C H A P T E R 41 
PATHOBIOLOGY OF SICKLE CELL DISEASE
Robert P. Hebbel and Gregory M. Vercellotti
Since it was recognized as the “first molecular disease,” sickle cell
anemia caused by homozygosity for the mutant sickle beta globin
gene has provided the classic paradigm for single-gene disorders.
Predominant clinical features include hemolytic anemia, episodic
painful events, chronic organ deterioration, disparate acute and
chronic complications, and a foreshortened life span. The genesis of
clinical sickle cell disease is complicated, and an understanding of its
pathophysiology integrates concepts from multiple disciplines,
includes contributions from the red blood cell (RBC) membrane and
the vascular wall endothelium, and recognizes the likely participation
of multiple genetic influences. This chapter addresses the pathophysi-
ology that underlies the sickle cell disease syndromes described in
Chapter 42.
EARLY YEARS OF SICKLE CELL DISEASE RESEARCH
Sickle disease syndromes were known in folk medicine for centuries
in parts of Africa, but the eponymous RBC was first reported in
the medical literature in 1910 when Herrick described a young
Grenadian man with recurrent pain, anemia, and sickle-shaped red
corpuscles in the blood (Fig. 41.1). In 1940, Ham and Castle pos-
tulated that sickle disease pathophysiology resulted from a “vicious
cycle” involving mutually promotive erythrostasis and RBC sickling
with adverse viscosity changes. In 1949, Neel validated the Men-
delian autosomal dominant inheritance of sickle cell anemia, and
Pauling demonstrated presence of an abnormal hemoglobin (Hb) in
patients and carriers. This was followed by observation of the poor
solubility of deoxygenated sickle Hb (HbS) and the reversible sol-gel
transformation of HbS solutions. In 1957, Ingram identified the
underlying amino acid substitution. Thereafter, increasingly detailed
investigations began to reveal the striking complexities of sickle cell
disease pathobiology.
GENETIC CONSIDERATIONS
Molecular Context
The sickle mutation in the HBB gene is a GAG→GTG conversion
that creates a β6Glu→Val substitution and thereby forms βS globin
chains. Genes for other β-globin variants are allelic to the βS gene
and have a codominant impact. Examples include genes for the
normal β chain (βA), β mutants (e.g., βC, β° or β+ thalassemia), and
deletional hereditary persistence of fetal Hb (HPFH). Compound
heterozygosity for βS and each one of these results in well-defined
clinical syndromes, such as HbAS (i.e., sickle trait), HbSC disease,
HbS–β-thalassemia, and HbS-HPFH. Eight percent of African
Americans have a βS gene, 3% have βC, 1.5% have β-thalassemia,
and 0.1% have HPFH. Among African Americans, about 1 in 600
births results in the homozygous state, sickle cell anemia (HbSS), and
about 1 in 400 results in some form of sickle cell disease, which
additionally includes the compound heterozygous variants other than
sickle trait. Worldwide, about 75% of sickle cell anemia births now
occur in sub-Saharan Africa, 15% in India, 5% in the Americans,
4% in the Eastern Mediterranean, 1% in Europe.
The HBB gene resides in a cluster of β-like genes within which
are various nonexonic polymorphic sites. Different combinations of
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these define discrete β-locus background haplotypes, referred to as
the Senegal, Benin, Bantu, Cameroon, and Arab–India haplotypes
(Fig. 41.2). Each designation refers to an ethnographic region in
which the sickle mutation achieved high gene frequency (typically
peaking at 0.10 to 0.15). In most cases, the sickle gene resides on one
of these five major haplotypes.
Origin, Selection, and Dispersion of the Sickle Gene
The residence of both βA and βS alleles on the distinct regional β
cluster haplotypes suggests that the sickle mutation arose indepen-
dently in the five regions. The βC mutation arose only once. Historical
and biologic data argue that frequency of the βS gene greatly expanded
in Africa about 3000 years ago and in South Asia about 4000 years
ago, following the introduction of iron tools. That led to adoption
of an agricultural system that promoted both increased human habi-
tation density and favorable breeding conditions for the mosquito
vector, Anopheles, which in turn enabled development of endemic
Plasmodium falciparum. In this context, high fixed βS gene frequen-
cies were reached because of a balanced polymorphism, such that
heterozygotes (HbAS) have an adaptive advantage over either homo-
zygote. Thus the Old World geographic distributions of the sickle
gene and historical endemic malaria are notably concordant (see Fig.
41.2), suggesting that the sickle gene represents “a biologic solution
to a cultural problem.”
In hyperendemic areas, falciparum malaria uniformly infects the
young and is the primary cause of death for children with sickle cell
anemia. However, those with sickle trait are less likely to develop
high-level parasitemia or to have severe malaria, an effect largely
exerted early in childhood. At the level of the RBC, this protection
reflects steps after initial parasite invasion. One proposed mechanism
links protection to the instability of HbS, immune status, and splenic
function. Infection of sickle trait RBCs with P. falciparum leads
sequentially to augmented Hb denaturation, clustering of membrane
protein band 3, attraction of band 3 autoantibody, complement
binding, and enhanced erythrophagocytosis, even of the early ring
forms. Thereby, an accelerated clearance of parasitized RBC by the
spleen could protect those with sickle trait, while HbS homozygotes
would lose this protection because of acquiring functional asplenia.
In synergy with this scenario, presence of HbS (via a different mecha-
nism) impairs microvascular endothelial cytoadherence of infected
RBC, thereby diminishing cerebral symptoms and impeding the
sequestration that protects parasitized RBC from splenic exposure.
The protective benefit of HbAS is lost if there is concurrent alpha
thalassemia (which lowers proportion of HbS). Yet, the blunted
malarial susceptibility in sickle trait reflects a complex interrelation-
ship among the sickle gene, host biology, and environmental factors.
Malarial severity is affected by polymorphisms in nonglobin genes
such as CR1 (complement receptor 1), CD36, TGFB1 (transforming
growth factor β), and HMOX1 (heme oxygenase 1); a polymorphism
in TLR4 (toll-like receptor 4) prevalent in sub-Saharan Africa exerts
a protective effect. Both carbon monoxide (CO) and nitric oxide
(NO) blunt severity of experimental malaria. And certain microRNA,
enriched in HbS-containing RBC, can inhibit P. falciparum growth.
Eventually, the sickle gene spread geographically by means of
commerce, migration, and the slave trade. This dispersion has been
tracked by analyses of regional β haplotypes, a biologic marker that
571
 572 Part V  Red Blood Cells

A B C D
Fig. 41.1  SICKLE RED BLOOD CELL (RBC) MORPHOLOGIES. Blood smears prepared under differing
conditions, using antecubital blood from the same sickle cell anemia patient. (A) Venous blood at a PO2
~40 mm Hg was fixed immediately to document RBC shapes occurring in vivo. Several RBC morphologies
are evident, including two granular (raisin-like) cells, five somewhat elongated cells, and two highly elongated
and curved cells. (B) Unfixed blood was fully oxygenated. Most cells resumed normal shape, but one elongated,
irreversibly sickled cell remains present. (C) The oxygenated cells from (B) were then partially deoxygenated,
upon which they assumed classic holly-leaf forms typical of rapid deoxygenation. (D) The partially deoxygen-
ated cells from (C) were then fully deoxygenated (PO2 ~ 0 mm Hg) and display the more elongated shape
having fewer spikes that is assumed by sickle RBC that have deoxygenated more slowly. The physical–chemical
basis for these shapes is presented in Fig. 41.5. (Reproduced with permission from Obata K, Mattiello J, Asakura K,
et al: Exposure of blood from patients with sickle cell disease to air changes the morphological, oxygen-binding, and sickling
properties of sickled erythrocytes. Am J Hematol 81:26, 2006).

Senegal

Arab-India
Benin
Cameroon Bantu

Malaria
Sickle cell gene

Fig. 41.2  SICKLE GENE AND MALARIA. The five regions in which the sickle gene achieved high allelic
frequency are superimposed on shading that identifies the Old World distribution of the sickle gene and of
historic, endemic malaria. (Reproduced with permission from Friedman MJ, Trager W: The biochemistry of resistance
to malaria. Sci Am 244:154, 1981; and from Nagel RL, Steinberg MH: Genetics of the βS gene: origins, epidemiology,
and epistasis in sickle cell anemia. In Steinberg MH: Forget BG, Higgs DR, Nagel RL, editors: Disorders of hemoglobin:
Genetics, pathophysiology, and clinical management, Cambridge, 2001, Cambridge University Press, p 711.)

largely corroborates predictions of gene flow derived from historical

Relationship of HbS Molecular Behaviors to Disease Features
records. As a generalization, it spread on the Benin haplotype to
North Africa and then across the Mediterranean. All three major Altered dimer assembly → RBC Hb composition
African haplotypes are present in the western Arabian Peninsula; but Hb phenotype and diagnosis
on the eastern side, the sickle gene tends to be on the Arab-India Polymerization risk
haplotype. This is also true in India, although sub-Saharan haplotypes HbS instability → Membrane defects
are represented as well. In the Americas, the βS gene is mostly found RBC dehydration
on the Benin, Senegal, and Bantu haplotypes. Hemolysis
Malaria resistance
HbS polymerization → Sickling
ABNORMAL MOLECULAR BEHAVIORS OF Vasoocclusion
Hemolysis
SICKLE HEMOGLOBIN
Because the β6Glu→Val substitution entails a loss of negative charge and
gain in hydrophobicity, HbS exhibits three abnormal molecular
behaviors of direct relevance to pathophysiology.

