ay contr ibute to this heterogen ei ty are notknow n. I t do es seem l ikely, how ever , that these (pre deter min ed) dense cel lshave a shor ten ed l ife- time in the c i rculat ion (M cCurdy and Sherm an, 1978)and may be dispropor t ionately involved in the pathogenesis of the acutepainful crises of s ickle cel l disease, the chronic haem olyt ic anaem ia, andot he r vaso-occtusive comp lications of this disease (Billett et al, 1988).Th e oxyg en aff ini ties of s ickle cells vary f rom about 40to 60 m m Hg valu es o f1 5o, indicating a m ark ed reduct ion in affinity as com pared to norm al cells


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70 C T N O G U C H I ET A L(Ps0 = 26 m m H g) (Ma y and Hu ehn s, 1975; Winslow, 1976). Th is chang e inaffinity is not p rima rily due to the type of H b as the basic oxy gen affinities ofHbS an d Hb A are com parable (Pennelly and Noble , 1978). R athe r , theredu ced effective oxygen affini ty is due to th e energetic l inkage betw eenpolym erization and oxygen binding (M ay and H uehn s, 1975). Since only thedeoxy -HbS molecules enter the poly me r (Hofr ichter , 1979), oxygen bindingma y be thought of as compet ing with the b ind ing energy of haemoglobintetram ers in the p olym er (Sunshine e t a l , 1982). T hus effective reduction inoxygen-binding energ y leads to the m ark ed r ight-shift of oxygen equil ibr iumme asure me nts (May and H uehn s, 1975). Th e physiological consequen ces ofthis phe no m en on are less c lear as this shift faci l ita tes oxygen delivery to thetissues. I t should be note d that problems in oxygen delivery due to theanae mia o f sickle cel l disease do n ot appear to be a m ajor l imitat ion of thesediseases as even severe decrements in haemoglobin level are of ten welltolerated (Serjeant, 1992).

R H E O L O G Y O F S I C K L E E R Y T H R O C Y T E SIt is generally accepted that the impaired flow properties of sickle celleryth rocy tes ( i .e . their abno rmal rheolog y) are due to th e acute and chroniceffects of the intracellular polymerization of HbS as s ickle erythrocytestransverse the c irculat ion f rom the lungs to the t issues and back (Mo hand asand Evan s, 1989). During m uch of the three-qua r ters of a century af ter theoriginal description of sickle cell disease, most characterization of thedisease re la ted to the m orpho logy of the eryth rocytes of patients , both as thecells appe ared in the per ipheral blood and af ter deoxygen ation withnitrogen or chemical agents (Stuar t and Joh nson, 1987). Durin g this per iod,l ight microscopy has been supplemented by birefr ingence measurementsand electron m icroscopy (Harr is e t a l , 1956; Dyke s e t a t , 1979; Edels te in ,1981) . These s tudies identif ied bundles of polymerized haemoglobinmolecules within s ickled erythrocyte s and led to th e realizat ion of therela t ionship between the physical s ta te of the HbS molecules and themo rpholo gical abnorm alities of the cells (Briehl et al, 1990; Ho riuchi et al,1990; Kaul and Xue, 1991).Ho wev er , the l ikely behavio ur of cel ls in the microcirculat ion canno t bepredicted f rom such observations . Thus, the advent of direct rheologicalme asure me nts of s ickle cells has b een of great imp ortance (Ballas, 1991;Mo rris et al, 1991; Phillips et al, 1991). Filtration m eas ure m ent s throu ghvar ious micropores show that even oxygenated s ickle erythrocytes areslightly m or e r igid than n orm al cells (Gr ee n et al, 1988; Ke idan e t al, 1989;Mackie and Hochmuth , 1990) . This phenomenon is p robably due to theeffects of the high intracellular haemoglo bin concentrat io n in the dense cellsubfractions and to the membrane abnormali t ies discussed ear l ier . Therehas been some specula t ion tha t abnormal haemoglobin membrane in te r -actions m ay also co ntr ibute to the re la tive r igidity of sickle erythro cytes butthis is sti ll no t cer ta in . U po n deoxy genation there is a ma rke d decrea se in thef i lterabili ty of s ickle eryth rocytes due to the forma tion o f intracellular HbS


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SICKLE CELL DISEASE PATHOPHYSIOLOGY 7

Z0l -


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72 C . T. N O G U C H I E T A L

(~-like Genes

~,-like Gen es 5

DeveloementalPedo~

Hemoglobins

v; wal

e' G A wB 6 B_- .:/ L { m = -.10

Embrvonic

Hb Gower 1( ~ )Hb Gower 2

Hb Portland

I I II I I20 30 ~0 50 60

Fetal I

HbF I HbA2 HbA(~) ; (~ ) t ( a2) (~2)

