HI I AM DR.GAYATHRI. JUNIOR RESIDENT MD TRANSFUSION MEDICINE, SCTIMST TRIVANDRUM, KERALA, INDIA

1. RBC MEMBRANE
&
METABOLISM

2. Discovery: 17TH century by ANTONJ VAN
LEEUWENHOEK
Presence of iron in blood: LEMERY in 17TH
century
Functional significance of RBC as oxygen
transporter:FELIX HOPPE-SEYLER IN 1877
Late 20TH century: Mechanism of oxygen,
carbon dioxide & nitrous oxide exchange
Lipid bilayer hypothesis: first proposed in
1925 and refined by Danielli and Davson in
1935
Uniquely anuclear ,highly specialized
of cells
Lacks cytoplasmic organelles as
nucleus, mitochondria, or ribosomes,
hence unable to synthesize new protein,
carry out the oxidative reactions
associated with mitochondria, or
undergo mitosis
 More than 95% of the cytoplasmic
protein is hemoglobin

3. “
EM VIEW OF: A) RBC
B) WBC
C) PLATELET

4. ◎At rest when suspended in isotonic solution:
flattened, bilaterally indented sphere
(biconcave disc)
◎In fixed, stained blood smears: appears
circular, with a diameter of about 7 to 8 μm
and an area of central pallor corresponding to
the indented regions
◎disc shape is well suited to erythrocyte
function; (surface to volume ratio maximum)
thereby facilitating gas transfer and cascading
in the microcirculation

5. “
Volume: 94 +/-14 fl

6. ◎Changes its shape with size of the vessel &
under shearing stress without remodelling
◎Due to cytoplasm(very low viscosity) &
plasma membrane(elasticity & viscocity)
◎Spherical: hypotonicity, discocyte-echinocyte
transformation, discocyte-stomacyte
transformation.

7. DISCOCYTE-ECHINOCYTE
TRANSFORMATION
◎Decrease in IC ATP
◎Increase in IC Calcium & pH
◎Cell exposed to stored plasma
◎Anionic detergents
◎Lysolecithin/Fatty acid
◎Washed cell when kept between glass slide &
coverslip
STAGE I STAGE II
STAGE III STAGE IV
IRREGULARLY
CONTOURED DISC
CRENATION OVER
FLAT SURFACE
SPICULES OVER
SURFACE
SPHERO-ECHINOCYTE

8. DISCOCYTE-STOMACYTE
TRANSFORMATION
◎Low pH
◎Cationic detergent
STAGE I
STAGE II

9. STAGES OF SHAPE CHANGE TYPICAL OF
PROLONGED STORAGE

10. ◎1935: Danielli & Davson gave lipid bilayer
model

11. ◎Two electron-dense
(osmophilic) layers
approximately 2.5 nm
thick separated by an
electron-penetrable
layer about 2.0 nm
thick, for a total
thickness of some 7.0
nm.

12. ◎Material following hemolysis due to
membrane rupture: stroma & if membrane
intact: cell ghost
Chart Title
protein
lipid
carbohydrate5240
8

13.  Phospholipids
a) Phosphatidylcholine (lecithin)
b) Phosphatidylethanolamine (cephalin)
c) Sphingomyelin
d) Phosphatidylserine
e) Lysolecithin
 Cholesterol
 Glycolipid
Chart Title
phospholipid
cholesterol
glycolipid
49.547.1

14. ◎Helps in regulating the SHAPE of RBC &
mechanical membrane stability in case of shear
stress
◎Certain provide bilipid layer attachment to
cytoskeleton (spectrin– phosphatidylserine
interaction)
◎Mobility of lipids: Outer layer >inner layer
◎ sphingomyelin, fluidity
◎Phospholipid composition: phosphatidylcholine
(PC) and sphingomyelin in outer leaflet and
phosphatidylinositol(PI),
phosphatidylethanolamine(PE), and
phosphatidylserine (PS) in inner leaflet