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 Chapter 41  Pathobiology of Sickle Cell Disease 573

Hemoglobin S Charge and Tetramer Assembly

Formation of Hb tetramers requires proximate assembly of stable
dimers from unlike monomers (e.g., α + β → αβ), an event governed
by electrostatic attraction. The normal α and β chains are positively
and negatively charged, respectively. In heterozygous states for
β-globin mutants, β-chain competition for dimer assembly is a
determinant of the relative proportions of the Hb variants.1 Mutant
β chains with lowered negative charge form αβ dimers more slowly;
the relative rates for dimer association are αβA >αβS >αβC, with αβA
dimers formed about twice as rapidly as αβS dimers. This explains
why those with sickle trait typically have only 40% HbS and why the
proportion of HbS exceeds this in HbSC disease. It also explains the
effect of concurrent α-thalassemia on the proportion of HbS in sickle
trait; as availability of α chains becomes limiting, the percentage of
HbS typically drops from 40% to 35% (one α deletion), 30% (two
α deletions), or less than 25% (three α deletions).
Hemoglobin S Stability and Oxidant Formation
HbS is modestly unstable, observed in vitro as instability to various
applied stresses. Two stresses that are most clearly physiologic involve
Hb oxidation.2 HbS has an abnormal redox potential compared with
HbA that may underlie its only modestly (~40%) increased auto-
oxidation rate. Yet, HbS exhibits markedly (~340%) augmented
instability and oxidation upon interaction with aminophospholipids
characteristic of the membrane’s inner leaflet. Its behavior once it
enters the plasma environment (caused by intravascular hemolysis) is
unknown. Although the physical–chemical mechanism of the desta-
bilizing role of the β6 valine in HbS is not known, this instability
leads to accumulation of various Hb and iron forms at the cytosol–
membrane interface.2 The resulting occurrence of abnormal, oxidative
biochemistry promotes a number of prominent defects of the sickle
RBC membrane.
Hemoglobin S Solubility and Hemoglobin S
Polymerization
Oxy-HbS, oxy-HbA, and deoxy-HbA have very high solubilities, but
deoxy-HbS aggregates into densely packed polymers, a process that is
fully reversible with reoxygenation.3,4 This abnormal property causes
the eponymous RBC shape change from polymer-mediated distor-
tion, the fundamental basis for disease promotion in sickling disorders.
Polymer Structure
Deoxygenation transforms soluble HbS into a highly viscous and
semisolid gel that behaves thermodynamically similar to a crystal in
equilibrium with a solution of individual tetrameric Hb molecules.
Even complete deoxygenation does not convert all deoxy-HbS to
polymer. The insoluble phase is a collection of domains of aligned
polymers, the basic unit of which is a double strand in which two
strings of deoxy-Hb tetramers make multiple contacts with each other
(Fig. 41.3).
Each HbS tetramer has two βS chains, the β1 and β2. Deoxy-HbS
undergoes a slight structural shift so that the A helix β6Val “donor”
site of the β2 chain in one tetramer can contact an EF helix “acceptor”
site (formed mainly by β85Phe, β88Leu, and β70Ala) in the β1 chain of a
tetramer in the neighboring single string. This critical, lateral associa-
tion can be made only when HbS is in its deoxy conformation; the
EF helix hydrophobic pocket is not a favorable acceptor site for the
charged β6Glu of the βA in HbA. In HbS, the β6Val in the β1 subunit
is located so it cannot participate in such contacts. However, the β2
chain of the second single string can form chemically similar β6Val-
dependent contacts with the β1 chain of the first single string. There
are multiple additional axial and lateral contacts, but these are largely
the same for deoxy-HbA and deoxy-HbS and are not themselves
sufficient to stabilize a polymeric structure.

a1 b1
a2 b2 b1 a
1
b2 a
a1 b1 2

a2 b2 b1 a
1
b2 a
a1 b1 2

a2 b2 b1 a
1
b2 a
2

=b6Val

A B C D E F G
Fig. 41.3  DEOXYGENATED HEMOGLOBIN S (HbS) POLYMER. (A) Electron micrograph of a fiber of
polymerized HbS obtained from a sickled red blood cell. (B) Electron density surface map, modeled from
authentic HbS fibers, shows pairings that create double strands plus a helical twist. (C) Model of the HbS
fiber, with Hb tetramers rendered as solid spheres. (D) Protein backbone shows tetramer staggering in the
HbS crystal. (E) Schematic representation of a double strand, emphasizing that only one of the two β6 valine
residues in each HbS tetramer participates in critical lateral contacts. (F) Sickled red blood cells, showing
various morphologies (top to bottom): granular, holly leaf shaped, classically sickled, and smoother and irrevers-
ibly sickled. (G) Electron microscopy of sickled RBC cytoplasm reveals highly ordered polymer domains, as
seen from the side (bottom) and on end (middle), or highly disorganized domains (top). (A and C, Reproduced
with permission from Dykes G, Crepeau RH, Edelstein SJ: Three-dimensional reconstruction of the fibres of sickle cell
hemoglobin. Nature 272:506,1978; B, reproduced with permission from Carragher B, Bluemke DA, Becker M, et al:
Structural analysis of polymers of sickle cell hemoglobin. J Mol Biol 199:315,1988; D, reproduced with permission from
Harrington DJ, Adachi K, Royer WE, Jr: The high resolution crystal structure of deoxyhemoglobin S. J Mol Biol 272:398,
1997; F and G, courtesy Dr. James G. White and reproduced with permission from White JG: Ultrastructural features of
erythrocyte and hemoglobin sickling. Arch Intern Med 133:545, 1974.)

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 574 Part V  Red Blood Cells

In the physiologic form of the polymer, the component strings of 35

Hb molecules in a double strand are half-staggered and have a slight
twist, creating a fiber that is approximately 21 nM in diameter and
30
is composed of one central and six peripheral double strands. The
crystal formed in vitro lacks the twist, but its molecular structure is
known in great detail. 25

Hb solubility
Role of Hemoglobin S Solubility 20
HbF
The RBC’s hydration state dominates the physical-chemical behavior 15 HbA2
of HbS. The solubility of deoxy-HbS (approximately 16 g/dL, HbA
measured under laboratory conditions) is much lower than the RBC HbC
mean cell Hb concentration (MCHC). So, even partial cellular 10
deoxygenation can raise deoxy-HbS concentration above its solubility 0 0.2 0.4 0.6 0.8
limit, allowing polymerization to occur. The biophysical effect of A Fraction Hb X
macromolecular crowding (boosting a protein’s activity far above that
predicted from concentration alone) confers nonideal behavior upon
cytoplasmic constituents, augmenting likelihood for polymerization 0.7
at any given degree of deoxygenation. SS
In vitro studies carried out under (nonphysiologic) equilibrium
conditions of stable oxygen tension and long-time scale corroborate
crystallographic identification of critical amino acids involved in

Polymer fraction
atomic contacts by revealing the influence of other Hbs on HbS solu-
bility (Fig. 41.4).3 When different Hbs are mixed together, the tetra- 0.35 +
mers dissociate into dimers that intermix and randomly assemble in +
+