Chromosome 16

3 Chromosome 11Kilobases

igure 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 ~-globin gen e is actively ranscribedearly during development, followed by a switch to the adult-like a-globin genes during earlygestation, which persists during fetal and adult life. The B-like, embryonic ~-globin gen e istranscribed during the first trimester in utero followed by a switch to the two 3,-globin geneswhich persist during fetal developm ent and another switch at birth to the adu lt 13-globinand8-globin. Haem oglobin tetramers resulting from combination of these c~- and B-like globinchains are also indicated. From Berg and Schechter (1992).s t e m c el ls in c u l t u r e a n d a n i m a ls p r o d u c e d e x p r e s si n g t h e t e s t g e n e h a s m a d eth is goa l ap pea r a t t a ina b le (G rosv e ld e t a l, 1987) . Th ese s tud i e s advan ced inpa ra l l e l t o bas ic s tud ie s o f g lob in gene ex p ress ion ( Ber g and S chech te r ,1992) u s ing va r ious an ima l ce l l s ( and the t ransgen ic mouse ) a s repo r t e rs y s te m s . T h e s e s t u d ie s l e d t o t h e c h a r a c t e r i z a t i o n o f t h e i m p o r t a n t p r o m o t e re l e m e n t s f o r e a c h o f th e g l o b i n g e n e s , t o t h e e x i s t e n c e o f v a r i o us e n h a n c e r sa n d s i l en c e rs a n d , m o s t i m p o r t a n t l y , t o t h e f u n c t i o n o f a r e g i o n u p s t r e a m o fthe 13-globin gene c lu s t e r (F igu re 8 ) , no w kno w n as the l ocus con t ro l r eg ion( L C R ) ( Or k i n , 1 99 0) . T h i s D N A r e g i o n , a n d a n a n a lo g o u s o n e f o r t h eo t -g lob in gene c lu s t e r ( Ja rman e t a l , 1991 ) , i s r equ i red fo r h igh - l eve l ,p o s i t i o n - i n d e p e n d e n t e x p r e s s i o n o f t r a n s f e r r e d g l o b i n g e n e s i n t r a n s g e n i cm o d e l s . On t h e b a si s o f t h e s e s t u d ie s , f o u r o r m o r e l a b o r a t o r i e s h a v ep r o d u c e d t r a n s g e n i c m i c e e x p r e s s i n g t h e h u m a n ~ S - g e n e ( o r a n a l o g u e se n g i n e e r e d f o r t h e p r o t e i n t o b e e v e n l es s so l u b le i n t h e d e o x y g e n a t e d s t a te )and , i n some cases , human a -genes (Greaves e t a l , 1990 ; Ryan e t a l , 1990 ;Rubin e t a l , 1991; Trudel e t a l , 1991) .

Un f o r t u n a t e l y , d e s p i t e t h e r e l a t iv e l y h i g h e x p r e s s i o n o f t h e h u m a n ~s_g e n e s in t h e s e t r a n s g e n i c m i c e , n o n e o f t h e a n i m a ls a p p e a r s t o b e a v e r y g o o dm o d e l f o r t h e h u m a n d i s ea s e . A f u n d a m e n t a l p r o b l e m w i t h a ll o f t h e s es t ud i e s is t h e c o n t i n u e d p r e s e n c e o f t h e c o m p l e x o f m o u s e g l o bi n s wh i chc o m b i n e w i t h t h e h u m a n p r o t e i n s t o g iv e a v a r i e t y o f h a e m o g l o b i n s p e c i e swh o s e s o l ub i li t y a n d o x y g e n - b i n d i n g p r o p e r t i e s a r e v e r y d if f e r e n t f r o m Hb S(R yan e t a l, 1990 ). As d i scussed ea r l i e r , fo r t he d i sease s ick l e ana em ia t o beman i fe s t , o ne need s ce ll s i n wh ich the vas t bu lk o f t he ha em og l ob in is t hepo lymer i z ing spec i e s . In add i t i on i t i s no t c l ea r t ha t t he mouse , wi th i t s


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SICKLE CELL DISE SE P THOPHYSIOLOGY 7

character is t ic microvasculature , oxygen metabolism and erythrocyteproper t ies , is a goo d m odel for hum an physiology. I t is poss ible , how ever ,tha t fur ther gene t ic engineer ing- - inc luding the very impor tan t knock outof the m ouse haem oglobins th rough hom ologous recombina t ion techniques(Shesely e t a l , 1991 )--and even the use of othe r animals as models (Wall ,1989; Wall e t a l , 1991) m ay eventually lead to the d evelo pm ent of an anim alm ode l for s ickle cell disease. These animals would be very valuable for thestudies of the vascular pathophysiology, in vivo behaviour of the s ickleerythro cytes , the effects of various pharmacological approach es to th erap yand perh aps gene express ion and gene transfer therapies .

