Pathophysiology of Sickle CellAnemia: Hemoglobin S

Polymerization

Constance Tom NoguchiLaboratory of Chemical Biology

National Institutes of Diabetes, Digestive and Kidney DiseasesNational Institutes of Health

Red Blood Cells from Sickle Cell Anemia

OXY-STATE DEOXY-STATE

• Deoxygenation of SS erythrocytes leads tointracellular hemoglobin polymerization, loss ofdeformability and changes in cell morphology.

Hemoglobin: The Oxygen Transporter

OXYHEMOGLOBIN DEOXYHEMOGLOBIN

Sickle Cell Mutation

+O2

-O2

+O2

-O2

5'α1α2ζ

3'Chromosome 16

5' 3'Chromosome 11

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ε γ γ δ βG A ������������

α αβ βCCT GAG GAG

-Pro-Glu-Glu-5 6 7

CCT GTG GAG-Pro-Val-Glu-5 6 7

βA βS

Normal (HbA) Abnormal (HbS)

*

Deoxyhemoglobin S Polymer Structure

A) Deoxyhemoglobin S 14-stranded polymer (electron micrograph)

D) Charge and size prevent 6ββββ Glu from binding.

C) Hydrophobic pocket for 6ββββ Val

B) Paired strands of deoxyhemoglobin S (crystal structure)

Dykes, Nature 1978; JMB 1979Crepeau, PNAS 1981 Wishner, JMB 1975

Determinants of Hemoglobin SPolymerization

• Intracellular hemoglobin composition

• Intracellular hemoglobin concentration

• Oxygen saturation

SS erythrocytesMCHC ~ 32 g/dlDeoxyHbS solubility ~16 g/dl

Hemoglobin S Polymerization in SS Erythrocytes

• Maximal at 0% O2 sat (oxygen saturation).• Detected at high O2 sat, particularly in dense cells.• Variable due to heterogeneity in corpuscular hemoglobin

concentration.

Unfractionated SS erythrocytes

Po

lym

eriz

ed

hem

og

lob

in (

g/d

l)

Fractionated SS erythrocyteslight middle dense

29.5 g/dl 32.7 g/dl 41.7 g/dl40

20

00 50 100 0 50 100 0 50 100

% Oxygen Saturation

0.7

0.35

00 50 100

Po

lym

er F

ract

ion

% Oxygen SaturationNoguchi, PNAS 1980 Noguchi, JCI 1983

SS Dense Cells Impair FiltrationEven at High Oxygen Saturation

Hiruma, Noguchi et al., 1995

(Cell suspension: Hct ~8%)

Mixtures of HbS with Other HemoglobinsIncrease Deoxyhemoglobin Solubility

• HbA or HbCincreases deoxyHbsolubility in mixtureswith HbS.

• HbF or HbA2 havean even greater“sparing” effect.

Fraction HbX

Hem

og

lob

in S

olu

bili

ty (

g/d

l)

HbF

HbA2

HbC

HbA

Poillon, Kim, Rodgers, Noguchi, Schecter, PNAS 1993

Hemoglobin Polymerization inAS and SS Erythrocytes

0 50 100% OXYGEN SATURATION

0.7

0.35

0

PO

LYM

ER

FR

AC

TIO

N

AS

SS

Polymerization tendency of AS hemoglobin (αααα2222ββββΑΑΑΑββββS) hybrid:0.5 times hemoglobin S (αααα2222ββββS

2222)

Noguchi, PNAS 1980; Biophys J 1984

Impaired Filtration and Polymer Fraction• Filtration is impaired in AS erythrocytes, but at markedly

lower O2 sat compared with other sickle due to lowerpolymerization tendency.

Imp

aire

d F

iltra

tio

n

Polymer Fraction

Imp

aire

d F

iltra

tio

n

Oxygen SaturationHiruma, Noguchi et al., 1995

(Cell suspension: Hct ~8%)

Defective Urine Concentrating Abilityin Sickle Cell Trait

• High osmolality and low O2 sat of the renal medulla areconditions that favor polymerization.

• Hemoglobin polymerization correlates inversely with urineconcentrating ability.

• αααα-Thalassemia reduces %HbS, and polymerization potential.

Uri

nar

y O

smo

lalit

y (

mO

sm/k

g H

2O)

5020 35 300

1000

Percent Hemoglobin S

400 500 600 700 800 900

(αααααααα////αααααααα )(−α/−α/−α/−α/αααααααα )(−α/−α−α/−α−α/−α−α/−α)

Gupta, Kirchner, Nicholson, Adams, Schechter, Noguchi, Steinberg, JCI 1991

Hemoglobin Polymerization inSickle Trait Erythrocytes

• Although disease manifestation is not generally associatedwith sickle trait individuals, AS erythrocytes can undergohemoglobin polymerization at extreme deoxygenation ordehydration.

• As in SS cells, determinants of polymerization potential inAS cells are intracellular hemoglobin composition that ismodified as a function of αααα-globin genotype, intracellularhemoglobin concentration and oxygen saturation.

• In the kidney, the hyperosmolality and low oxygen tensionof the renal medulla results in a urine concentrating defectassociated with HbS polymerization determined by % HbS.

Therapeutic Approaches forSickle Cell Disease

PRODUCTION CIRCULATION OBSTRUCTION

BONE MARROW

MICROVASCULATURE���������������������������������������������

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INTRACELLULAR HbS POLYMERIZATION

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-O2

+O2

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• Strategies to reduce hemoglobin S polymerization– Increase hemoglobin F

• Hydroxyurea, butyrate, erythropoietin

– Prevent sickle cell dehydration• Clotrimazole, Mg+2 pidolate