+
a binomial distribution to reform tetramers. This clarifies the impact

+
+
of naturally occurring, intracellular Hb mixtures. In mixtures of HbS AS +

+ +
+
and HbA, overall solubility is improved because the hybrid αβS/αβA

+
tetramer integrates into polymer only one half as well as the αβS/αβS

+
+

+
tetramer (Fig. 41.4A). Addition of HbF to HbS has a greater sparing + + +

effect because neither the αγ/αγ nor the hybrid αβS/αγ tetramer can
be incorporated into polymer. In this regard, HbC has the same effect
as HbA, and HbA2 has the same effect as HbF (see Fig. 41.4A). This
sparing effect of HbA is such that much lower Hb oxygen saturation
is required for polymer to form in HbAS than in HbSS RBCs
(Fig. 41.4B).
Kinetics of Polymerization
Laboratory measurements of polymerization kinetics, enabled by
inducing (nonphysiologic) near-instantaneous and complete conver-
sion of HbS from R (oxy) to T (deoxy) state, reveal a delay until
polymer forms explosively.4 This inherent delay time is inversely
related to an extremely high power of the initial Hb concentration;
it is approximately 10 ms at Hb of 40 g/dL, but it is 100,000 seconds
at Hb 20 g/dL (Fig. 41.5A). HbS solutions and sickle RBCs behave
similarly in this regard. Delay times must vary enormously from cell
to cell because they are dominated by the marked heterogeneity in
MCHC (i.e., shorter delay for more dehydrated cells) and are influ-
enced by the presence of any non-S Hb (i.e., longer delay for presence
of HbA, C, or F) (Fig. 41.5E). Admixture of 20% to 30% HbA with
HbS (simulating HbS-β+-thalassemia) increases the delay time
10 to 100 fold, and admixture of 20% to 30% HbF with HbS
increases it by 103- to 104-fold.
The mechanism of such polymer formation is hypothesized to
proceed by a two-step, double-nucleation process (Fig. 41.5F).
Accordingly, the initial homogeneous nucleation takes place in bulk
solution, during which small numbers of tetramers associate, with
accumulation not favored until a critical nucleus size develops (esti-
mated to be 30 to 50 tetramers). Only then can new tetramers be
added lengthwise to form a large polymer. After this occurs, hetero-
geneous nucleation causes explosive, autocatalytic polymer formation
as new fibers form and extend on the surface of the preexisting
polymer. It is the time until this explosive formation occurs that labo-
ratory experiments detect as the inherent delay time. It is believed
that the striking irreproducibility of long delay times (Fig. 41.5B)
reflects stochastic formation of a single (or at least very few) homo-
geneous nucleation event(s) in cells that slowly polymerize and that
+
+ ++
0
0 50 100
B Oxygen saturation (%)
Fig. 41.4  DEOXYHEMOGLOBIN S SOLUBILITY, DEFINED BY
STUDIES UNDER EQUILIBRIUM CONDITIONS. (A) Admixture of
other hemoglobins with hemoglobin S raises overall solubility in absence of
oxygen. The x-axis indicates the proportion of admixed nonsickle Hb. (B)
The hemoglobin oxygen saturation required to initiate intracellular polymer
formation (i.e., polymer fraction) is much lower for HbAS RBC than for
HbSS RBC. (A, Reproduced with permission from Poillon WN, Kim BC, Rodgers
GP, et al: Sparing effect of hemoglobin F and hemoglobin A2 on the polymerization of
hemoglobin S at physiologic ligand saturations. Proc Natl Acad Sci U S A 90:5039,
1993; B, reproduced with permission from Schechter AN, Noguchi CT: Sickle hemo-
globin polymer: Structure-function correlates. In Embury SH, Hebbel RP, Mohandas
N, Steinberg MH, editors: Sickle cell disease: Basic principles and clinical practice,
New York, 1994, Raven Press.)
short delay times (Fig. 41.5D) reflect simultaneous formation of
multiple nucleation sites in cells that polymerize rapidly.
Polymerization Under (Patho)physiologic Conditions
In physiology, sickle RBCs are neither at equilibrium with constant
oxygen tension nor undergoing instantaneous or complete deoxygen-
ation. Rather, irrespective of the inherent delay time, the rate of
deoxy-HbS polymer growth in vivo is limited by the rate at which
RBC deoxygenation develops during microvascular passage. Since
this transit time is on the order of ~1 second, it probably effectively
renders irrelevant any inherent delay times of less than ~1 second
(Fig. 41.5G).4 Thus kinetic considerations argue that most RBCs in
patients with sickle cell anemia are unlikely to sickle during their
passage through the microcirculation unless something, such as
RBC–endothelial adhesion, slows their transit.
Predictability is complicated by the marked heterogeneity among
sickle RBCs in MCHC and HbF content, as well as the natural
biologic variability in capillary transit times. A good qualitative

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 Chapter 41  Pathobiology of Sickle Cell Disease 575

Concentration (g/dL) Fibers Cells 40

[Polymer]
20 30 40 30 S/S disease
5 20
4 “Sickle” 10

Number of cells
B 0 150 200 0
Log delay time (sec)

3 20 S/C disease

[Polymer]
2 10
0
1 “Holly leaf” A/S trait
40
0
C 0 5 10
20

[Polymer]
“Granular” 0
:3 :2 :1 0 1 2 3
Log delay time (sec)
0.5 0.6 0.7 0.8
0 0.5 1
Log [concentration (mM)]
A D Time (sec) E

Log time to reach 10% (sec)

Homogeneous nucleation
2
Deoxygenation
1
Critical in 1 sec
nucleus 0

:1

:2
Instantaneous
:3
0.5 0.6 0.7 0.8
F Heterogeneous nucleation
G Log concentration (mM)
Fig. 41.5  KINETICS OF HEMOGLOBIN S POLYMERIZATION AFTER NEAR-INSTANTANEOUS
AND COMPLETE DEOXYGENATION. (A) Extreme dependence of delay time on hemoglobin concentra-
tion. (B−D) Kinetic progress curves for polymer formation show that long delay times are highly variable (B),
but very short delay times are highly reproducible (D). To the right is a representation of domains and cor-
responding RBC morphology postulated to result from these different scales of polymerization rate (see Fig.
41.1B–E; and Fig. 41.3F). (E) Delay times for individual RBCs are influenced by substituent hemoglobins.
(F) A double nucleation process underlies polymer formation, with unfavored homogeneous nucleation (top)
followed by explosive heterologous nucleation (bottom). (G) Physiologically, the finite rate of deoxygenation
effectively caps the polymer growth rate and eliminates the relevance of delay times that are short relative to
deoxygenation rate (<1 second). (A–E, Reproduced with permission from Eaton WA, Hofrichter J: Hemoglobin S
gelation and sickle cell disease. Blood 70:1245, 1987; F, reproduced with permission from Ferrone FA, Hofrichter J, Eaton
WA: Kinetics of sickle hemoglobin polymerization II. A double nucleation mechanism. J Mol Biol 183:611, 1985; G,
reproduced with permission from Ferrone FA: Oxygen transits and transports. In Embury S, Hebbel RP, Mohandas N,
Steinberg MH, editors: Sickle Cell Disease: Basic Principles and Clinical Practice, New York, 1994, Raven Press.)

correspondence between polymerization in solution and within
RBCs argues that the fundamental polymerization mechanism (Fig.
41.5F) is not altered by membranes. Yet, emerging evidence indicates
that the abnormal sickle RBC membrane can accelerate nucleation,
in essence eliminating the inherent delay time. A similar effect would
be exerted by any preexisting polymer not completely melted during
prior pulmonary transit (expected for fewer than 1% of RBCs).
However, neither of these effects would alter the physiologic con-
straint that bulk polymer growth rate can only parallel RBC deoxy-
genation rate.
In vitro, sickle RBC can become classically sickled or assume holly
leaf or granular forms, depending on deoxygenation rate (slow to
rapid, respectively), which determines the number of nucleation
domains created (Fig. 41.5B–D; and see Fig. 41.1B–E). In the
microcirculation, granular forms are most likely to occur; in contrast,
frankly sickled forms are most likely to develop during venous return
to the heart. The RBC shape per se is not a determinant of RBC
deformability, but rigidification caused by polymer can impede
microvascular passage. This would develop dynamically during
microvascular transit.
HbF and Its Protective Effect
In sickle cell anemia, HbF in RBC lysates averages ~5% to 8% (range
1% to 25%). However, this HbF is not distributed evenly amongst
RBCs.5 Rather, its heterocellular expression is evident in the presence
of F cells (RBC particularly enriched in HbF) that comprise anywhere
between 2% and 80% of all RBCs. For most patients, only the small
proportion of their F cells that contain at least ~10 pg HbF (roughly
one-third of RBC Hb content) are expected to be protected from
polymerization under physiologic conditions.5 Nonetheless, on
average, F cells remain better hydrated and exhibit better survival.
Alternative Ligands: Carbon Monoxide
and Nitric Oxide
Patients with sickle cell anemia can have nontrivial elevations of
CO-Hb levels (reportedly as high as 7.6% in children) because of
hemolysis. Hb that is partially liganded with CO is shifted to the
R state conformation but has lost a portion of its oxygen carrying