SICKLE ERYTHROCYTE MEMBR NEThe e ry throcyte mem brane p lays an impor tan t ro le in regula ting the ion andwate r con tent o f the cell and in maintaining the cell volum e and intracellularhaemo globin concentra t ion (Agre , 1992). The acquired mem bran e defec tsand their potentia l pathogenic role in s ickle cel l disease have been sum-mariz ed recently (Hebbel , 1991). Som e SS erythroc ytes susta in me m bra nedam age w hich rende rs them p oor ly deform able (For t ier e t al , 1988). Th esem ay ar ise through a var ie ty of mechan isms including oxidative dam age tom em bran e pro teins . A num ber of these e ry throcytes suffe r impairedt ranspor t o f K and Ca 2, a re p erman ent ly deform ed even in the presenceof com plete oxygenation and, as noted above, are terme d ir revers iblysickled cells (ISCs). ISCs have an inc reased Ca 2 con ten t, an inc reasedin t race l lu la r haemoglobin concentra t ion and an increased tendency towardhaemolysis (Ortiz e t a l , t986) . I t has been proposed that ISCs have anoncova len t rea r rangement of the spec t r in -ac t in cy toske le ton , resu l t ingfrom repe ate d cycles of s ickling, and producing abno rmally r igid cells. Th eir revers ib le membrane damage may inc lude abnormal membrane pro te inphosph orylat ion or som e oth er nonco valent modif icat ion, a calcium effecton th e cyto skeleton or a direct interaction with haem oglobin S (Fairbanks e tal, 1983; Apo vo e t al, 1989). E xce pt for hae mo lytic ana em ia, a correl ationbet we en ISCs and disease sever i ty has not bee n de mo nstrated . I t is poss iblethat the increased tendency toward haemolysis of the ISC results in i tsaccelera ted remov al f rom th e c irculation and m inimizes i ts role in micro-vascular obstruction.The ro le of mem branes in the po lymer iza t ion process remains uncer ta in(Noguchi and Schechter , 1985; Eat on and Ho fr ichter , 1987). M easu rem entsof pol ym er form ation in the intact erythroc yte us ing NM R (discussed above)suggest tha t the amo unt of po lymer is de te rmin ed primar i ly by the haem o-globin concentrat ion and composit ion and by the oxygen saturat ion, andtha t contr ibu tions of the m em bran e to am ount of haemoglobin po lym er isnot s ignif icant (Noguchi e t a l , 1980) a l though the membrane may affectpo lym er a l ignment .On e in tegra l mem bran e pro te in , Band 3 , has been shown to b ind haemo-globin in the region of the 2,3-diphosphoglycerate pocket (Walder e t a l ,1984). Th e role of this interaction in haemog lobin S polym erization (Liu e t


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74 C T NOGUCHI ET ALal, 1991) and the role of the mem bran e o r membran e-associated proteinsserving as nucle ation si tes for polym erization thus affecting kinetics bu t notthermo dynam ics) h ave bee n suggested, but have been dif ficul t to dem on-strate G otd be rg et al , 1981; Mizu kam i et al , 1986).Hb S i s more mechanical ly uns tab le than H bA and m ore read ily dena turedHebbet , 1990) . Several membrane changes , par t icular ly oxidat ive damageto the m emb rane , have be en a t t r ibu ted in par t to the increase o f depos i t ionof na t ive o r dena tured haemoglob in o r Heinz bodies on or near the innersurface of the m em bran e. The generat ion of superoxide f ree radical resul t-ing f rom dena tu red or uns tab le HbS , inc lud ing f rom re leased haem groupsor i ron a toms , has bee n proposed as an impor tan t m echanism for mem branedam age He bb el , 1990).Sickle erythro cytes have been o bserv ed to adhere to endo thel ium in t issuecul ture and in perfusion s tudies Francis and Johnson, 1991). Fibr inogen,fibronectin an d possib ly oth er acu te-phase reactan ts can facili tate thisadherence which appears to be sensi t ive to the suspending medium butoxygen inde pend ent Mo hand as and Evans, 1985; Smith and LaCel le , 1986;Wic k et al , 1987). Since adh eren ce of eryth rocy tes to end oth elium isvar iable and also depen den t on shear forces present in the microcirculat ion,the significance of these findings in the evo lution o f vaso-occlu sion remainsuncer ta in .Repea ted cyc les o f haemoglob in S po lymer iza t ion lead to a number o fme mb rane changes Ohnishi , 1983). These may include rearrange men t ofphospho l ipid with an increase of aminopho sphol ipids on the oute r surface ofthe mem brane comp ared to normal e ry throcy tes , pa r ti cula rly upon deoxy-genatio n Fran ck et al , 1985). De cre ase d phos pholip id diffusion in sickleerythro cytes has a lso been ob served Zacho wski e t a l , 1985) .The ISC or SS e ry throcy tes wi th damaged membranes which a repermanent ly deform ed even in the p resence of fu ll oxygena t ion a re foundpredo mina nt ly in the dense cell f ract ion Berr ies and Milner , 1968). Thechange in ionic f luxes that occurs wi th erythrocy te deoxygen at ion Joiner e ta l , 1988) and HbS polymerizat ion may be responsible for the dense cel lformat ion and the eventua l format ion of the ISC. Alternat ively , i t may beme mb ran e dam age that accelerates the cat ion and water loss which gives r iseto the increase in cell dens ity Ho riuchi et al , 1988). O wing to the increase incell density or intracellular ha em oglo bin c onc entratio n, i t is l ikely that the secells, a long with the general den se cel l populat ion, wil l contain H bS polym ereven at the high oxygen saturat ions found on the ar ter iolar s ide of thecirculation Nog uchi and Schechter, 1981). The ISC and dense cell fractionshave a disp roport ionate effect on f i lt ra t ion o f SS erythrocytes and appe ar toincrease the haemolyt ic anaemia in sickle cell d isease Mc Cur dy andSherman, 1978; Keidan et al , 1989; Mackie and Hochmuth, 1990).

