Red Cell Physiology & Pathophysiology of Sickle Cell Disease

1. Red Cell Physiology &
Pathophysiology of
Sickle Cell Disease
DR. G D ARJUNA SAMARANAYAKA

2. SCIEPRO/Science Photo Library/Getty Images

3. Physiology of Red
Cells

5. Production begins hemocytoblast –
multipotent stem cell
Control step - proerythroblast
Cell near the end of development ejects
nucleus and becomes a reticulocyte
Develop into mature RBC within 1-2 days
Negative feedback balances production with
destruction
Controlled condition is amount of oxygen
delivery to tissues
Hypoxia stimulates release of erythropoietin –
promote maturation

7. Sites of hematopoiesis

8. EM photograph of the bone
marrow

9. Functions of Erythrocytes
 Gas transport & exchange
 Primary function
 O2
 CO2
 pH control of blood
 Hemostasis

10. Structure
 Biconcave shape
 Greater surface area for gas exchange
 Increase flexibility – squeeze through capillaries
 Rigid but flexible cytoskeleton
 Increase flexibility
 Mature erythrocytes has no nucleus, Golgi apparatus and
mitochondria
 Glycolysis for ATP production
 no O2 consumption during transportation

12. Haemoglobin
 Main oxygen transporter
 Hemoglobin is a protein
 Hemoglobin = haem + globin
 Globin - protein
 Haem
 Iron + Protoporphyrin IX

13. Protoporphyrin IX
 4 pyrrole rings - Tetrapyrrole
 Bridges – Methine (CH)
 Side chains -8
 Methyl (CH3) - 4
 Vinyl (CHCH2) - 2
 Propionic acid - 2
 (CH2CH2COOH)

14. Iron
 Ferrous form (Fe2+).
 Iron attached to nitrogen atom
of each pyrrole ring.
 Iron can bind with
 Oxygen
 Carbon monoxide.

15. Globin
 Made-up of 4 polypeptide chains
 2 alpha like chains
 2 beta like chains
 Each polypeptide chain has one
bound haeme molecule
 1 Globin molecule has 4 haeme
molecules
 Can bind 4 oxygen molecules

16. Haemoglobin Production
 In the early stages of erythrocyte maturation
 Nucleus, Ribosomes, Golgi apparatus & mitochondria are present
 Nucleus – code for globin chains
 Mitochondria - aerobic generation of energy - insertion of
ferrous iron into protoporphyrin IX – haem production
 Ribosomes - globin and other protein synthesis

17. Synthesis of globin
 Arranged in two clusters
 β cluster – β, δ, γ, ε globin chains - Chromosome 11
 α cluster – α & ζ globin chains – Chromosome 16

18. Types haemoglobin

20. O2 binding to hemoglobin
 Oxygen binds reversibly to Hb
 Upon O2 binding to an active site of hemoglobin there is a
conformational change in the Hb molecule
 ‘Cooperation’ - binding of oxygen to one site of the four subunits will
increase the likelihood of the remaining sites to bind with oxygen as well.

21. O2 binding to haemoglobin
 Allostery
 Regulation of an enzyme by binding an effector molecule at
a site other than the enzyme's active site
 Allosteric effectors
 H+
 CO2
 2,3-bisphosphoglycerate

22. Allosteric effect & cooperative effect– Hemoglobin vs
Myoglobin
Exponential vs sigmoid curve
Cooperativity Enhances Oxygen
Delivery by Hemoglobin.
cooperativity between O2-binding
sites, hemoglobin delivers more
O2 to tissues than would a
noncooperative protein (about
1.7x)

23. Effect of 2,3 - BPG
 2,3-biphosphoglycerate – by product of glycolysis
 Binds the central cavity of the Hb molecule only when its in the
tensed state (low affinity state)
 increase stability of T-state
 Therefor decrease oxygen affinity – increases oxygen release at
tissues