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 576 Part V  Red Blood Cells
capacity. It is difficult to predict whether this produces a net benefit
or loss. RBC and Hb appear to participate in NO transport to
the microcirculation, although both magnitude of the effect and
mechanisms involved are debated. NO is asserted to improve RBC
deformability and impair HbS polymerization. Reaction of NO with
oxy-Hb causes Hb oxidation to met-Hb and reciprocal consumption
of NO.6
ABNORMALITIES OF SICKLE RED BLOOD CELLS
Even oxygenated sickle RBCs exhibit a variety of cellular and mem-
brane abnormalities that contribute directly to pathophysiology.
Some are the consequence of proximate polymer formation, while
others result from oxidative biochemistry. An overarching theme in
sickle disease pathobiology is that individual sickle RBC exhibit
remarkable heterogeneity in various cellular characteristics. The strik-
ing variability in hydration status and HbF content is particularly
important.
Membrane Iron and Oxidant Generation
An abnormal oxidative biochemistry takes place at the cytosol–
membrane interface of the sickle RBC.2 The avidity of HbS for
bilayer lipid, and perhaps its modestly enhanced auto-oxidation in
solution, result in augmented formation of superoxide and met-Hb.
This, in turn, can become denatured and lose its heme to the lipid
bilayer, where it is easily destroyed by lipid hydroperoxides to
liberate “free” iron. Forms of iron associated with the membrane
are catalytically active, generating highly reactive oxidants. Also,
membrane “free” iron can form a redox couple with soluble oxy-Hb
to promote further hemoglobin oxidation, denaturation, and deposi-
tion. The sickle RBC membrane thereby acquires abnormal amounts
of various iron forms: Hb, denatured hemichrome, free heme, and
nonheme iron.2 A large portion of sickle RBC oxidant generation
and stress is from enhanced nicotinamide adenine dinucleotide
phosphate (NADPH)-oxidase activity, probably in reticulocytes
and exhibiting a responsiveness to certain plasma substances, e.g.,
endothelin-1.
Of equal importance to excessive oxidant generation, the mem-
brane location of catalytic iron establishes in sickle RBC a unique
oxidant risk (not present in normal RBC) because it effectively targets
oxidative damage to membrane components. Further, the juxtaposi-
tion of iron with bilayer lipid allows reinitiation of peroxidative chain
reactions, effectively bypassing protection by vitamin E. Deficient
levels of antioxidants (e.g., vitamin E, glutathione, ascorbic acid) in
sickle RBC, caused by oxidative consumption and dietary insufficien-
cies, contribute. The result is abnormal oxidation of membrane
protein thiols and peroxidation of membrane lipids. Among the
many sickle membrane defects, evidence for an oxidative origin or
contribution is strongest for Band 3 clustering, abnormal membrane
stiffness, formation of irreversibly sickled cells (ISCs), aberrant cation
homeostasis, tendency toward microvesiculation, abnormal mecha-
nosensitivity, and erythrophagocytosis.2
Cation Homeostasis and Dehydrated Cells
For normal RBCs, MCHC averages ~32 g/dL and varies from 27 to
38 g/dL, with fewer than 1% of cells having MCHC greater than
38 g/dL. In contrast, the MCHC of sickle RBCs averages ~34 g/
dL and ranges from 23 to 50 g/dL, with up to 40% of cells having
MCHC greater than 38 g/dL. This extreme density heterogeneity
results from reticulocytosis (low-density, low-MCHC cells) and dehy-
drating mechanisms (higher density, high-MCHC cells) (Fig. 41.6).
The most dramatic ion-handling abnormality of the sickle RBC
is sickling-induced permeabilization of the RBC membrane to cations
(Na+, K+, Ca2+). Since this depends on cell deformation, it probably
partly reflects the sickle RBC’s exaggerated leak susceptibility to
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A B C D
Fig. 41.6  MARKED HETEROGENEITY IN SICKLE RED BLOOD
CELL HYDRATION. Compared with normal RBCs (A) studied by discon-
tinuous density-gradient centrifugation, RBCs from a sickle patient with four
α genes (D) include cells of unusually low density (mostly reticulocytes) and
abnormally high density (dehydrated cells). Sickle patients with three and two
α genes are shown in (C and B), respectively. (Reproduced with permission from
Embury SH, Clark MR, Monroy G, Mohandas N: Concurrent sickle cell anemia and
alpha-thalassemia. J Clin Invest 73:116, 1984.)
deformation (mechanosensitivity).7 Sickling induces calcium influx
and a slight acidification, occurring stochastically and only in some
cells at any one time. This results in net potassium and water loss
mediated mostly by activation of a Ca2+ activated (Gardos) K+ channel
and potassium chloride (KCl) cotransport. The latter can be activated
by lowered pH, endothelin-1, thiol oxidation, and a membrane
interaction effect of hemoglobins that are relatively positively charged
(HbC > HbS). It is influenced by macromolecular crowding of
cytosolic proteins caused by the high MCHC. Even at steady state,
sickle RBCs contain increased Ca2+ because it is sequestered in
cytoplasmic inside-out membrane vesicles, providing evidence of
prior cytosolic Ca2+ transients.
These aberrancies lead to decrements in RBC hydration and
deformability.7 However, hyperdense RBCs—mostly ISCs—are not
necessarily older cells with longer histories of sickling and unsickling.
Rather, they can develop via a rapid reticulocyte-to-ISC transforma-
tion, with those RBC having lower HbF levels being particularly
susceptible. It is unclear whether this rapid induction of cation loss
or the gradualism of classic interpretations is the dominant mecha-
nism underlying sickle RBC dehydration. Dehydrated RBCs have
diminished deformability and increased propensity for polymeriza-
tion, the mutually promotive effects of dehydration and sickling
comprising a vicious cycle. RBC dehydration is particularly likely to
be exaggerated by the renal medullary environment and possibly by
nocturnal arterial desaturation accompanying disordered sleep.
Deformability, Fragility, and Vesiculation
Even oxygenated sickle RBCs are poorly deformable.7 The dominant
cause of this is the abnormally high cytoplasmic viscosity of dehy-
drated cells. Additional factors include abnormal stiffness of the RBC
membrane caused, in part, by thiol oxidation and, in part, by a poorly
understood direct effect of hemoglobin upon the membrane. Upon
RBC deoxygenation in vitro, there is a temporal correspondence
between appearance of polymer-induced shape change and deteriora-
tion of deformability, as measured by micropipette and laser dif-
fractometer. On the other hand, filtration studies found decreased
deformability before morphologic change, and viscometry reveals a
large deterioration in bulk viscosity caused by deoxygenated dense
discocytes that show little shape change.
Sickle RBCs are somewhat mechanically fragile, which may be a
consequence of dehydration and a weakening of critical skeletal
associations caused by oxidative protein damage. The tendency of
sickled RBCs to lose membrane microvesicles reflects separation of
 Chapter 41  Pathobiology of Sickle Cell Disease 577

Hemoglobin
Anti–
precipitate
band 3
Bivalently
bound 30 µm
anti–
band 3
Band 3
cluster
Band 3
Fig. 41.7  BAND 3 AND IMMUNOGLOBULIN COCLUSTERING.
Denatured Hb on the RBC membrane, is associated with clumping of Band
3, and opsonization by naturally occurring anti-Band 3 antibody. Clusters of
band 3 are colocalized with immunoglobulin on the membranes of sickle red
blood cells (left). The drawing shows the colocalization scheme (right).
(Reproduced with permission from Schluter K, Drenckhahn D: Co-clustering of
denatured hemoglobin with band 3: Its role in binding of autoantibodies against band
3 to abnormal and aged erythrocytes. Proc Natl Acad Sci U S A 83:6137, 1986.) Fig. 41.8  RBC ADHESION TO ENDOTHELIUM. RBCs adhere to the
vascular wall endothelium under flow conditions in the microcirculation of
a rat infused with human cells. Immobile RBCs are on walls of the postcapil-
lary venule, and the smaller feeder microvessels (small arrows) have no flow
Major Sickle RBC Membrane Defects because of the logjam of RBC. (Reproduced with permission from Kaul DK, Fabry
ME, Nagel RL: Microvascular sites and characteristics of sickle cell adhesion to vascular
Membrane iron deposits → endothelium in shear flow conditions: Pathophysiological implications. Proc Natl Acad
Band 3 clumping → Ig attraction → erythrophagocytosis
Sci U S A 86:3356, 1989.)
Oxidative reactions targeted at membrane →
Thiol oxidation →
ISC formation
↓ Deformability and ↑ fragility
PS externalization contributes to various sickling-induced RBC responses, e.g.,
Cation leak microvesiculation.
Microvesiculation
Lipid peroxidation →
Mechanosensitivity Irreversibly Sickled Cells
Erythrophagocytosis
Abnormal cation homeostasis → The sickled RBCs seen on a typically-obtained blood smear are
RBC dehydration → ↓ deformability mostly ISCs (see Fig. 41.1). Their permanent shape abnormality is
Abnormal microrheology →
↓ Deformability
caused not by retained polymer but rather by membrane retention of
PS externalization → an elongated shape, explained by thiol oxidation of β-actin such that
Coagulation acceleration the spectrin–actin-4.1 complex exhibits abnormally slow dissocia-
Erythrophagocytosis tion. Otherwise, ISCs are similar to other equally dense RBC in
Adhesion to endothelium, monocytes, and macrophages having high MCHC, poor deformability, externalized PS, and low
Enhanced mechanosensitivity → ↑ responsiveness to deformation HbF content. ISC counts on average are higher in male patients,
perhaps reflecting their average lower levels of HbF. The fundamental
requirements for ISC formation seem to be RBC dehydration, pro-
longed deoxygenation, and assumption of a fixed membrane shape.
the bilayer from the underlying skeleton due to spicules of polymer- Perhaps there is a prior “conditioning” residence in the
ized hemoglobin, with enhanced susceptibility caused by protein microcirculation.
thiol oxidation. The clinical importance of ISCs lies in their ability to prompt
diagnosis of a sickling disorder when seen on blood smear and in
their short life span that contributes to overall hemolytic rate. They
Membrane Proteins and Lipids would contribute to the RBC logjam involved in occlusion, but it is
unclear whether ISC count correlates with vasoocclusive manifesta-
Sickle RBC membrane protein function is adversely affected by thiol tions. Although still adhesive to endothelium, ISCs are less so than
oxidation and possibly other oxidative protein modifications.2 other sickle subpopulations, but they exhibit greater adherence to
Ankyrin interactions with spectrin and Band 3 are abnormal, gly- macrophages.
cophorin and Band 3 exhibit decreased mobility, and thiol-oxidized
β-actin displays abnormal associations in the spectrin–actin-4.1
complex. Band 3 is abnormally clumped from binding of denatured Endothelial Adhesivity
HbS, which enables attraction of naturally occurring anti-Band 3
immunoglobulin (Fig. 41.7). Oxygenated sickle RBCs are abnormally adhesive to vascular endo-
Normal enforcement of bilayer phospholipid asymmetry is thelial cells (Fig. 41.8). About 20 candidate mechanisms have been
impaired in sickle RBCs. A scramblase that moves phosphatidylserine implicated, most involving adhesion molecules on endothelium,
(PS) outward is activated by calcium transits, and a translocase that adhesive structures restricted to reticulocytes or present on all RBCs,
restores PS inwardly can be inhibited by thiol oxidation. RBC sickling and with or without bridging by adhesogenic plasma proteins.8 Some
promotes PS externalization, especially in ISCs, but also in some mechanisms require RBC signaling responses to plasma factors for
reticulocytes. Other changes include presence of peroxidation activation. Involvement of mixed cell interactions with endothelium
byproducts such as malondialdehyde (MDA) that can cross-link has been proposed. Most described candidate mechanisms involve
proteins. Notably, the increased presence of bilayer lipid hydroperox- adhesive reticulocytes and are high affinity, identified using flowing
ides appears to account for the sickle RBC membrane’s abnormal conditions. Yet, in the biologic context microcirculatory blood flow
mechanosensitivity, evident in its enhanced cation leak response can be intermittent and occurs within vessels of constraining diam-
to deforming stress. Presumably, this deformation susceptibility eters enabling greater potential contact surface area. It seems probable