P T H O PH Y S IO L O G Y N D T H E R PE U T IC S T R T E G IE SThe fol lowing may serve as a br ief synopsis of the current pathophysiologicalmo del o r paradigm for unders tanding the c l inical manifes ta t ions o f s ickle


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SICKLE CELL DISEASE PATHOPHYSIOLOGY 75cell disease. A s the re d cell t raverses the body to deliver oxyg en f rom thelungs to th e t issue, the par t ia lly deoxy gena ted HbS can polym erize as aconsequence of the reduced so lubil ity of deoxygena ted HbS which is on lyabout ha l f tha t o f the haemoglobin concentra t ion wi th in the matureeryth rocy te Schech ter e t a l , 1987). Th e haem oglobin S aggregation whichdramatically increases the intracellular viscosity can dis tor t th e mo rpholo gyof the red ce l l, bu t the m ore imp or tan t change is the m arked decrease in theabili ty of these ceils to f low through the c irculat ion and microvasculature .O the r cel lular changes such as decreases in cel l water result ing in denseceils) , a l terat ion of ion balance, decreased membrane deformabil i ty andm em bra ne dam age result ing in ISCs) can also occur as consequ ences ofHbS polym erization and a small decrease in HbS s tabili ty Heb bel , 1990,1991). I t is likely that obstruc tion can occur bo th in t he capillary beds as wellas in th e precapil lary ar ter ioles, result ing in com prom ised oxygen deliveryand blo od f low. The decreased deformabil i ty of the erythro cyte andobstruction to f low eventually leads to pre m ature cell des truction result ingin anaem ia) and t is sue dam age . Ad heren ce of s ick le cel ls to the endothe l iumm ay exace rbate th ese processes by al lowing deoxy genation to increase.Sickle cel l anae mia includes a broad spectru m of sever i ty Ser jeant , 1992;Bu nn and F orget , 1986) Table 2). A num ber of factors have been identified

able 2. Possible mo difiers of sickle cell disease.GeneticHbF : h ereditary persistence of HbF, H bF levels , F-cellset-Thalassaemia: coexistence of ao d -a ) or - a /- e t) genotypes13-Thalassaemia: hete roz yg ou s for 13 -, 13+-thalassaemiaDo uble heterozygosity: C, D o r O-A rab haemoglobinopathyellularIntracellular haemog lobin concentration: MCHC , density distributionOther intracellular compon ents: 2,3-disphosphoglycerate, pH, Ca2Erythrocyte m emb rane abnormalit ies: ISCs, ionic pumps, l ipids, proteinsAdhesio n: endothelial , leukocyte, platelet

PhysiologicalHu mo ral factors: yon Will ebran d factor, integrins, cytokinesMicrovasculature: arterio veno us shunts, collateral circulation, vessel tone ,tissue oxyge n levels, am bien t PO2, oxygen extraction, nitric oxide ?)HbF , fetal haemoglobin; MCHC , mean corpuscular haemoglobin concentra-tion; ISC , irreversibly sickled cells. Modified from Schechter et al 1987).

which can mo dify the course of s ickle cel l disease. Th ese include a largenu m ber of othe r genetic var iables that can be co- inher i ted with the sicklecell gene such as other mutant haemoglobins and thalassaemia) ; cel lularfactors which affect cel l volume, haemoglobin concentrat ion, pH, ionbalance and oth er cel lular consti tuents ; an d othe r physiologic factors such asvascular tone. Un ders tan ding th e biophysical nature of s ickle cel l anaem iaand the factors which affect disease sever i ty has given rise to a num ber oftherap eutic s tra tegies De an an d Schech ter , 1978; Schechter e t a l , 1987;Stam atoya nnop oulos and Nienhuis , 1992).


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76 C. T. NOGUCHI ET ALTable 3. Approaches to specific therapy of sickle cell disease.

Inhibition of polymerizationHaemoglobin modificationNon-covalent reagentsNon-specific alteration of solventStereospecific competitors (peptides, modifiedamino acids) } 1' deoxy HbS solubilityCovalent reagentsAmino-terminal residues (cyanate, pyridoxal)Side chains (glyceraldehyde,acetylationagents)Diphosphoglycerate-binding ite

rythrocyte modificationIncrease cell volumeHyponatraemia(DDAVP) |Membrane modifiers(Cetiedil)}Modify on transport |Decrease 2,3-diphosphoglycerateGenetic modificationIncrease y gene expressionDNA m ethylation nhibitors (5-azacytidine)S-phase cell cycle nhibitors (hydroxyurea)Differentiating gents (butyrate, phenylacetate)Gene therapyViral-mediated gene transfer into erythroid stem cellsSite-specific homologousrecombination nto stem cellsBone marrow transplantation

ecrease mierovascular entrapmentVasodilatorsCell adhesion nhibitors

~ 02 affinityor ~' deoxy-HbSsolubility

MCH(S)CandMCHC

~ MCH S)C

Modified from Schechter et al (1987).