15. ◎Loss of phospholipid asymmetry results in
exposure of PS(apoptopic marker) on the red cell
surface promotes red cell removal from the
circulation
◎Phospholipid asymmetry is maintained by the
ATP-dependent flippase (aminophospholipid
translocase) activity which counteracts
phospholipid scrambling in which PS moves from
the inner to the outer cell surface
◎Flippase activity decreases during storage, but
can be corrected by rejuvenation of the red cells
◎Phospholipid scrambling is normally low during
storage, but can be enhanced by photodynamic
treatment for pathogen inactivation

16. FATTY ACID COMPOSITION
◎SATURATED
•Palmitic
•Stearic
•Others
◎UNSATURATED
•Oleic
•Linoleic
•Arachidonic
•Others
Chart Title
UNSATURATED
SATURATED
47 53
PALMITIC
STEARIC
OLEIC
LINOLEIC
ARACHIDONIC
24.5
19
16
11.2
10.3

17. CHOLESTEROL
◎Increases in membrane cholesterol content
decrease membrane deformability
◎ Abnormally high levels of cholesterol lead
to distortions in red cell shape; bizarre
spicules form (“spur cells”), deformability of
the cells is reduced, and they are destroyed in
the spleen.

18. GLYCOLIPIDS
◎Ceramide glycolipids:
with one glucose(GL-1: 5%), one glucose & one
galactose(GL-2: 14%), and one glucose & two
galactose(GL-3: 12%), one glucose & 3 galactose
(GL-4: 69%) molecules attached
◎Fucose-containing ceramide glycolipids, in
trace amounts: surface antigenic activity
corresponding to the A, B, H, and Lewis blood
groups

19. ◎Solubilization of membrane proteins done by adding
detergents to cell ghost
◎Separation & analysis: high resolution by means of
electrophoresis in polyacrylamide gel
◎Main proteins are:
Peripheral:- SPECTRIN, ANKYRIN, PROTEIN KINASE,
ACTIN & G-3PD
Integral:- GLYCOPHORINS(Sialoglycoproteins),
ANION CHANNELS & GLUCOSE TRANSPORTERS
◎Surface glycoprotein CD47 inhibits phagocytosis
(decreased in old RBCs & during storage)

20. TRANSMEMBRANE
PROTEINS
◎Two prominent ones-: Glycophorin A (GPA)
and Anion channels
◎Sialic acid residues attached to glycophorins:
60% negative charge of membrane; GPA also
bears blood group antigens & also binding site for
many pathogens
◎AE1(Anion Exchange Protein)/ Band3:
traverses the membrane 12 times; the
extracellular domain is highly glycosylated- bears
CHO blood group antigens & ABO system
antigens
Within the membrane, AE1 exists
predominantly as a dimer ;its function appears to
be Cl–HCO3 exchange
AE1 also interacts with the erythroid
cytoskeleton by binding ankyrin and binds NO,
possibly facilitating its transit across the
erythrocyte plasma membrane
Role of band 3 in anion and CO2 transport

21. Rh PROTIENS
◎only 100,000 copies per cell
◎RhD protein is the most immunodominant
determinant of red cells outside the ABO antigens.
Complete absence results in multiple erythrocyte
defects and mild hemolytic anemia.
◎ The proteins that carry the D, C (or c), and E (or e)
(Rh antigens are highly homologous to one another)
traverse the membrane multiple times
◎Minor role in CO2 transport
Interaction between integral proteins & cytoskeletal
proteins

22. CYTOSKELETAL PROTEINS
◎most abundant of the peripheral proteins:
spectrin-actin cytoskeletal complex
◎The complex includes large α and β spectrin
polypeptide chains and the smaller actin chain
◎ It preserve erythrocyte integrity in the face of
the shear stresses of the circulatory system and
spleen
◎Protein 4.1: promote spectrin-actin interaction
◎Protein 2.1/Ankyrin: serves as a mode of
attachment of the cytoskeleton to the membrane
 Other proteins include protein (band) 4.9,
tropomyosin, tropomodulin, and adducin:
play vital roles in formation and stability of
the cytoskeleton

24. ◎In Stored blood : spectrin oxidation occur
with time, leading to loss of membrane surface
area by formation of lipid vesicles comprising
membrane detachment from cytoskeleton.
◎Other cytoskeletal proteins may also be
affected by oxidative processes that alter their
ability to interact with members of the
spectrin-actin meshwork underpinning the
erythroid membrane.