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 578 Part V  Red Blood Cells
that low-affinity adhesive mechanisms would gain relevance in that
situation. Indeed, an unanswered question is whether or not RBC
adhesion occurs via a single, dominant mechanism in vivo. To date,
only RBC/endothelial adhesion mediated by αvβ3 and P-selectin have
been verified in vivo in the sickle mouse, in which blockade of
P-selectin inhibits adhesion of both RBCs and white blood cells
(WBCs) to endothelium and effects improvement in blood flow.
Participation of sickle RBC adhesion in pathophysiology is gov-
erned, in part, by endothelial activation state. For example, adhesion
events mediated by endothelial vascular cell adhesion molecule 1
(VCAM-1), αvβ3, and P-selectin are activated, respectively, by tumor
necrosis factor (TNF), platelet activating factor, and thrombin. Each
of these endothelial stimulants is elevated in sickle blood. However,
there is a multitude of biologic modifiers in the sickle context that
can influence endothelial surface features (see Box on Complex Sickle
Milieu). Additional influencing factors would include: the reticulo-
cyte count; flow and shear rates; vessel diameter, geometry and
vasomotion; marginated WBCs; mixed blood cell interactions with
endothelium; concurrent processes (e.g., degree of platelet activation
or dehydration); and possibly even environmental exposure to endo-
thelial toxins such as tobacco smoke.
Macrophage Interaction
Sickle RBCs are readily phagocytosed by macrophages because of
RBC membrane modifications by malondialdehyde, PS externaliza-
tion, and opsonization by immunoglobulin. The latter process is
triggered by abnormal clustering of membrane protein Band 3 (see
Fig. 41.7) and possibly by modification induced by malondialdehyde.
The most dense cells have the most surface immunoglobulin and
higher PS externalization, and they exhibit the greatest interaction
with macrophages and potential for erythrophagocytosis.
THE ROLE OF RED BLOOD CELLS IN
DISEASE PATHOGENESIS
Vascular Occlusion
Notwithstanding the conceptual simplicity of the sickling phenom-
enon, when acute microvascular occlusion occurs, causing an acute
painful episode, it is a complex and evolving process. It seems prob-
able that similar events, but of less severe degree and remaining at a
subclinical level, are a recurrent or even near-constant feature of sickle
vascular pathobiology. The current understanding of microvascular
vasoocclusion in sickle cell anemia does carry lingering enigmas.9
Insofar as sickling is responsible, risk factors would include any-
thing that would increase RBC dehydration and MCHC (e.g.,
insufficient clinical hydration, injudicious use of diuretics), foster
arterial oxygen desaturation (e.g., lung disease, sleep-disordered
breathing), prolong microvascular transit time (e.g., inflammatory
milieu), increase blood viscosity (e.g., transfusion, clinical dehydra-
tion), right shift the oxygen binding curve (e.g., acidosis), or disturb
vascular dynamics (e.g., cold, aberrant neurochemical responses,
vasomotive rhythms). The extraordinary heterogeneity amongst
sickle RBC undoubtedly confers enormous variability in behavior of
individual RBC as they lose oxygen while traversing the microcircula-
tion single-file. Although such behavioral heterogeneity is perhaps the
dominant feature of sickle disease pathobiology, it currently is
immeasurable at the level of microcirculatory physiology. At the
sensitivity of epidemiology, HbF level is inversely related to frequency
of vasoocclusive painful crises.10
Enabling vasoocclusion, adhesion of sickle RBC to endothelium
allows greater deoxygenation by slowing microvascular flow. Indeed,
in transgenic sickle mice, vascular occlusion is a two-step process.11
This model holds that adhesion of less dense (reticulocyte-enriched)
sickle RBCs to endothelium in the postcapillary venule initiates
vasoocclusion, after which logjamming by dense, poorly deformable,
and sickling cells creates retrograde propagation (Fig. 41.8). Thus,
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determinants of RBC adhesivity and endothelial activation play an
important role in vasoocclusion pathobiology. Consistent with this,
clinical vasoocclusive severity correlates with the endothelial adhesiv-
ity of sickle RBCs in vitro.
Impairment of microvascular flow also derives from the dimin-
ished deformability of dehydrated sickle RBC. Dense cells (especially
ISCs) can have difficulty entering the microvasculature, e.g., at
bifurcations. Whether RBC adhesivity and poor deformability
perhaps exert combined or synergistic effects within the smallest
vessels has not been studied, nor has any role for dynamic change of
rigidity as deoxygenation progresses during microvascular transit.
Clinical vasoocclusive severity in humans correlates with preservation
of RBC deformability rather than with impairment thereof,7 perhaps
because more dense cells tend to be misshapen and less able to make
close adhesive contacts with endothelium.
Hemolytic Anemia
RBC life span in sickle cell anemia averages about 15 days but
with marked interindividual variability (from ~7 to ~30 days); in
HbSC disease, the average is about 30 days.12 All four fundamental
mechanisms that can underlie RBC removal in hematologic
disease—erythrophagocytosis, fragmentation, trapping, and osmotic
lysis—probably contribute (Fig. 41.9, bottom). These are conse-
quences of the proximate aberrancies of the sickle RBC discussed
earlier (Fig. 41.9, middle) that result from the specific molecular
behaviors of the mutant HbS (Fig. 41.9, top). Although speculative,
the illustrated, integrated synthesis of extant research data presents a
plausible mechanistic blueprint.12
Although complex, the routes to accelerated RBC removal seem-
ingly resolve into two contributory mechanistic cascades: one from
polymer formation that underlies three terminal processes (trapping,
fragmentation, osmotic lysis) that cause intravascular hemolysis; and
one from HbS instability that leads to erythrophagocytosis and
extravascular hemolysis. Notably, intravascular hemolysis seems to
account for only one-third of overall hemolysis, while two-thirds
seemingly is explained by extravascular hemolysis. The influence of
the instability-based cascade is most evident in enhanced erythropha-
gocytosis of sickle RBC, promoted by denatured Hb causing Band 3
clumping causing attraction of immunoglobulin.
The shortest survival is exhibited by the sickle RBCs that are most
dehydrated and that have the lowest amounts of HbF,13 consistent
with the polymerization-based abnormalities (see Fig. 41.9, left side).
Yet, it is not known whether these two RBC features fully explain
the very wide range of hemolytic rates. Presumably, the sickle RBCs’
fragility is related, and sickled RBCs do lose Hb via microvesiculation
when sickling is reversed. Improved RBC hydration caused by con-
current α-globin gene deletion improves RBC survival.14 Sickle RBC
survival drops substantially during acute painful episodes, but whether
this precedes or follows vasoocclusion onset is not known. The only
biomarkers so far documented to correlate strongly and quantitatively
with measured RBC lifespan in sickle cell anemia are the (uncor-
rected) reticulocyte percentage and HbF level.12
Consequences of Hemolysis
Hemolysis exerts complex effects. Some are indirect, stemming from
expanded erythropoiesis, such as enhanced production of highly
adhesive reticulocytes and augmented elaboration of placental growth
factor. This growth factor activates blood monocytes and promotes
augmented production of endothelin-1 (ET-1) that can exert multiple
effects relevant to sickle pathobiology: induce vasoconstriction, cause
nociceptive hypersensitization, activate RBC NADPH oxidase activ-
ity, and prompt release of inflammatory mediators. Other hemolysis
effects derive directly from RBC components released into the blood.
Arginase diminishes plasma arginine, possibly impeding endothelial
nitric oxide synthase (eNOS). A robust elaboration of PS-positive
RBC microparticles exerts a signaling impact upon endothelial cells,
 Chapter 41  Pathobiology of Sickle Cell Disease 579