24. Effect of CO2 & pH
 Bohr effect
 Increases in the carbon dioxide partial pressure of blood or decreases in
blood pH result in a lower affinity of hemoglobin for oxygen
 Carbaminohemoglobin & H+ stabilizes T state hemoglobin by
formation of ion pairs.
 Allows unloading of oxygen in peripheral tissues
⇌
CO2 + H2O H2CO3 H+ + HCO3
−
⇌
CO2 + Haemoglobin ⇌ Carbaminohemoglobin

27. Pathophysiology of
Sickle cell disease

30. Basic pathogenesis – HbS polymerization
 First disease to demonstrate genetic mutation
can lead to production of abnormal protein.
 “First molecular disease”
 Mutation substituting thymine for adenine in
the sixth codon of the beta-chain gene
 GAG to GTG
 Coding of valine instead of glutamate in
position 6 of the Hb beta chain
 glutamic acid - hydrophilic
 Valine – hydrophobic
 Formation of hydrophobic patch on the Hb
molecule

31.  When Hb is in tensed state - another
hydrophobic pocket is exposed
 In both normal Hb and HbS
 Formed by ßPhe85 and ßLeu88
 In tensed state (deoxygenated)
 Normal Hb -> no hydrophobic patch -> no
polymerization
 HbS - > polymerization

32.  Polymerization -> helical fiber formation
 Polymerization is the root cause for pathogenesis
 Fibers group together
 stiffen the red cell
 repeated and prolonged sickling involves the membrane
 give rise to the characteristic shape - Sickle

33. Factors affecting polymerization
• Oxygen saturation
• Intracellular Hb composition
• Intracellular Hb concentration

34. Oxygen saturation
 Decreased SpO2 increase the likelihood of sickling
 High altitudes
 Diseased pulmonary vascular bed - frequent infarcts and lung infections
 High O2 consumption – exercise, fever, acidosis
 Maximum polymerization at 0% saturation
 2,3-BPG, low pH & temp, increased CO2 -> stabilize T state -> increase
sickling

35. Intracellular Hb composition
 Presence of other hemoglobin types reduce the chances polymerization
 Reduce with the concentration
 HbF > HbA2> HbA > HbC
 Disperse among HbS -> reduce contact

37. Intracellular Hb concentration
 Higher haemoglobin concentrations increase polymerized Hb
concentration
 Therefor increase number of sickled cells

38. Intracellular Hb concentration
 Repeated sickling -> Membrane damage -> activation of ion channels ->
Dysregulation of cation homeostasis
 K-Cl co-transport system
 Ca-dependent K-channel (Gardos channel)
 Loss of intracellular K+ -> cellular dehydration
 Increase in intracellular Hb concentration -> increase chances of sickling

40. Basic pathogenesis - Erythrocytic membrane
changes
 Methemoglobin formation
 Fe 2+ -> Fe3+
 Denaturing of Hb -> hemichromes
 Oxidative stress - > membrane alterations
 The normal asymmetry of membrane phospholipids is
disrupted (reversal)
 Promote coagulation
 Proteins of the cytoskeleton express outside
 Sp. protein band 3 (Band 3 anion transport protein)
 Anti-band 3 IgGs accumulate on the protein band 3
aggregates, inducing erythrophagocytosis by
macrophages
 Membrane changes cause microparticle formation ->
cell membrane loss
 Leads to stiffening and increased fragility of the SS-
RBCs

41.  After recurrent episodes of sickling
 membrane damage occurs
 cells are no longer capable of resuming the biconcave shape upon
reoxygenation.
 irreversibly sickled cells (ISCs).
 Cause vaso-occlusion
 5-50% of RBCs permanently remain in the sickled shape
 Membrane alterations -> trap in RES -> extravascular hemolysis - >
anemia

42. Is sickling the only cause of
vaso-occlusion
 “Delay time” – time gap between trigger event
& sickling of red cells
 If there is a marked delay time – red cells can
escape microvasculature before starts sickling
 If the delay time is shorter than the transit time -
> vaso-occlusion
 Current data suggest that delay time is
actually longer than the transit time
 Therefore, the passage must be delayed by
other causes which contribute to the
development of sickling within the
microvessels.

