CCSS, NGSS

RED BLOOD CELL

MEMBRANE
Presenter: Dr. Lekshmi G Y (1st year PG resident)

Moderator: Dr. Girija Nandini Kanungo

Introduction on RBC Membrane

 Semipermeable lipid bilayer

supported by a mesh-like protein
cytoskeleton structure

• Accounts for 1% of total weight of

the red cell

• Plays an important role maintenance

of erythrocyte integrity

• Provides erythrocytes the flexibility,

durability, and tensile strength
(a) Scanning electron microscopy image of a human red blood cell, (b) The
shape of a human red blood cell with average geometric parameters, and (c)
schematic organization of the red blood cell membrane.
Biochemical Composition

• 52% Protein
• 40% Lipid
• 8% Carbohydrate

Planes of Interaction

• Vertical Interaction
- stabilize the lipid bilayer membrane
• Horizontal Interaction
- support structural integrity of RBC
maintains the biconcave shape of RBC
Membrane Lipids

Lipid components of RBC membrane:

• Phospholipids 60%
• Non-esterified cholesterol 30%
• Glycolipids 10%

 Primarily provides a permeability barrier between the external

environment and the red cell cytoplasm
Phospholipids
• Essentially phosphatidylcholine (PC),
phosphatidylethanolamine (PE),
sphingomyelin (SM) and
phosphatidylserine (PS)

• Minor components such as

phosphatidylinositol (PI),
PI-monophosphate (PIP),
PI4,5-bisphosphate (PIP2),
phosphatidic acid (PA),
lysophosphatidylcholine (Lyso-PC) and
lysophosphatidylethanolamine (Lyso-PE)
 At the physiologic pH, the majority of phospholipid content is electrically
neutral, although PS, PA and PI are negatively charged.
Glycolipids

• Mainly based on sphingosine, such as glycosphingolipids

• Sugar residues present in glycolipids are responsible for numerous functions

such as adhesiveness to the extracellular space

• This explains the location of glycolipids almost exclusively, on the outer

leaflet of the bilayer
Lipid-rich microdomains/Lipid rafts

 Characterized by having a low density and being insoluble in cold nonionic

detergents
 Also known as: detergent-resistant membranes (DRM), Triton-insoluble
membranes (TIM) and triton-insoluble floating fractions (TIFF)

• Rich in cholesterol, sphingomyelin, and glycosylphosphatidylinositol (GPI)-

anchored proteins
• Have a high level of organization and contain over 4% of total RBC
membrane protein
• Has lower fluidity than bulk plasma membrane
• Thicker than nonraft bilayer domains.
Trans-asymmetry

→ lipids are asymmetrically distributed across the bilayer

• The external layer is rich in glycolipids and choline phospholipids.

• The internal cytoplasmic layer of the membrane is rich in amino
phospholipids

 There is constant exchange of phospholipids by a flip/flop mechanism

between the two lipid leaflets
• Maintained by an ATP dependent transport system
• Two important enzymes responsible for the translocation of phospholipids:
Flippase and Floppase
Trans-asymmetry

• Flippase is responsible for translocating PS and PE from the outer to the

inner leaflet
• Floppase catalyzes the translocation of the other lipids from the inner to the
outer leaflet of the bilayer

 Phospholipid flipping is seen in a variety of disorders, including sickle cell

disease, thalassemia, and diabetes

 Exposed phospholipids trigger conversion of prothrombin to thrombin and

activate the coagulation cascade.
Bilayer Couple Effect

→ The shape of the lipid bilayer is responsive to very slight variations (<0.4%) in
the surface area of its inner and outer halves