β6 glu→val

αβ dimer assembly Hb polymerization Hb instability

Hb oxidation
% HbS
Sickling
+ Mechano-
sensitivity Membrane • O2−
MCHSC HC, heme, Fe H2O2
• OH
LOOH
K+ & H2O PSout
loss
? Vesicles
−S−S− MDA HC/B3
Dehydration C5b-9 clustering
Fragility

+ Ig + Ig
↓ deformability

Trapping Fragmentation Osmotic lysis Erythrophagocytosis

Fig. 41.9  MECHANISMS LEADING TO HEMOLYSIS IN SICKLE CELL DISEASE. This integrated
synthesis proposes how the molecular behaviors of hemoglobin S (HbS) (top) cause development of multiple
RBC abnormalities (middle) that lead to the four mechanisms of accelerated RBC destruction (bottom).
(Reproduced with permission from Hebbel RP: Reconstructing sickle cell disease: A data-based analysis of the “hyperhemolysis
paradigm” for pulmonary hypertension from the perspective of evidence-based medicine. Am J Hematol 86:123−154,
2011.)

accelerates thrombin generation, promotes pulmonary sequestration

of WBCs, and protects disgorged cell-free Hb from scavenging pro-
teins. The oxy-Hb liberated from lysing sickle RBCs can consume
NO,6,15 thus augmenting the diminished NO bioavailability primar-
ily caused by endothelial dysfunction. It has been argued that this
phenomenon is the specific cause of multiple specific complications
of sickle disease, based upon correlations between them and indirect
hemolytic biomarkers.16 However, this assumption is belied by the
extreme complexity associated with hemolysis. It is very likely that
biodeficiency of NO in sickle disease contributes to sickle disease
complications, but it is improbable that it is a sole causal factor.12
Deficiency of NO does restrain its normal braking effect on platelet
activation and inflammation, as well as its vasodilatory and superoxide
buffering functions.
Once HbS in plasma oxidizes to met-HbS, it is abnormally likely
to lose its heme2 and thereby: oxidize blood lipids, induce endothelial
activation, stimulate blood monocytes and endothelial cells to
produce TNF-α and express tissue factor, and trigger disgorgement
of neutrophil extracellular traps, among other effects. Some cell
responses to heme are mediated by its binding to TLR4; e.g., in sickle
mice this causes enhanced endothelial surface expression of P-selectin
and triggers vascular stasis.17 In the sickle context any potential effects
of liberated cell-free Hb would be augmented because of the greatly
limited levels of scavenging proteins, haptoglobin and hemopexin.
UNIQUE SYSTEMS BIOLOGY OF SICKLE CELL ANEMIA
Sickle cell anemia is unique amongst human diseases because of the
extraordinary complexity arising from concurrent disturbance of
multiple biologic processes, such that blood cells and the vessel wall
are exposed to a broad spectrum of abnormal inputs (see Box on The
Complex Sickle Milieu). Remarkably, this complexity derives from
only two proximate events, vasoocclusion and hemolysis. Although
it is not possible to identify the proportionate contributions and
importance of the resulting disparate aberrancies, two themes emerge.
The Complex Sickle Milieu
Anemia (altered wall shear stress, tissue hypoxia, HIF-1 signaling)
RBC abnormalities (rigidity, sickling, abnormal adhesivity)
Hemolysis (plasma Hb and heme, RBC microparticles, arginase)
Blood cell activation (signaling and adhesive interactions with
endothelium)
Systemic inflammation:
Mediators (TNF, IL-1β, MCP-1, prostaglandins, leukotrienes,
others)
Endothelial glycocalyx degradation
Activated monocytes, granulocytes, and lymphocyte subsets
Microparticles (endothelial, monocyte, platelet)
Mast cell activation (neuroinflammatory mediators)
NETs
Oxidant stress (activated WBC, xanthine oxidase, NADPH oxidase,
mitochondrial, others)
Growth factors (VEGF, PlGF, erythropoietin, others)
Dehydration (vasopressin)
Hemostatic system abnormalities:
Coagulation activation (thrombin, others)
Fibrinolysis (D dimer, others)
Platelet activation (platelet microparticles, plasma TSP, others)
Vasoactive agents (hypoxia, ET-1, thromboxane, vasopressin,
prostaglandins, adrenergic agonists, peroxides, CO, NO, others)
Adhesogenic proteins in plasma
Ischemia/reperfusion physiology
Endothelial cell activation and dysfunction
Endothelial cell injury (from occlusion, oxidants, oxidized plasma
lipid, mechanic forces)
Inadvertent effects of therapies (iron overload, opioid angiogenesis
signaling)
CO, Carbon monoxide; ET, endothelin; Hb, hemoglobin; HIF, hypoxia inducible
factor; IL, interleukin; MCIP, monocyte chemotactic protein; NETs, neutrophil
extracellular traps; NO, nitric oxide; PlGF, placenta growth factor; RBC, red
blood cell; TNF, tumor necrosis factor; TSP, thrombospondin; VEGF, vascular
endothelial growth factor; WBC, white blood cell.

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 580 Part V  Red Blood Cells
HbS RBC
polymer sickling
↓pO2
deoxyHbS Vascular
↑[HbS]
Stasis
+ Hemolysis
↑ RBC & WBC adhesion Free heme &
to endothelium hemoglobin
Vascular
Occlusion
Systemic
Endothelial Systemic
Dysfunction Inflammation Ischemia
reperfusion
Fig. 41.10  ISCHEMIA/REPERFUSION AS THE CORE DRIVING
FORCE IN SICKLE CELL ANEMIA. As the consequence of vasoocclusion,
ischemia–reperfusion provides an incessant driving force causing systemic
inflammation with microvascular dysfunction. The adhesion biology result-
ing from activated/dysfunctional endothelial cells creates a positive feedback
loop that slows microvascular transit and enables polymer formation. (Modi-
fied from Hebbel RP: Reconstructing sickle cell disease: A data-based analysis of the
“hyperhemolysis paradigm” for pulmonary hypertension from the perspective of
evidence-based medicine. Am J Hematol 86:123, 2011.) Hb, Hemoglobin; PS,
phosphatidylserine.
First, vasoocclusion and hemolysis are interrelated and mutually
promotive. Second, there is an apparent unifying explanation for
emergence of this panoply of biologic mediators.
Ischemia-Reperfusion Physiology
The abnormal adhesion of sickle RBC to endothelium creates a posi-
tive feedback loop that slows microvascular transit and thereby
enables deoxygenation, polymerization, sickling, and occlusion.
Experimental studies in transgenic sickle mice indicate that the
enabling, proximate instigation of endothelial activation derives from
ischemia-reperfusion (I/R) injury physiology, a process that would
comprise an incessant driving force for systemic inflammation (Fig.
41.10).18 This combination of inflammation and endothelial dys-
function can provide a unifying explanation for the multitude of
vascular biologic abnormalities in sickle cell anemia, and it establishes
a contextual and generative explanation for several of the specific
clinical complications.
The complicated biology of I/R injury occurs when a proximate
vascular occlusion causing ischemia is followed by reperfusion that
reintroduces oxygen to the formerly ischemic area. In the unique
sickle context, the initiating occlusive event(s) would be microvascular
and multifocal, and they would happen recurrently. As revealed by
studies of sickle transgenic mice, this triggers the classic, reperfusion-
dependent, early I/R events of localized and rapid onset of oxygen
radical generation, nuclear factor kappa-B (NFκB) activation, TNF-α
production, and WBC activation. Research on I/R generally illustrates
that its pathobiology thereafter explosively arborizes to become vastly
complex. Its hallmark, however, is conversion of this localized process
into a sterile inflammatory response that is robust and systemic. It
initiates widespread microvascular dysfunction and organ accumula-
tions of inflammatory cells by infiltration from blood plus activation
of tissue resident macrophages and mast cells. This can lead to disease
remote from the initiating occlusion, and it can explain emergence
of macrovascular disease from inciting microvascular events.
INFLAMMATION
Many agents comprising the exceedingly complex sickle milieu (see
Pink Box) exert proinflammatory effects; a number interact with each
other in ways that alter the nature or complexity or magnitude of
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responses. As one example, the growth factor angiopoietin-2, released
from Weibel-Palade bodies, sensitizes the endothelial cell toward
TNF-α and consequent adhesion molecule expression. It is not pos-
sible to identify proportionate contributions of the many specific
inflammatory inputs, but ligand-triggered TLR4 signaling is emerg-
ing as an important contributor.17
In summation, sickle cell anemia is a chronic, systemic inflamma-
tory state.19 Characteristic features include: leukocytosis; activation
of granulocytes, monocytes, lymphocyte subsets, and mast cells;
elevated levels of proximate inflammatory mediators, acute-phase
reactants, and distal effectors; abundance of soluble adhesion mol-
ecules; activation of coagulation; presence of microparticles from
multiple activated cell types; and generation of excess oxidant via
multiple mechanisms. Importantly, this systemic inflammatory state
is perpetual, with footprints of active inflammation apparent even
between acute clinical events. Clinically, leukocytosis in sickle cell
disease is a risk factor for mortality, clinical and silent stroke, and
acute chest syndrome, and it helps predict which babies will develop
a severe clinical course. Conversely, several clinical complications are
themselves overtly inflammatory, in particular acute painful episodes
and acute chest syndrome.
Endothelial Activation and Dysfunction
The vascular endothelial cell receives and responds to disparate
inputs, both sensing and modifying its environment. Although
normally adaptive, the endothelial response can become maladaptive.
In the sickle context, the extreme complexity of the sickle milieu (see
Box on The Complex Sickle Milieu) molds endothelial cell function,
leading to a harmful state. The core inflammatory and oxidative input
causes a high level of endothelial activation with adverse consequences
such as coagulation activation, degradation of the endothelial glyco-
calyx (a critical determinant of endothelial cell homeostasis), and
endothelial dysfunction. An often-overlooked principle predicts that
endothelial dysfunction in the sickle context creates unique risk (see
Mortality and Sudden Death, later).
Aberrant Vasoregulation
Sickle cell anemia involves deficient endothelial-dependent, flow-
mediated conduit artery dilation.20 Beyond the dominant role of I/R
and inflammation-induced endothelial dysfunction, there are several
additional contributing processes: TNF-α induction of endothelial
L-arginase, limiting L-arginine availability to eNOS; NO consump-
tion by excess superoxide and cell-free oxyHb; elevated asymmetric
dimethylarginine; plus other aberrancies (e.g., microparticles, abnor-
mal wall shear stress, elevated phospholipase A2).12 The proportionate
contributions of these processes is unknown. NO bioavailability
seems to be higher for females than males, who exhibit substantial
non-NO regulation of flow.
Perfusion patterns are complex in sickle cell anemia but can gener-
ally be described as impaired microvascular flow and augmented
macrovascular flow.21 Aberrancy of both endothelial-dependent and
endothelial-independent vasoregulation is apparent, and the milieu
is replete with a multitude of vasoactive substances. A state of vascular
instability may derive from tonic upregulation of both vasoconstric-
tive and vasodilatory systems, in addition to exaggerated α1-adrenergic
vessel wall responsiveness. Sickle humans reveal disruption of auto-
nomic regulation (e.g., with augmented risk for hypoxia-induced
perfusion decrements).22 Systemic blood pressure in patients with
sickle cell anemia is lower than in nonanemic controls, yet is higher
than in comparably anemic β-thalassemics.
Coagulation Activation
The interplay between inflammation and coagulation often seen in
biomedicine is generally highly evident in sickle cell anemia, with

