FFP - Basic Principles of Haematology 2023.pptx

RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1 – Basic Principles of Haematology; Haemopoiesis and Haemoglobin Dr. Patrick Walsh 4th October 2023 Learning Outcomes 1. Des...

RCSI Royal College of Surgeons in Ireland Coláiste Ríoga na Máinleá in Éirinn FFP1 – Basic Principles of Haematology; Haemopoiesis and Haemoglobin Dr. Patrick Walsh 4th October 2023 Learning Outcomes 1. Describe the composition of whole blood and its components 2. Summarise the sites, steps & factors involved in haemopoiesis with particular reference to erythropoiesis 3. Describe the characteristics and life cycle of erythrocytes 4. Discuss the role of haem in oxygen binding 5. Describe the structure and function of myoglobin (Mb) 6. Describe the structure and function of haemoglobin (Hb) 7. Discuss the co-cooperativity and the allosteric regulation of haemoglobin 8. Outline the metabolic capability of red blood cells  What is blood? One of the largest organs distributed throughout the entire body Blood circulates through the body’s heart, arteries, veins, capillaries 70kg man = 5.6L – 7- 8% body weight Temperature = 38 C Slightly alkaline pH 7.35-7.45  Blood composition Plasma 55 % (~ 3.5 L) – Liquid component of blood in which the cells are suspended – Complex aqueous solution – Gases, salts, proteins, carbohydrates & lipids Formed elements 45% – Red cells (erythrocytes) 99% – Platelets < 1% – White cells (leukocytes) < 1% Whole blood allowed to clot – clot removed – remaining fluid is SERUM – serum does not contain coagulation factors  Functions of Blood Carries TO the body tissues… Carries AWAY from the tissues… oxygen waste matter nutrients carbon dioxide hormones water solutes heat Haemopoiesis - production & development of new blood cells Erythropoiesis - Red blood cell (RBC) production Leucopoiesis – White blood cell (WBC) production Thrombopoiesis – Platelet production >500 times more RBCs than WBCs in circulation Haemopoiesis Sites of haemopoiesis In children, haematopoiesis occurs in the marrow of the long bones such as the femur and tibia. In adults, it occurs mainly in the pelvis, cranium, vertebrae, and sternum. In some cases, the liver, thymus, and spleen may resume their haematopoietic function, if necessary. This is called extramedullary haematopoiesis. Maturation, activation, and some proliferation of lymphoid cells occurs in the spleen and lymph nodes.  Key features of haemopoiesis Single stem cell produces >106 mature cells, and accounts for < 0.1% of all cells in bone marrow Stem cells grow and divide in bone marrow Lose Cell Adhesion Molecules (CAMs) – Allow cells to leave marrow & enter circulation Require Growth factors: – Erythropoietin – colony stimulating factors – Interleukins – thrombopoietin Erythropoiesis Typically one proerythroblast gives rise to about 16 mature RBCs Erythrocytes (RBCs) Anucleate, discoid shape ~120 day life-span 1% destroyed per day Anaemia: A decrease in Hb concentration below the reference range for the age & sex: 11.5 – 16.0 g/dL (female) 13.5 – 17.5 g/dL (male) Inherited haemolytic anaemias: Glucose-6-phosphate dehydrogenase (G6PD) deficiency Sickle cell anaemia  Life cycle of RBCs The life span of an RBC is ~120 days Senescent RBCs removed by macrophages Haemoglobin components are recycled: globin - amino acids reutilized iron reutilised Haem excreted in bile Aerobic Metabolism Anaerobic [O2 not used] Metabolism Aerobic [O2 used] Aerobic metabolism = most efficient Multi-cellular organisms need to transport O2 to all tissues for aerobic metabolism and to store O2 Specialised proteins for this: Myoglobin Haemoglobin Haem group Oxygen binding by proteins depends on the haem group. Not part of the polypeptide chain Tightly bound to the protein Essential for haemoglobin activity [Fe2+/ferrous] Iron is held in position by 4 N’s Fe2+ can make 2 more bonds… Protoporphyrin IX +Fe2+ = haem Structure of myoglobin O2 reservoir within heart & skeletal muscle cells 153 aa 17kDa compact protein Structure: 75% a- helix 8 helices (labelled A-H) Non-helical regions (AB, BC etc.) Exterior hydrophilic Interior