25. MEMBRANE TRANSPORT
PROTEINS
◎Nonpolar substances diffuse through the
membrane
◎Polar solutes cross the membrane at
specialized transport proteins, including the
anion transporter (AE1/ band 3), glucose
transporter, urea transporter,Ca2+-ATPase, Na-
K-ATPase, the GSSG (oxidized glutathione)
transport ATPases, Amino Acid transporters
and a water channel aquaporin-1( 85% of the
osmotic water permeability)

26. ION EXCHANGE
◎Rapid exchange: mediated by the band 3
anion-exchange protein and plays an
important role in the chloride–bicarbonate
exchanges that occur as the red cell moves
between the lungs and tissues
◎Slower ionic diffusion: accounting for net
loss or gain of anions
◎d isomers of glucose, mannose, galactose,
xylose & arabinose are transported easily
whereas fructose is not transported under
physiological conditions

27. ◎Glucose enters the erythrocyte by facilitated
diffusion, mediated by a transmembrane protein
GLUT1(5% of erythrocyte membrane protein)
◎Potassium is the predominant cation and sodium
is a relatively minor constituent, whereas the
relationship is reversed in plasma; preservation of
these gradients is the result of the cation transport
process(passive diffusion & active transport)
Active Na+ and K+ transport depends on the
activity of the membrane protein Na-K ATPase
3Nai
+ + 2Ko
+ + ATPi → 3Nao
+ + 2Ki
+ + ADPi + Pi
 Urea transporter: transports urea rapidly
across the membrane and helps preserve red cell
osmotic stability and deformability

28. ◎The Na-K-ATPase is highly sensitive to changes in
temperature and scarcely functions at 4ºC
◎ During cold storage, Na diffuses into the cells
and K leaks out until a new equilibrium is reached
◎ The leakage of K is further increased by
irradiation
◎ Increased K content in plasma or in the additive
solution of stored RBC units presents a potential
hazard to neonates
◎Citrate in plasma increases K toxicity
◎Reducing RBC supernatant volume results in less
K leakage before equilibrium is reached

29. MEMBRANE
ASSOCIATED ENZYMES
◎Externally Oriented: Hydrolytic enzymes
( glycosidases and acid phosphatases), AChE
◎Membrane Bound: Production of ATP – 3
enzymes:-aldolase, glyceraldehyde 3-
phosphate dehydrogenase (G3PD), and
phosphoglycerate kinase
◎These three enzymes convert fructose
diphosphate to 3-phosphoglycerate with the
production of ATP
Use of ATP: protein kinases, and adenosine
triphosphatases (ATPases)
ATP cAMP
Protein kinases: phosphorylated structural
proteins have lower-affinity for their target proteins
Eg: dephosphorylation due of ATP depletion, stress
develops; more rigid spectrin-actin network and
reduced membrane deformability
 ATPases (Na-K ATPase, Ca-Mg ATPase, and Mg
ATPase) : Na-K ATPase maintains the high internal K
and low internal Na concentration
ADENYLYL CYCLASE

31. GLUCOSE
HEXOKINASEATP
ADP
GLU-6-PHOSPHATE
FRUCTOSE-6-PHOSPHATE
PHOSPHOGLUCOSE
ISOMERASE
FRU 1,6 BIS PHOSPHATE
PHOSPHO FRUCTO KINASEATP
ADP
ALDOLASE
DIHYDROXY ACETONE-P GLYCERALDEHYDE 3 PTPI
NAD+
NADH
G-3 P DEHYDROGENASE
1,3 BISPHOSPHO GLYCERATE

32. 1,3 BISPHOSPHO GLYCERATE
3- PHOSPHO GLYCERATE
2-PHOSPHO GLYCERATE
2- PHOSPHOENOL PYRUVATE
PYRUVATE
LACTATE
PHOSPHOGLYCEROKINASEADP
ATP
MONO PHOSPHOGLYCERATE
MUTASE
ENOLASE
PYRUVATE KINASEADP
ATP
LACTATE DEHYDROGENASENAD+
NADH