chronic activation of coagulation, fibrinolysis, and platelets.23 This is
accompanied by increased whole blood tissue factor (TF) and its
increased expression on endothelial cells and blood monocytes. Such
abnormalities can follow signaling via heme/TLR4 or TNF or various
other perturbing factors. Presence of PS on released microparticles
and on some sickle RBC promotes accelerated thrombin generation.
Oxidation appears to create resistance to cleavage of ultra-large forms
of von Willebrand factor by ADAMTS13. There is excessive con-
sumption of antithrombotic proteins. The extant biodeficiency of
NO would contribute to TF expression and augmented platelet
activation because it normally inhibits these. Products of platelet and
coagulation activation undoubtedly exert signaling and perturbing
effects on endothelium.
Genesis of Clinical Disease
For the individual patient, evident clinical disease may not parallel
or accurately predict the extent of underlying overall disease burden.
It has been argued that some clinical complications (pulmonary
hypertension, leg ulcers, priapism, stroke) are caused by hemolysis,
while others (acute chest syndrome, acute painful episodes) are caused
by vasoocclusion.17 That different processes underlie different clinical
complications is entirely possible, but evidence for there being such
dramatic dichotomy of phenotype pathogenesis is very indirect.12 It
is important to recall that effects of the proximate inputs (hemolysis
and vasoocclusion) are undoubtedly modulated by the complex sickle
milieu, and that multiple biologic systems are concurrently activated.
Also, the vessel wall is exposed lifelong to the great variety of stressors
and perturbing substances of the complex sickle milieu. Few data in
the research literature, whether from general medicine or study of
sickle disease, can be extrapolated with any confidence whatsoever to
chronicity of this magnitude and complexity.
Pain Syndromes
In adults, a higher frequency of acute painful episodes is associated
with increased mortality,10 presumably reflecting derivation of both
from severity of underlying disease activity. The immense further
disturbance of multiple biologic processes during such episodes
augments risk for complications like acute chest syndrome and
sudden death. Pain frequency is higher in association with higher
hematocrit and lower HbF level, consistent with the concept that
acute painful episodes involve ischemic pain from vasoocclusion.
However, pain in sickle disease appears to be multimodal and
complex. There probably is a substantial inflammatory contribu-
tion to both acute and chronic sickle pain, involving many factors
including inflammatory neuropeptides and mast cells.24 It probably
involves a state of nociceptive hypersensitization. The chronic, daily
pain affecting many patients seems to be both inflammatory and
neuropathic in origin.
Chronic Vasculopathy
The macrovascular component of sickle vascular disease is a chronic
inflammatory vasculopathy. Histopathology from the large and
medium vessels at the circle of Willis, where some sickle children
develop occlusive disease, is characterized by intimal hyperplasia,
fibrotic and proliferative changes, and damage to internal elastic
lamina. Pathology of arterial lesions at other sites is described simi-
larly. This typical arterial wall response to inflammation occurs, in
the sickle case, without the fatty streak and foam cells of atheroscle-
rosis, probably reflecting the absence of hyperlipidemia plus the
different spectrum of proximate inflammatory inputs. It is quite
possible that the specific generative stressors for vasculopathy could
vary somewhat from organ to organ, but it seems likely that universal
contributors include inflammation, endothelial dysfunction, growth
factor excess, and aberrant wall shear stress.
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Chapter 41  Pathobiology of Sickle Cell Disease 581
Thrombosis
The hypercoagulable state suggested by blood biochemistries is
accompanied by a thrombotic diathesis.23 Arterial thrombosis occurs
in areas of vasculopathy in the circle of Willis in association with
childhood ischemic stroke, and in lungs exhibiting pulmonary
hypertension. Incidence of venous thromboembolism is increased,
and sickle cell anemia is a complicating condition for pregnancy.
Downstream consequences of the coagulation system activation may
play a greater role than is currently appreciated.
Stroke and Cerebrovascular Disease
Several stroke syndromes occur in sickle disease and may have differ-
ing pathogeneses. Ischemic stroke with clinical deficit develops in 5%
to 10% of children with sickle cell anemia, with arterial wall changes
that narrow vascular luminal diameter within the circle of Willis
being the strongest risk factor and presumptive cause. Completion of
the actual stroke tends to involve thrombosis at the site of vessel wall
disease. Despite this, most clinical research has focused upon stroke
events per se rather than upon separate risks for the apparent funda-
mental underlying processes, arterial wall disease and thrombotic
diathesis. Concurrent α-thalassemia or HbSC disease lowers the risk
of this stroke type, and HbF level is not protective. It can be suspected
that elevated growth factors are participants in arterial wall disease
and the sometimes-associated Moyamoya. In a seemingly separate
syndrome, “silent” strokes (but associated with time-dependent
degradation of neuropsychologic function) accumulate throughout
childhood and are believed to derive from inadequate microvascular
blood flow eventuating in multifocal microinfarcts. Yet another
syndrome, hemorrhagic stroke, can complicate the vessel fragility of
Moyamoya and sometimes aneurysm; otherwise, risk factors and root
causes of hemorrhagic stroke are unknown.
Pulmonary Disease
The spectrum of sickle pulmonary complications includes chronic
restrictive lung disease, pulmonary hypertension, asthma, infection,
in situ and embolic thrombosis, and acute chest syndrome (ACS).
The acute inflammatory syndrome of ACS is associated especially
with infection in children and fat embolism (probably from marrow
infarction) in adults, but other presumptive triggers are similarly
implicated. Risk factors include leukocytosis, lower levels of hemo-
globin and HbF, and a history of asthma. The nature of ACS and its
occurrence and timing during acute vasoocclusive episodes raise the
question whether it might be an event of remote organ injury com-
plicating I/R physiology.18 Regardless of specific proximate trigger, in
this syndrome it is likely that inflammation, adhesion biology, NO
deficiency, and endothelial permeability conspire to augment the
deleterious effects of hypoxia on the lung. Mast cell activation,
another consequence of I/R, may contribute to pulmonary suscepti-
bility generally and to pathobiology of ACS and asthma specifically.
Pulmonary hypertension occurs in about 6% of adults with sickle
cell anemia,25 in half from pulmonary venous disease, in half from
pulmonary arterial disease. Genesis is almost certainly multifactorial,
as the sickle context involves multiple features associated with pul-
monary hypertension in the general population: absence of splenic
function and presence of activated platelets, an inflammatory state,
and endothelial dysfunction. It is difficult, however, to distinguish
between causes and consequences of this pathobiology. Further, the
pulmonary arterial wall in sickle cell anemia is awash with suspect
mediators, and the biodeficiency of NO emasculates a normal braking
mechanism on inflammation, proliferation, and vasoconstriction.
Notably, both children and adults with sickle cell anemia not infre-
quently exhibit sleep-disordered breathing with nocturnal hypoxia,
the major pulmonary vasoconstrictor. This justifies suspicion that
long-term, repeated exposure to this major stressor plays a role in
development of pulmonary hypertension in sickle disease. There may
 582 Part V  Red Blood Cells
be a substantial problem in sickle cell anemia of transient elevations
of pulmonary pressure unrelated to histopathologic pulmonary
hypertension, as discussed later under “Mortality and Sudden Death”.
Kidney Disease
In sickle cell anemia renal hyposthenuria is a nearly universal problem
arising from medullary hypoperfusion resulting from the harsh renal
medullary environment; its hypoxia, acidosis, and hyperosmolarity
all would promote HbS polymerization and adverse changes in blood
viscosity. Indeed, this even occurs in sickle trait as well. The renal
cortex, on the other hand, exhibits hyperperfusion, believed to
underlie glomerular dysfunction with albuminuria.21 Patients can also
develop acute renal injury, papillary necrosis and chronic renal
disease, the latter being a significant contributor to mortality. Hema-
turia from papillary damage is very common.
Mortality and Sudden Death
Mortality rate is increased in sickle cell anemia patients having higher
rates of acute painful episodes,26 with notable risk indicators includ-
ing low HbF, high WBC count, chronic renal failure, and elevated
tricuspid regurgitant jet velocity. Among apparent causes of death at
autopsy, cardiopulmonary disease is most prominent.27 In 1994, a
large study from the United States identified a median survival of 42
years for men and 48 years for women with sickle cell anemia; the
comparable ages were 60 years and 68 years, respectively, for HbSC
disease.26 This reflected large past improvement after introduction of
prophylactic penicillin for children, revealing the earlier dominance
of pneumococcal sepsis in disease natural history. Further improve-
ment has derived from chronic use of hydroxyurea, an agent that
boosts HbF level, lowers WBC count, improves RBC hydration and
survival, and blunts inflammatory biology and RBC adhesivity.
Unfortunately, survival is much worse in parts of the world with less
access to medical care and greater abundance of comorbidities (e.g.,
malaria, diarrheal disease).
Sickle cell anemia entails risk for sudden death, tending to occur
during painful episodes or other acute events. Although unexplained,
this suggests acute cardiac catastrophe. A candidate mechanism may
be occurrence of transient, explosively abrupt elevations of pulmonary
artery pressure, even in absence of histopathologic pulmonary hyper-
tension.12 This derives from an important principle: the underlying,
preexisting endothelial dysfunction of sickle disease enables and
predicts exaggerated vasoconstrictive responses to unrelated potential
vasoconstrictors such as hypoxia, augmented NO consumption and
inflammatory signaling. Each can fluctuate in short time scale, e.g.,
during acute painful episodes, and thereby might create risk for
sudden death from rapidly elevated right heart pressure.
Sickle Trait
Sickle trait typically is associated with renal hyposthenuria. More
seriously, it comprises risk for exertional sudden death, probably
precipitated by effects of dehydration on RBC and blood viscosity.
At an epidemiologic level, trait also involves heightened risk for
venous thromboembolism, chronic renal disease, and thromboembo-
lism complicating pregnancy. It may predispose toward ischemic
stroke.
BASIS OF PHENOTYPIC DIVERSITY
Factors aside from the beta globin contribute to the remarkable
interindividual diversity of clinical phenotype and severity in sickle
cell anemia. Both can be significantly influenced by environment,
nutrition, socioeconomic status, endemic infectious agents, and avail-
ability of medical care. Therefore, phenotypic variability is most
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evident in countries where greater survival and stability derives from
accessible medical care, lower infant mortality, and lesser childhood
infection burden. The innumerable questions regarding phenotypic
diversity are of great importance. In sickle cell anemia, why do some
children, but not others, develop stroke? Why do only a small fraction
of adults develop pulmonary hypertension, even though all have
ongoing hemolysis? Why does pain severity vary so widely irrespective
of globin genotype? And so on.
Level of Hemoglobin F
HbF level varies amongst individuals as a quantitative trait and is
determined approximately 80% by genetics.5 After its decline over
the first 6 months of life, most of the antisickling protection from its
high level at birth is lost, but its level still varies among sickle adults
over a 20-fold range. HbF level reflects the number of F cells, the
amount of HbF per F cell, and the preferential survival of F cells.13
Known determinants account for perhaps one-half of its variance: a
polymorphic XmnI site (11p) upstream of the Gγ gene; the HBSIL-
MYB intergenic region (6q23); SNPs in TOX (8q12.1); and poly-
morphisms of BCL11A (2p16), a transcriptional silencer of the HBG
gene.5 HbF is somewhat higher levels in females, hinting at a con-
tributing locus on the X chromosome. Polymorphisms in trans-acting
enhancers of BCL11A account for some, but not all, of the regional
variations in HbF level. Among the African autochthonous haplo-
types, the Benin haplotype is associated with higher HbF level;
however, it is twice again as high among those with the Arab-India
haplotype.
α-Thalassemia
The normal genotype is αα/αα, but about 30% of African Americans
have a single α deletion (–α/α), so concordance with sickle cell
disease is common; and homozygosity for the allele is seen (–α/–α).
Its prevalence elsewhere varies regionally. An α-gene deletion has
minimal effect on the HbF level but results in improved RBC hydra-
tion (see Fig. 41.6), a lower ISC count, improved RBC survival, and
less severe anemia.14 Perhaps loss of one α gene nudges α/β chain
balance toward normal, given the mild instability of βS globin.2 Yet
there is no amelioration of pain severity, and some complications
(osteonecrosis, retinopathy) increase, possibly because of the increased
blood viscosity.
β-Globin Alleles
Compound heterozygosity for the sickle gene and another β allele
can affect clinical phenotype, with amelioration to, e.g., in the direc-
tion of amelioration by admixing βA or γ chains with βS. However,
HbSC disease presents a unique case caused by the lesser electronega-
tivity of HbC compared with HbS.28 Rather than simulating sickle
trait (see Fig. 41.5A), presence of HbC stimulates of KCl cotransport,
causing RBC dehydration and increasing concentration of HbS.
Combined with a concurrent augmentation of HbS proportion
(~50% versus ~40% in HbAS), this creates a sickling disorder only
somewhat less severe than sickle cell anemia. Although pain and
anemia are lessened a bit, there is an increased propensity for retino-
pathy and osteonecrosis. It has been assumed that this derives from
the somewhat higher blood viscosity when anemia is less severe.
Other less common β gene alleles can likewise interact with HbS and
affect clinical phenotype via impact on polymerization.
Unexplained Phenotypic Diversity
Beyond these well-defined influences, there is still enormous unex-
plained variability in the clinical phenotype of sickle cell anemia. The
disparate biologic processes that participate in vasoocclusion,