• Processes that expand the outer bilayer (or

contract the inner) will produce uniform
membrane spiculation, called echinocytosis
• Conversely, relative expansion of the inner
bilayer will lead to membrane invagination
and cup-shaped red cells called
stomatocytosis
Renewal of Membrane Lipids
 Mature human red cells cannot synthesize membrane lipids de novo
 Depend on lipid exchange and acylation of fatty acids as the mechanisms for
phospholipid repair and renewal
• Free cholesterol (FC) in red cell membranes
exchanges readily with the unesterified FC in
plasma.
• Damaged chains are removed by phospholipase
A 2 , producing a lysophospholipid.
• FC is also in part converted into esterified
cholesterol (EC) by the enzymatic process of
lecithin-cholesterol acyltransferase (LCAT). The
EC, which is produced de novo by the reaction,
is transferred out to the plasma.
RBC membrane proteins

Classification:

 Based on protein function:

1. Cytoskeletal proteins (eg: spectrin, actin, protein 4.1)
2. Integral structural proteins (eg: band 3, glycophorins)
3. Anchoring proteins (eg: ankyrin, protein 4.2)

According to the easiness with which they can be separated from membranes:
1. Peripheral proteins (eg: spectrins)
- easily isolated by high- or low-salt or high-pH extraction
2. Integral proteins (eg: band 3 and glycophorins)
- can be extracted only by harsh reagents since they are strongly
embedded into the lipid bilayer
RBC membrane proteins
Characteristic Function
Peripheral proteins
1. Spectrin cytoskeletal protein that lines Responsible for biconcave shape
the intracellular side of the of RBC
membrane
2. Actin Abundant protein in cell participates in protein- protein
membrane interactions

3. Ankyrin are a family of adaptor protein Interacts with band 3 protein and
spectrin to achieve linkage
between bilayer and cytoskeleton.
4. Protein 4.1 is a major structural element. Stabilises actin-spectrin
interactions.
5. Protein 4.2 is an ATP-binding protein regulate the association of protein
3 with ankyrin.
RBC membrane proteins
Characteristic Function
7. Trophomyosin Heterodimeric protein Stabilizing the actin filaments.
Integral proteins
1. Glycophorin Sialic acid rich glycoproteins imparts a negative charge to
the cell, reducing interaction
with other cells/ endothelium
2. Band 3 protein Anion exchanger 1 Exchanges bicarbonate for
chloride (chlorine shift).
Contain antigens for several
blood groups

3. Rh Proteins Encoded by RHD and RHCE genes Critical to maintaining the

and requires presence of Rh- structure of the erythrocyte
associated glycoprotein (RhAG) for membrane
antigen expression
RBC membrane proteins

Schematic model of the red cell membrane.

RBC adhesion molecules

• RBCs are generally considered non-adhesive cells.

• However, several studies have reported the expression of adhesion molecules

in RBC such as CD44, CD47, CD58, LW/ICAM-4, RAGE and Lu
• Role: cell– cell and cell–tissue interactions.
Additionally, they have been implicated in a large range of biological functions
such as erythropoiesis (differentiation, maturation, enucleation and release of
RBCs), self recognition mechanisms, red cell turnover and cell aging

 The several adhesion molecules are differentially expressed at distinct stages of

the life of RBCs
Blood Group Antigens on RBC Membrane

 Blood group antigens are either sugars or proteins, and they are attached to
various components in the red blood cell membrane.

• The antigens of the ABO blood group are carbohydrates. They are produced by
a series of reactions in which enzymes catalyze the transfer of sugar units.

• Apart from the sugar antigens, the RBC membrane contains three types of
protein that carry blood group antigens: single-pass proteins, multi-pass
proteins, and glycosylphosphatidylinositol (GPI)-linked proteins.
Red cell deformability

• RBC can maintain the its discoid shape, and yet allowing cytoskeleton
rearrangements that permit it to pass through capillaries, and then restore
again its normal shape without cell fragmentation

• RBC shape is ultimately determined by membrane proteins, especially spectrin

network, and also by the lipid bilayer content

• Minimal changes in the surface area (inner or outer) could be liable for several
abnormalities either in morphology or function

Modes of deformation of RBCs.