T h e s t ro n g d e p e n d e n c e o f Hb S p o ly me r i z a t io n o n h a e mo g lo b in c o m-pos i t ion , haemoglob in concen t ra t ion , oxygen sa tu ra t ion and the in t r ins icso lub i li ty o f deoxy gena ted HbS have p rov ided severa l ra t iona l approa chesfor the rap y in sickle cel l an aem ia (T able 3; Figures 9 and 10) .H ae moglob in S mod i f i c a t ionEar ly a t tem pts a t the rapy fo r s ick le cel l di sease focused on increas ing thedeo xyg ena ted HbS so lub i li ty (De an and Schech te r , 1978) . The goa l is no t toe l imin a te Hb S p o ly me r i z a t io n c o mp le t e ly , b u t t o r e d u c e th e p o ly me r i z a t io npo ten t ia l to leve ls observed in the more mi ld s ick le syndromes such asHbS/f3+- tha lassaemia (wi th 70% HbS) , o r be t te r s ick le t ra i t (wi th 40%Hb S ) , wh e re t h e so lu b il it y o f d e o x y g e n a te d h a e mo g lo b in f ro m in d iv id u a lswi th s ick le t ra i t is abou t 1 .5 t imes tha t o f pure deo xyg ena t ed HbS (Sunsh ineet al , 1978; Noguchi et al , 1981; Noguchi et al , 1988) (Figure 11).Ag e n t s k n o wn to d i s ru p t h y d ro p h o b ic i n t e r a c t io n s su c h a s u r e a we rein i t ia l ly p ropo sed to min im ize in te rac t ions wi th the 13S6valine (M ura yam a,1966; Coopera t ive Urea Tr ia l s Group , 1974) . Other agen ts known to a l te rso lven t e f fects , such as e thano l , a ro mat ic a lcoho ls and ac ids and a lky lureas


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SICKLE CELL DISEASE PATHOPHYSIOLOGY 77I io O-lo o~ Iocto~o o_olP oo~oOoOoO0 lip 0 0 0i o Io~o^o_o o21 ooo_oo~o~1S I__lOio~t~ol_3o~O~ ioo ooo- IOoORoO~:x:)o'OOo o ib S : I O 0 % 12= oollt ~ I I -00'T- i lO l lO01i -: oi oo-o-

o . oo . .oo . 0

SFHbF=25%HbS=75%

oOto_O#oO_Ol P O_oo O-o oI lo l l ~ oO~llol l io o o on ' lo l lI O~lo ~ - i o lo l l i o oOT oo~ - t~ o o 1~3 oi S= R] E6~.~- o: elI u o I l l ~ O l

..O300CLO0(o~o~ oYo_-, oOoNOpoQp~0 0 o0 00 ~

ASHbA=60HbS=40

I~ eeo e o?~lI:Oo_?,oi100 O0J "l l" l l l l~-I~ll-- Ileo lo ipOeo-e I

0 40

O0_l lOl l~ 0 Io~iOi~o_i5o~o-o~.'~(:~@O00u C)"P~O OjOOq0~0 I Iu 0 (

70Oxygen saturation

po?.poo moo qI~ cY,-~Oo ~ o 0 o lI~ , -w~oouoo 0 I

100

Figure 9. Representat ion of haemoglobin polymerizat ion at equi l ibr ium for a haemoglobinconce ntration of 34 g/dl. Oxy gen saturation varies (from left to right) from 0 , 40 , 70 and100 . For il lustration, haem oglob in comp osition includes 100 haem oglob in S (top) to mo delhomozygous SS individuals , 75 haemoglob in S with 25 haemoglobin F (middle) to mod el apancel lular dist ribut ion of a high level haemoglobin F and 40 haemoglobin S with 60haemoglobin A (bottom) to model sickle trait individuals. Polymer formation is maximal for100 haem oglob in S, decreas es with increasing oxygen saturation and is stil l presen t at 70oxygen saturat ion. Increasing levels of haemo globin F reduces polym er formation and at 25haem oglobin F begins to approach levels associated with sickle trait with 60 haem oglob in Aand 40 haemoglobin S.

were foun d to increase deox ygen ated HbS solubili ty W aterm an et al , 1974;Ross an d Su bram ania n, 1977; El bau m et al, 1978), bu t at levels too low fortherapeutic benef i t . Inhibitors based on the s tructure of the HbS polymerwe re designed as stereospecific inhibitors Sch echt er et al, 1987). A m on gthese are the aromatic amino acids and shor t peptides including theoligop eptide m imick ing the 13S6valine region Ku bota and Y ang , 1977;No guch i and S chec hter, 1979; Vo tan o et al, 1984). W hile an increase insolubil i ty was observed in some of these agents , other shor t peptidesdecrea sed deox yge nated HbS solubili ty, presumably because of the non-ideal behav iour due to m olecula r c rowding of the c oncentra ted HbS so lu tionrequire d for polym erization Noguchi e t al , 1985). Fu r ther increases inefficacy and perhaps specific ity are necessary for these com poun ds a nd theirchemical analogues to be of potentia l therapeutic value. In general , thecovalent and non-covalen t inhibitors of haemoglob in S polym erization mu stbe po ten t e no ug h to be use ful at levels well below their toxicity, be specificenou gh to affect only deoxy-HbS solubil i ty , and be tak en up by the erythro-cyte or be able to be d elivered to the ery throcy te us ing such techniques asl ipofusion Kum pati , 1987) or extracorpo real t reat m ent Coo perative U rea