macrovascular vasculopathy, hemolysis, and specific complications
highlight the certainty that phenotypic heterogeneity will be influ-
enced by underlying genetic variations affecting adhesion biology,
cation homeostasis, inflammatory signaling, vasoregulation, and so
on. Indeed, the spectrum of potential foci at which genetic variation
might exert effects and be relevant to sickle disease phenotypic
diversity is as vast and complex as human biology itself.
The single-nucleotide polymorphisms (SNPs) that have been
detected in association with specific clinical complications are far too
numerous to describe here.29 Of course, much work is still needed to
discern whether such associations are actually informative vis à vis
pathogenic specifics. Several SNPs seem particularly interesting. A
TNF (-308) promoter polymorphism is associated with large vessel
stroke in children. Polymorphisms affecting the HO-1 promoter
create heterogeneity in GT repeat lengths (the shorter of which enable
greater HO-1 responsiveness, e.g., to heme) are described as being
associated with lower hospitalization rate for ACS in children. Inter-
estingly, a TLR4 polymorphism prevalent only in sub-Saharan Africa
leads to a greater inflammatory TNF-α responsiveness to TLR4
ligands that is protective in malaria, and could well impact sickle
biology. A wholly different approach to this general problem was
provided by examination of gene expression by, and inflammatory
response of, endothelial cells derived from sickle children.30 Those
from children with circle of Willis disease exhibited suggestive inflam-
matory gene expression, plus an actual exaggerated NFκB response
to stimulation with inflammatory mediators. Certainly, the contribu-
tion of various nonglobin genetic influences will continue to be
identified as modern and creative approaches are being applied to the
problem of phenotypic diversity in sickle cell anemia.
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