33. GLUCOSE
GLU-6-PHOSPHATE
FRUCTOSE-6-PHOSPHATE
FRU 1,6 BIS PHOSPHATE
DIHYDROXY ACETONE-P
1,3 BISPHOSPHO GLYCERATE
GLYCERALDEHYDE 3 P
6-PHOSPHOGLUCONATE
G-6-P DEHYDROGENASE
NADP+ NADPH
RIBULOSE-5-P
NADP+
NADPH
6PG
DEHYDROGENASE

34. PENTOSE PATHWAY
◎Source of NADPH
◎ NADPH maintains glutathione (GSH) in its
reduced form (eliminate peroxide, protection
of protein sulfhydryl groups and detoxification
processes)
◎Ribulose-5-phosphate needed for
production of phosphoribosyl pyrophosphate
(PRPP)essential for the synthesis of adenine
nucleotides required for ATP synthesis
◎Pentose pathway accelerated when NADPH
is oxidized to NADP (oxidative stress) and as
well as when Hb in R state(O2 bound)

35. 1,3 BISPHOSPHO GLYCERATE
3- PG
2-PG
2- P E P
PYRUVATE
DIPHOSPHOGLYCERATE MUTASE
2,3 BISPHOSPHO GLYCERATE
DIPHOSPHOGLYCERATE PHOSPHATASE
phosphate

36. ◎Production of large quantities of 2,3-DPG is a
unique feature of glycolysis in the red cell
◎This is to stabilise low affinity state of Hb(T state)
◎Both reactions are catalyzed by the same enzyme
and are balanced at physiologic pH.
◎ At higher pH, the enzyme acts only as a mutase
◎At low pH, the enzyme acts as phosphatase
◎2,3-DPG is made at the expense of ATP(shunt
bypasses 1 ATP-making steps)
◎ In storage systems, a high pH can shut down ATP
production, while a lower pH leads to a burst of ATP
production driven by breakdown of 2,3-DPG.

37. 2,3 DPG
◎Storage source of phosphate
◎Membrane shape and deformability are
controlled by the ATP-driven cytoskeleton
◎Free 2,3-DPG increases cell flexibility by
weakening the links between the membrane and
the cytoskeleton, and facilitates gas exchange by
allowing the red cell to slip into narrow capillaries
and splenic sinusoids
◎Because some capillaries in the microcirculation
have a diameter of only half that of a RBC, loss of
flexibility and deformability is a serious storage
lesion responsible for removal of rigid cells

38. 2,3 DPG + Hb
T state of HbR state of Hb
Hb
FREE 2,3 DPG
BAND 3
PROTEIN
4.1
PROTEIN
4.2
ANKYRIN

39. GLYCOPHORIN C
PROTEIN
4.1
ACTIN
SPECTRIN

40. ALTERNATE SUBSTRATES FOR
RBC METABOLISM
◎RBCs are capable of metabolizing other sugars like
fructose, mannose, galactose, and the three-carbon
sugar dihydroxyacetone but none proven to be
useful in the design of blood preservatives.
◎Nucleosides: such as inosine to support ATP
synthesis by action of nucleoside phosphorylase
Inosine + Pi → Ribose-1-P + Hypoxanthine
◎R-1-P(without ATP expenditure) is then readily
converted to F-6-P by the pentose shunt:- generation
of ATP through Glycolysis

41. REGULATION OF GLYCOLYSIS
I) Feedback mechanisms
◎Negative feedback mechanisms between
glycolytic pathway and 2,3-DPG & ATP
II) pH
◎HK and PFK inhibited by hydrogen ions
◎Accumulation of lactic acid & acidity of first-
generation additive solutions in blood storage
slows glycolysis
III) Pentose Shunt Activity : facilitatory effect