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100 HbSoo Io o g o

75 HbS and 25 HbF

[] [Do[3 DGNDI O ~ 0 [] Hybrid ~ [~) 02formation 0 i_.1 ]]O g O ,1+02 ., - , 1 1 2~ ~ O~ o o O o o . ,

O = HbS ][] = HbF]] = HbS/HbFhybridigure 10. Sparing effect of haemoglobin F (HbF). Deoxygenation of a pure solution ofhaemoglobin S ne ar physiologic conditions, or o f cells of average haemoglobin content andlittle Hb F results in a polymer fraction of about 0.7 . Mixture of HbS and Hb F containhaem oglob in tetramers of H bS (a213s2), HbF (ot2~2) and the hybrid haemoglobin (a213s'y).Thesparing effect of HbF results from the fact that neither HbF nor the hybrid haemoglobin(u213s~) enters into the polym er phase unlike HbA in which the hybrid haem oglob in (a213s13)can enter the polym er, but at reduced tendency. Hence, for a mixture of 75% HbS and 25%Hb F near physiologic conditions, deoxygenation results in a polymer fraction of 0n ly 0.4.Factors which increase Hb F tend to cause a reduction in HbS, for reasons that are not entirelyclear, and th is fact should also be considered as part o f the sparing effect.

T r i a l s G r o u p , 1 9 74 ) ) w i t h a s u f fi c ie n t ly l o n g r e s i d e n t t i m e i n th e e r y t h r o c y t er e l a t ive to t he e ry th r oc y te l i fe span (Sch ech te r e t al , 1987).

Sickle erythrocyte modificationsT h e s t ro n g d e p e n d e n c e o f H b S p o l y m e r i z a t io n o n c o r p u s cu l a r h a e m o g l o b i nc o n c e n t r a t i o n s u g g e s t s th a t i n c r e a s i n g c el l w a t e r o r d e c r e a s i n g t h e i n t r a -c e l l u l a r H b S c o n c e n t r a t i o n w o u l d p r o v i d e s o m e r e d u c t i o n i n t h e p o l y -m e r i z a t i o n p o t e n t i a l o f t h e e r y t h r o c y t e ( I z u m o e t a l , 19 8 7) . P r e l i m i n a r ys t u d i e s u s in g m e d i c a l l y i n d u c e d h y p o n a t r a e m i a b y s tr i ct r e g i m e n o f f lu i dl i m i t a t i o n a n d th e a d m i n i s t r a t i o n o f d e s m o p r e s s i n a c e t a t e r e s u l t e d in t h eo s m o t i c s w e ll i ng o f t h e e r y t h r o c y t e w i t h a r e p o r t e d r e d u c t i o n i n th ef r e q u e n c y o f c r is e s ( R o s a e t al , 1 9 80 ). A l t h o u g h l i m i t e d in s c o p e , t h e s e e a r l yr e s u l ts i l lu s t r a te t h e p o t e n t i a l o f i n c r e a s i n g ce ll w a t e r o r d e c r e a s i n g M C H C( m e a n c o r p u s c u l a r h a e m o g l o b i n c o n c e n t r a t i o n ) a s a t h e r a p e u t i c s t r a t e g y .S e v e r a l o t h e r p h a r m a c o l o g i c a l a g e n t s w h i ch a f f e ct r e d c e ll m e m b r a n e a n de r y t h r o c y t e i o n b a l a n c e h a v e b e e n p r o p o s e d a s ' m e m b r a n e a c t i v e 't r e a t m e n t s ( B e n j a m i n e t a l , 1 9 8 0 ; C l a r k e t a l , 1 9 8 2 ; A s a k u r a e t a l , 1 9 8 4 ;Johnson e t a l , 1989 ; Ohni sh i e t a l , 1989 ; V i toux e t a l , 1989 ; Jo ine r , 1990) .


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S I C K L E C E L L D I S E A S E P A T H O P H Y S I O L O G Y 79