43. Mechanisms Participating In the
Vaso-occlusive Event
 Retardation of the blood flow through the microcirculation
 Adhesion of young red cells on the endothelial wall
 Activation of the endothelial cells
 Activation of the passing-by leucocytes and platelets and adhesion on the
endothelial wall
 Vasoconstriction

44. Increased adhesion of sickle red blood
cells to the endothelium
 Due to haemolysis -> decresed Hb -> increased reticulocyte
production
 “Stress reticulocytes”
 Coming out prematurely from the bone marrow because of the
anemic stress
 Express adhesion proteins that normally keep them in the
marrow
 Stress reticulocytes bind to the endothelium of post-capillary
venules
 Slow down the blood flow mature SS-RBCs kept a longer time
in a hypoxic environment.
 Entrapment of irreversible sickle cells and to the complete
occlusion of the micro-vessels

45.  SS- RBCs express receptors
 induce signaling pathways ->
modify their functions.
 Some extracellular stimuli activate
adhesion mechanisms.
 Epinephrine -> Lu/BCAM
 BCAM - Basal cell adhesion
molecule (CD-239)
 CD-36
 CD-47
 CD-44

46. Activation of endothelial cells
• VCAM-1 - vascular cell
adhesion molecule 1
(CD 106)
• ICAM-1 - Intercellular
Adhesion Molecule
1(CD – 54)
• BCAM - Basal cell
adhesion molecule (CD-
239)

47. Activation of the leucocytes and
platelets
 Adherent leukocytes in post-capillary venules - major factor causing circulatory slowing
down that initiates VOCs.
 Activators
 SS-RBCs are capable of abnormally interacting PMNs.
 Creation of a proinflammatory environment
 release of free hemoglobin and heme secondary to RBC lysis
 borderline activation of the coagulation system
 abnormally exposed phosphatidylserine - at the SS-RBC surface
 activated circulating endothelial cells – express tissue factor
 Lead to the production of proinflammatory cytokines -> generalized cell activation.
 PMNs
 Platelets
 Endothelial cells

48. Vasoconstriction
 Regulation of the vascular tone balance
between
 Vasoconstrictors - endothelin-1 (ET-1)
 vasodilators - nitric oxide (NO)
 In SCD NO level decrease due to free HB
 Haemoglobin is the most powerful NO
scavenger known
 destroys NO and generates free radicals
 reduced NO production by depletion of endothelial
NO synthase
 Balance shift towards vasoconstriction

49. Proposed model of sickle cell VOC
1. endothelial activation by SS-RBCs and other
inflammatory mediators
2. recruitment of adherent leukocytes
3. activation of recruited neutrophils and of other
leukocytes (eg, monocytes or NK cells)
4. interactions of sickle erythrocytes with adherent
neutrophils
5. vascular clogging by heterotypic cell-cell aggregates
composed of SS-RBCs, adherent leukocytes and
possibly platelets
6. increased transit time to greater than the delay time
for deoxygenation-induced hemoglobin
polymerization, propagating retrograde VOC
7. ischemia as a result of the obstruction that creates a
feedback loop of worsening endothelial activation

50. Summery
 Red cell has unique structure and molecules for gas transportation
 Pathogenesis of SCD involves complicated chemical pathways
 Novel/Experimental drugs utilize the understanding of the complex pathogenesis of
sickle cell disease.

51. References
1. Pathophysiological insights in sickle cell disease; Marie-Hélène
Odièvre, Emmanuelle Verger, Ana Cristina Silva-Pinto,* and Jacques
Elion; Indian J Med Res. 2011 Oct; 134(4): 532–537.
2. Treating sickle cell disease by targeting HbS polymerization; William
A. Eaton, H. Franklin Bunn; Blood 2017 129: 2719-2726
3. Vaso-occlusion in sickle cell disease: pathophysiology and novel
targeted therapies; Deepa Manwani and Paul S. Frenette;
Blood 2013 122:3892-3898
4. Ganong’s review of physiology

52. Thank You