42. IV) States of Hb
◎N-terminal cytoplasmic domain of Band 3
binds Hb, cytoskeletal proteins, and
glycolytic enzymes
◎Hb in T state & Band 3 interaction causes
release of glycolytic enzymes Glycolysis
◎ Hb in R state pentose shunt (enzymes
activity reduced in bound state with Band 3)

43. ADENOSINE NUCLEOTIDES
◎Equilibrium exist between ATP,ADP & AMP
◎As ADP increases, some is converted to AMP& it
inturn is deaminated in the AMP-deaminase reaction
◎Total adenine pool decreases during storage
leading to depletion of ATP (so adenine is added to
anticoagulant and/or the additive solution)
◎Adenine in storage system enters the cells & purine
nucleotides are synthesized through adenine
phospho- ribosyl transferase reaction
Adenine + PRPP → AMP + PP
◎PRPP synthesised from pentose pathway : for AMP
production

44. GUANINE NUCLEOTIDES
◎Guanine nucleotides: formed by action of
hypoxanthine-guanine phosphoribosyl transferase.
◎Main functions:
1) signal transduction of membrane shear into
secretion of local vasodilators cyclic AMP and ATP
2) high concen. of GTP inhibit RBC trans –glutaminase
(which has Factor XIII like activity that interacts with the
cytoplasmic domain of band 3 and with protein 4.1)
◎GTP concentration & transglutaminase is reduced
when RBCs age, which facilitate the removal of old RBCs
by binding them to fibrin clots

45. IN NUTSHELL…….
metabolize
GLUCOSE by the
glycolytic
pathway with its
pentose and 2,3-
DPG shunts
ATP
•maintain ion and glucose
concentration gradients
between the plasma and
erythrocyte
•secure red cell deformability
For optimal dissociation of
oxygen from Hb & as a phophate
depot
rise and fall of non-Hb-bound
2,3-DPG induces repetitive
changes in the membrane-
cytoskeleton architecture
(implications for red cell flexibility
and gas transport)
2,3DPG
Adequate levels of ATP,
NADH, NADPH, and 2,3-
DPG for these metabolic
functions are secured by the
glycolytic pathway with its
pentose and 2,3-DPG shunts

47. ◎Maintaining the Iron of Hb in a reduced state is a
prerequisite for effective oxygen transport(by NADH)
Fe3 Fe2
NADH NAD+
◎GSH is synthesised from precursor AAs &
nucleotides through salvage pathway
◎SH groups of Hb and membrane proteins is
protected from oxidation by maintaining adequate
amounts of reduced GSH by oxidation of NADPH to
NADP
GSH GSSG
MetHb reductase
PEROXIDASE
H2O2 H20
Fe3 Fe2

48. ◎Citrate dextrose: nutrient for red cells
◎Acid-citrate-dextrose : Shelf life of 21 days; Acid
pH(pH 5) does not help in maintaining 2,3-DPG
levels
◎Citrate-phosphate-dextrose: Alkaline pH and
PO4(phoshate) help in maintaining 2,3-DPG & shelf
life of 28 days
◎Citrate-phosphate-dextrose-adenine (CPDA-1):
Improved ATP synthesis & longer shelf life (35 days)
◎Citrate Phosphate Dextrose Adenine 2
(CPDA 2): more glucose content than CPDA-1

49. ◎Citrate phosphate Double Dextrose
(CP2D):100% more glucose than CPD and 60%
more than CPDA-1;used with an additive
solution (AS3)which doesnot contain glucose
◎Added nucleotides:
I) ADENOSINE : Restores ATP
II) INOSINE : generates ATP
III) GUANOSINE: Used in PAGGS-M which
provides 7 weeks of RBC storage with recovery
of 74 %

50. RBC STORAGE LESIONS
CHARACTERISTICS CHANGE
% VIABLE CELLS
GLUCOSE
ATP
pH
2,3 DPG
LACTIC ACID
PLASMA K+
PLASMA Hb
O2 DISSOCIATION CURVE SHIFT TO LEFT

51. REFERENCE
Rossi’s Principles of Transfusion
Medicine, Fourth Edition
Wintrobe’s Clinical Hematology
12th Edition