zo

ec-erILl

0n

(.)e-

1 .0 ......... . . . . 1 .0

0 00 0 .5 1 .0 0

OXYGEN SATURATION

c

0 0

J v i | v i i =

b

0 5OXYGEN SATURATION

i i i ~ r

d~ b ~

J

1.0

0 0 5 1 0 0 0 5 1 0OXYGEN SATURATION OXYGEN ATURATION

Figure 11. Predictions for expected polymer formation versus oxygen saturation for severalconditions reflecting potential therapies for sickle cell disease. Th e predictions were calculatedfrom total haemoglobin concentration and haemoglobin composition and oxygen saturation.(a) Polymer fraction predicted fo r pure haemoglobin S at 26, 30, 34, 38 and 42 g/dl. For a wholecell population of sickle erythrocytes, cell heterogeneity will skew the polymer curve to theright due to the higher polymerization potential of dense cells (see Figure 4). (b) Polymerfraction predicted for homozygou s sickle cell disease, without (SS) and with (S S /( -o d- a) )homozygous ~t-thalassaemia, and for sickle trait (AS) based on the m ean corpuscular haemo-globin concentration and mean percentages of haemoglobins S, F, A2 and A . (c) Polymerfraction predicted fo r haemoglobin concentration of 34 g/dl fo r Hb S and F mixtures forincreasing amounts o f HbF f rom 0 to 40%. (d) Polymer fraction predicted for haemoglobinconcentration of 34 g/dl with increasing solubility of deoxyhaemoglobin S from 16 to 26 g/dl.From Noguchi et al (1989).W h i l e s o m e o f th e s e a g e n t s m a y i n c r e a s e c e ll w a t e r , t h e i r e f fe c t s o n o t h e rs i ck l e e r y t h r o c y t e p r o p e r t i e s , c e ll s u r v i va l o r c li n ic a l b e n e f it r e m a i n t o b et e s t ed .

T h e i n c r e as e i n o x y g e n a f fi ni ty p r o p o s e d b y i n a c t iv a t io n o f 2, 3 -d i p h o s p h o g l y c e r a t e ( P o i l l on a n d K i m , 1 99 0) o r b y c h e m i c a l a g e n ts s u c h asc y a n a t e w h i c h m o d i f y h a e m o g l o b i n ( N i g e n e t al , 1 97 4) h as t h e p o t e n t ia l t od e c r e a s e t h e e x t e n t o f p o l y m e r i z a t i o n a t a n y o x y g e n t e n s i o n b y re d u c i n g t h e


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80 C T NOGUCHI ET AL

propor t ion of deoxygenated s ickle haemoglobin (Beddell e t a l , 1984;Franklin et al, 1986; A br ah am et al, 1991). Preli min ary tr ials with cyana teincluding extraco rporeal adminis tra t ion resulted in a d ecrease in haemolysisand ana em ia associated with sickle cell disease, but no significant redu ctio nin painful crises was ob serv ed (Balc erzak e t al, 1982; Lee et al, 1982).Because of the uncer ta inties regarding absolute oxygen delivery to thetissues , the therap eutic value of increasing HbS oxygen aff ini ty remains tobe demons tra ted .

enetic approachesThe approach to therapy of the d isease a imed a t augment ing fe ta l haemo-globin synthesis was predic ated on cl inical and epidemiological observationsof geographical group s of sickle cell patients dem onst ratin g a milde r clinicalcourse wh en the HbF level exc eed ed 20 (Noguchi e t a l , 1988). Tho ughprevious s tudies involving a few hund red patients conclu ded that H bF levelssubstantia l ly lower than th e 20 , w hich is com m only found am ong USsickle cell individuals imparted a minimal effect on disease manifestations(Powars et al, 1984), the more recent analysis of data obtained on severalthousan d patients in the Coo perative Study of Sickle Cell Disease suggestsan ameliorat ing effect on the pain ra te is demonstrable with only modestincreases in steady-state HbF (Platt et al, 1991). Biophysical studies havenow de lineated the quanti ta t ive bases for these observations. A n increase inthe propor t ions of non-S haemog lobins , par t icular ly Hb F, within a red cell isbenefic ia l since, as m ent ion ed above, i t ef fectively reduces th e intracellularconcentra t ion of HbS and the m ixed hybr ids of HbS and Hb F do not en te rthe polymer . This ' sparing ' ef fect con ferred by Hb F on H bS poly merizationtherefo re would tend to n ormalize the rheological proper t ies o f the s ickleeryt hro cyt e in which it is cont aine d (S chec hter et al, 1987) (Figures 9 and 10).The init ia l a t temp ts to ' reactivate ' the synthesis of HbF, were extensions ofobservations made in cel l cultures that eukaryotic gene express ion wasrela ted (a t least in par t) to the exte nt of me thylat ion within and arou nd thegene of inquiry (Fesel, 1985). Thus, 5-azacytidine (5-Aza C), a potentinh ib i tor o f D N A methyla t ion , was adminis te red to anaem ic pr imate models(DeS imo ne, 1982), and su bsequently to patients w ith B-thalassaemia (Ley etal, 1982) and sickle cell disea se (C ha rac he et al, 1983; Ley et al, 1983) and w asfound to increase dramatically the synthesis of HbF . Ind eed , baboons w howere main ta ined chronica l ly anaemic th rough regula r ph lebotomy, a f te rreceiving subcutane ous courses 5-Aza C , w ere able to increase consis tentlythe synthesis of H bF (up to 70-80 ) , while reciprocally decreasin g [3-globinsynthesis (DeSimone, 1982) . Phenotypically , these changes effectivelyrecapitula ted a reversal in the fe ta l- to-adult haemoglobin switch and,therefore , provided th e ra t ionale for i ts exper im ental applicat ion to patientswith [3-thalassaemia and sickle cell anaem ia.Following shor t courses of parente ral 5-Aza C, s ickle cell patients haveinvar iably shown an increase in percentage HbF and in F-cell numbers ,generally within 2-3 days f rom the ini t ia t ion of therapy and reachingmaxim al values of four to seven t imes baseline values (Chara che et a l , 1983;