Red cell membrane fluidity

Membrane lipid fluidity is in general dependent on several factors such as:

i. the class of phospholipids
ii. the degree of saturation of the fatty acids,
iii. the length of the acyl chains,
iv. the type of cholesterol (free or esterefied)
v. the presence or absence of amphipathic compounds such as
lysophosphatides
Red cell membrane Permeability

 Freely permeable to water and anions

 Relatively impermeable to cations such as Na+ and K+

• RBC volume and water homeostasis are maintained by controlling the

intracellular concentrations of sodium and potassium
• The permeability properties of the RBC membrane and the active RBC cation
transport prevent colloid hemolysis and control the volume of the RBCs.
• When RBCs are ATP-depleted, Ca2+ and Na+ are allowed to accumulate
intracellularly, and K+ and water are lost, resulting in a dehydrated rigid cell
that is subsequently sequestered by the spleen, resulting in a decrease in RBC
survival.
Disorders of red blood cell membrane

RBC membrane disorders can be regarded either as:

 abnormalities in vertical interactions of membrane components
-mainly in spectrin-ankyrin-band 3 interactions, the protein 4.1-GPC linkage
and also at the level of the interaction of skeletal proteins with membrane
lipids
-eg: Hereditary Spherocytosis
 abnormalities in horizontal interactions of membrane components
-usually concern either the self-association of spectrin to form its tetramers or
its interactions to protein 4.1 and/or actin
-eg: Hereditary Elliptocytosis, Southeast Asian Ovalocytosis, Hereditary
Pyropoikilocytosis
Hereditary Spherocytosis

• Haemolytic disorder characterized by numerous microspherocytes

• seen in the blood film
• Membrane abnormality caused by dysfunctional and/or deficiencies in one or
more of:
i. Band 3
ii. Ankyrin AD
iii. β-Spectrin
iv. α-Spectrin
AR
v. Protein 4.2
Hereditary Spherocytosis
Hereditary Elliptocytosis

• Characterised by elliptical red cells on the peripheral smear

• Decreased membrane stability
- weakened horizontal linkages in membrane skeleton due to either defective
spectrin dimer-dimer interaction or a defective spectrin-actin-protein 4.1
junctional complex
Metabolic pathways

RBC metabolic pathways include:

• Anaerobic glycolytic pathway
• Methemoglobin reductase pathway
• Pentose phosphate pathway
• Luebering –Rapport shunt
Metabolic pathways

 Glycolysis generates 90% of ATP

 Pentose phosphate pathway generates 10% of ATP needed by RBC

 Defect in methemoglobin reductase pathway affects post transfusion

RBC survival and function

 Luebering – Rapport pathway permits accumulation of 2,3-DPG

 2,3-DPG amount within RBC affects how well RBC functions after
tranfusion
Hemoglobin-Oxygen Dissociation Curve

Factors affecting ODC

 1. Carbon dioxide
 2. Protons (↓pH)
 3. Temperature
 4. 2,3 DPG

 Increase in any of the above shifts

curve to right and vice-versa
Respiratory movement in Hemoglobin

• The unloading of oxygen by hemoglobin is accompanied by widening of a

space between β chains and the binding of 2,3-DPG on a mole-for-mole basis,
with the formation of anionic salt bridges between the chains
→ known as the tense (T) form [lower affinity for oxygen]

• When hemoglobin loads oxygen and becomes oxyhemoglobin, the established

salt bridges are broken, and β chains are pulled together, expelling 2,3-DPG-
→ relaxed (R) form of the hemoglobin molecule, [higher affinity for oxygen]
References:

 Harmening 7th edition

 Rossi’s Principles of Transfusion medicine
 Mollison’s Blood Transfusion in Clinical medicine
 Sofia de Oliveira and Carlota Saldanha. An overview about erythrocyte
membrane, Clinical Hemorheology and Microcirculation 44 (2010) 63–74
Thank you