Red Cell Physiology & Pathophysiology of Sickle Cell Disease

Red Cell Physiology & Pathophysiology of Sickle Cell Disease

1. Red Cell Physiology & Pathophysiology of Sickle Cell Disease Dr. G D Arjuna Samaranayaka

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3. Physiology of Red Cells

4. 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

5. Sites of hematopoiesis

6. EM photograph of the bone marrow

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

8. 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

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

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

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

12. 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

13. 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

14. 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

15. 2 succinyl – CoA + 2 glysine ----> Pyrrole 4 Pyrrole ----> Protoporphyrin IX Protoporphyrin IX + Fe2+ -----> Heme Heme + Globin ----> Hemoglobin chain ( α or β) 2 α chains + 2 β chains ----> Haemoglobin molecule

16. Types haemoglobin

17. O 2 binding to hemoglobin Oxygen binds reversibly to Hb Upon O 2 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.

18. O 2 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 + CO 2 2,3-bisphosphoglycerate

19. Allosteric effect & cooperative effect– Hemoglobin vs Myoglobin Exponential vs sigmoid curve Cooperativity Enhances Oxygen Delivery by Hemoglobin. cooperativity between O 2 -binding sites, hemoglobin delivers more O 2 to tissues than would a noncooperative protein (about 1.7x)

20. 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

21. 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 ⇌ CO 2 + H 2 O H 2 CO 3 H + + HCO 3 − ⇌ CO 2 + Haemoglobin ⇌ Carbaminohemoglobin

22. Pathophysiology of Sickle cell disease

23. 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

24. When Hb is in tensed state - another hydrophobic pocket is exposed In both normal Hb and HbS Formed by ßPhe 85 and ßLeu 88 In tensed state (deoxygenated) Normal Hb -> no hydrophobic patch -> no polymerization HbS - > polymerization

25. 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

26. Factors affecting polymerization Oxygen saturation Intracellular Hb composition Intracellular Hb concentration

27. 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

28. 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

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

30. 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

31. Basic pathogenesis - Erythrocytic membrane changes Methemoglobin formation Fe 2+ -> Fe 3+ 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

32. 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

33. 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 .

34. 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

35. 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

36. 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

37. 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)

38. 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

39. 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

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

41. 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.

42. References 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. Treating sickle cell disease by targeting HbS polymerization; William A. Eaton, H. Franklin Bunn; Blood 2017 129: 2719-2726 Vaso -occlusion in sickle cell disease: pathophysiology and novel targeted therapies; Deepa  Manwani  and Paul S.  Frenette ; Blood 2013 122:3892-3898 Ganong’s review of physiology

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