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SICKLE CELL DISE SE P THOPHYSIOLOGY 8

Ley et al , 1983). S uch enha nc em en t in HbF production has been associatedwhen m easu red, with a decrea se in the propo r t ion o f dense red cells and inthe ra te o f haem olysis (Le y et al, 1983). O th er S-phase specific drugs such asarabinosylcytosine (Ara C) (V eith et al, 1985a), vinblastine (Veith et al,1985b) and m yle ran (Liu et al, 1990) have be en also found to be e ffective ins t imulating HbF production, but because of their perceived carcinogenicpotentia l , especial ly given the requis i te chronici ty of adminis tra t ion tomaintain e le vated Hb F levels in these patients , their use has been cur ta i ledexcept in individuals with end-s tage ~- thalassaemia (Low ery and N ienhuis ,1991).Hydroxyurea (HU) is a cy tos ta t ic agent , usefu l for the t rea tment ofmyeloproliferat ive disorders (Alte r and Gilber t , 1985) and hea d and n eckneoplasms (Donehower , 1992) , by vir tue of i ts inhibitory effect on theenzyme r ibonuc leo t ide reduc tase which resu l ts in the per turba t ion of D N Asynthesis in rapidly dividing cells . A lte r and Gilb ert (1985) f irst rep ort ed thatpatients with chronic my eloge nous leuka em ia treated pallia t ively with HUshowed substantia l e levations in Hb F levels above baseline, with those ondaily therapy at ta ining greater levels of HbF than those on intermittentt rea tment . Le tv in e t a l (1984) demo ns tra ted tha t H U could augment HbFlevels in a p rim ate m od el w hich led to its successful application to sickle cellpatients in small pilot studies (Platt et al, 1984; Ch arac he et al, 1987; Do ve rand Ch arache, 1989). T o date , near ly 100 SS patients have been e ntere d intoclinical tr ials with HU (G old berg et al, 1990; Ro dge rs et al, 1990; Ch arac heet a l , 1992), which has substantia ted this agent to be a pote nt inducer of HbFproduction.Several general and re levant conclusions can be drawn from these tr ia lswhich have included trea tm ent per iods f rom 3 mo nths (Rodg ers e t a l, 1990)to several years (Charache et al, 1992). First, under close medical super-vision most patients , but n ot a l l , wil l respo nd to H U with a t leas t doublingfrom baseline in the Hb F and F-ret iculocyte num bers . Th e 20-25 ofnon-respo nders (or poor respon ders) cann ot be dis t inguished at the presentt ime by curre nt haematolog ical , b iochemical or m olecular analyses. Secon d,in contras t to the respon se to 5-Aza C, increases in Hb F pro duction on H Uoccur more gradually (over weeks) with some patients not a t ta ining aplateau on a s table dose of H U for several mon ths . Third, th e HbF responseto H U occurs near m yelotoxic dosages and the m agnitude of this responsecan be quite var iable , with the best responders achieving maximal HbFlevels of 15-20 , invariably associated with a striking macrocyto sis.Anecdotal exper ience accumulated dur ing these tr ia ls suggests that asubstantial n um ber of these responders will exper ien ce fewer , less severecrisis and a n increa sed sense of well-being. The subjective nat ure o f thesesymptoms , however , d ic tates tha t H U should be v iewed as an exper imenta lagent pend ing t he out com e of con trolled c l inical tr ia ls .In view of the var iabil i ty of the response to HU, together with themyelotoxici ty observe d at the optimal do se of H U , othe r agents , includingcytokines which exer t an indep enden t e f fec t on H bF produc t ion have beenstudied for their potential synergistic effects. The observations thatrecom binant hum an e rythro poie t in (Epo) a lone (A1-Khatti e t a l , 1988) or in


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82 C. T. NOGUCHI ET ALcombinat ion wi th H U Mc Do nag h et al , 1989) may resul t in substant ialincreases in both F-cel l nu m ber and Hb F levels in a pr imate m odel ra ised thepossibil i ty that Epo may exert an addit ive or synergist ic effect to HbFstimulation. A n init ial rep ort involving a small num be r of sickle cell patientsshowed no addi t ive effect of Epo wh en adm inis tered to pat ients receivingdai ly H U Go ldberg et a l, 1990). M ore recen t ly, i t has bee n found that thet iming of the recombinant Epo wi th H U , the absolu te dose of Epo, and/orthe conco mitant adminis t ra t ion of oral i ron wi th Epo therapy are im portantvar iables that d eterm ine the response to this form of combinat ion therapyRodgers et al , 1993). Figure 12 demonstrates the HbF and F-reticulocyteresponse to H U alone and HU al ternat ing with Ep o given wi th supple-men tal i ron) .

P a t i e n t I H U > H U / E P O50 r . . . . ~ 20HU

. . . . . . ' - ~ - F-retics40 -'0- '- HbF

20 i ;:

o~0

sickle cell disease pathophysiology

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