hydrophobic except histidines E7 & F8 Schematic of O2 binding site Haem sits in a crevice near the surface lined with non-polar residues E7 = distal His F8 = proximal His Conformational change with O2 binding Deoxy – Mb Haem iron lies 0.3 Å out of plane Oxy – Mb Haem iron lies 0.1 Å out of plane Oxygenation moves the haem iron which moves His F8 Helix F moves & causes other elements of the structure to move Differences in Hb and Mb Mb is a storage protein binds O2 avidly, dissociates slowly Mb is not co-operative Mb is 1 polypeptide Haemoglobin 2 Major functions: Transports O2 to tissues. Transports CO2 and protons away from tissues. Structure: 4 polypeptide chains Each chain has a haem group = can bind 4 oxygens Subunits held together by non- covalent interactions  Gene expression of the a & b chains Genes for a & b chains: a-like chains – chromosome 16 z a2 a1 Hb Gower 1 Hb F Hb A2 Hb A (z2e2) (a2g2) (a2d2) (a2b2) e Gg Ag d b b- like chains – chromosome 11 Hb variant forms & gene expression Hb type Expression Chains HbA Adult a2b2 HbA2 Minor adult form a2d2 HbF Foetal a2g2 Hb Gower 1 embryonic z2e2 Hb Gower 2 embryonic a2e2 Hb Portland embryonic z2g2  Oxygen dissociation curve Hyperbolic shape Myoglobin = reversible binding All sites are full 100 of a single O2 % Saturation with O2 (Y) Haemoglobin All sites are empty 0 120 Partial pressure of O2 (pO2) (Mm Hg) Myoglobin has a higher affinity for O2 than haemoglobin PO2 (partial pressure of oxygen) reflects the amount of oxygen gas dissolved in the blood. O2 binding to Hb Sigmoid saturation curve Indicative of co-operative binding. i.e. ‘cross-talk’ between protein sub-units. O2 binding Hb vs. Mb Hb dissociates at a higher partial pressure than Mb. This allows delivery of O2 from Hb to Mb. Haem interactions Sigmoidal shape of O2 binding curve due to structural changes initiated at one haem and transmitted to other haem groups. Affinity of 4th O2 bound = 300x greater than 1st O2 bound. O2 O2 O2 O2 O2 O2 O2 O2 O2 Hb Hb Hb Hb Hb O2 O2 O2 O2 O2 Increasing affinity for O2 Structural changes due to Oxygenation Tense +4O2 Relaxed T - form R - form Deoxy-Hb -4O2 Oxy-Hb [Low affinity] [High affinity] ab dimer 1 Ionic bonds broken ab dimer 2 Hydrophobic interactions Allosteric Effects Haem-Haem interaction – cooperativity Bohr Effect 2,3 Bisphosphoglycerate The Bohr effect O2 is released more easily at low pH or increased pCO2. Results in decreased oxygen affinity, stabilizes the T (deoxy) form Differential pH gradients (lungs have higher pH than peripheral tissues) favours unloading of O2 in tissues and loading of O2 in lung Shifts curve right = P50 increased The Bohr effect maximises efficient Oxygen handling by Hb 2,3-BPG Present in erythrocytes at ~equimolar concentrations to Hb. Binds to deoxy-Hb only, decreasing its affinity for O2. R Binding stabilizes the taut (T) conformation. T Kumar & Clarke: Clinical Medicine  2,3-BPG How? 2,3-BPG forms salt bridges with positively charged residues on the  subunits in a central cavity. These extra salt bridges must be broken during oxygenation; the cavity narrows and squeezes the 2,3- BPG out. Hb from which 2,3 BPG has been removed has high oxygen affinity  2,3-BPG Deoxy-Hb + 2,3-BPG P50 = 26 Torr Deoxy-Hb alone P50 = 1 Torr No 2,3-BPG = high O2 affinity 2,3-BPG shifts curve right (allowing O2 release in tissues) At low [O2] or anaemia, 2,3-BPG increases = more O2 delivery to tissues 2,3-BPG If 2,3-BPG concentrations are elevated, what happens to the delivery of O2 in the tissues? 2,3-BPG If 2,3-BPG concentrations are elevated, what happens to the delivery of O2 in the tissues? Delivery of O2 to tissues goes up. More O2 dissociated at a higher pO2 2,3 BPG increases in hypoxia (COPD, emphysema), high altitude or chronic anaemia Presence of 2,3BPG reduces oxygen affinity of Hb shifting oxygen dissoc curve to the right Reduced affinity allows Hb to release O2 efficiently at pO2 in tissues A useful mnemonic… "CADET, face Right!" for CO2, Acid, 2,3-DPG, Exercise and Temperature – physiological states that increase tissue requirement for oxygen  push oxygen dissociation curve to the right…