Haematology; Haemopoiesis and Haemoglobin

Basic Principles of Haematology; Haemopoiesis and Haemoglobin

Dr. Roger Preston
School of Pharmacy & Biomolecular Sciences
Royal College of Surgeons in Ireland
Coláiste Ríoga na Máinleá in Éirinn

Learning Outcomes

• Describe the composition of whole blood and its components.
• Summarise the sites, steps & factors involved in haemopoiesis with particular reference to erythropoiesis.
• Describe the characteristics and life cycle of erythrocytes.
• Discuss the role of haem in oxygen binding.
• Describe the structure and function of myoglobin (Mb).
• Describe the structure and function of haemoglobin (Hb).
• Discuss the co-cooperativity and the allosteric regulation of haemoglobin.
• Outline the metabolic capability of red blood cells.

What is Blood?

• Blood is one of the largest organs distributed throughout the entire body.
• It circulates through the body’s:
  • Heart
  • Arteries
  • Veins
  • Capillaries
• For a 70 kg man, blood volume is approximately 5.6 liters, which represents 7-8% of body weight.
• Average blood temperature is 38 °C.
• Blood is slightly alkaline, with a typical pH range of 7.35–7.45.

Functions of Blood

Carries TO the body tissues:

• Oxygen
• Nutrients
• Hormones
• Water
• Solutes
• Heat

Carries AWAY from the tissues:

• Waste matter
• Carbon dioxide

Blood Composition

• Blood is composed of two main components:
  • Plasma:
  • Accounts for 55% of blood volume (~3.5 liters).
  • Liquid component in which blood cells are suspended.
  • Complex aqueous solution containing: Gases, salts, proteins, carbohydrates, and lipids.
  • Formed Elements:
  • Constitutes 45% of blood volume:
    • Red blood cells (erythrocytes) comprise 99% of formed elements.
    • Platelets account for less than 1%.
    • White blood cells (leukocytes) also account for less than 1%.
• Whole blood, when allowed to clot, results in serum after clot removal; serum lacks coagulation factors.

Haemopoiesis

• Definition: Haemopoiesis is the production and development of new blood cells.
• Two-thirds of cell production results in white blood cells, a process known as leucopoiesis.
• One-third of cell production results in red blood cells, through a process called erythropoiesis.
• There are >500 times more red blood cells than white blood cells in circulation.
• Initial stages of haemopoiesis occur within the first two minutes of life.

Key Features of Haemopoiesis

• Differentiated cells lose their ability to self-renew.
• A single stem cell can produce more than 10^6 mature blood cells while accounting for less than 0.1% of all cells in bone marrow.
• Stem cells undergo growth and division primarily in the bone marrow.
• They lose Cell Adhesion Molecules (CAMs), allowing them to leave the bone marrow and enter circulation.
• Hematopoiesis requires various growth factors:
  • Erythropoietin
  • Colony stimulating factors
  • Interleukins
  • Thrombopoietin

Sites of Haemopoiesis

• In children, hematopoiesis occurs primarily in the marrow of long bones, such as the femur and tibia.
• In adults, it mainly occurs in the pelvis, cranium, vertebrae, and sternum.
• In certain situations, the liver, thymus, and spleen may return to their hematopoietic roles; this is referred to as extramedullary haemopoiesis.
• Maturation, activation, and some proliferation of lymphoid cells take place in the spleen and lymph nodes.

Erythropoiesis

• An overview of erythropoiesis shows that typically one proerythroblast gives rise to about 16 mature red blood cells.

Erythrocytes (RBCs)

• Characteristics:
  • Anucleate (lack nucleus)
  • Discoid shape
  • Average life span of approximately 120 days
  • 1% of erythrocytes are destroyed daily.

Anaemia

• Defined as a decrease in hemoglobin (Hb) concentration below the normal reference range, which is:
  • 11.5 – 16.0 g/dL for females
  • 13.5 – 17.5 g/dL for males
• Inherited haemolytic anaemias include:
  • Glucose-6-phosphate dehydrogenase (G6PD) deficiency
  • Sickle cell anaemia

Life Cycle of RBCs

• Life span of an RBC: ~120 days.
• Senescent (aged) RBCs are removed by macrophages.
• Components of hemoglobin are recycled:
  • Globin portion is reutilized as amino acids.
  • Iron is recovered and reused.
  • Haem is excreted in bile.

Aerobic Metabolism

• Aerobic metabolism is the most efficient form of energy production.
• Multicellular organisms require oxygen (O2) transportation to all tissues for aerobic metabolism, which necessitates specialized proteins such as myoglobin and hemoglobin.

Haem Group

• The oxygen-binding capability of proteins depends on the haem group.
• It is not a part of the polypeptide chain but is tightly bound to the protein.
• Hemoglobin activity hinges on the presence of the iron in the haem group, specifically in its ferrous (Fe2+) state.
  • The iron is held in position by 4 nitrogen atoms (N).
  • Fe2+ can form two additional bonds.
• The reaction to form haem is:
  $ext{Protoporphyrin IX} + ext{Fe}^{2+} = ext{Haem}$

Structure of Myoglobin

• Myoglobin acts as an oxygen reservoir within heart and skeletal muscle cells.
• It comprises 153 amino acids and has a molecular weight of 17 kDa.
• Structural Composition:
  • Approximately 75% of its composition is in alpha helix form.
  • Contains 8 helices labeled A-H.
  • Features several non-helical regions (e.g., AB, BC).
  • Has a hydrophilic exterior and a hydrophobic interior, except in the regions around histidines E7 and F8.

O2 Binding Site Structure

• The haem group is situated in a crevice near the protein surface that is lined with non-polar residues.
  • E7 = distal histidine
  • F8 = proximal histidine

Differences between Hemoglobin (Hb) and Myoglobin (Mb)

• Myoglobin functions primarily as a storage protein for oxygen.
• It binds oxygen avidly and dissociates slowly.
• Myoglobin does not exhibit cooperative binding.
• Myoglobin is structured as a single polypeptide chain, while hemoglobin is a tetramer composed of four polypeptide chains.

Hemoglobin

• Major Functions:
  • Transports oxygen to tissues.
  • Transports carbon dioxide (CO2) and protons away from tissues.
• Structural Composition:
  • Composed of four polypeptide chains, each associated with a haem group capable of binding one oxygen molecule.
  • Hemoglobin can bind up to four oxygen molecules.
  • Subunits are held together by non-covalent interactions.

Gene Expression of Hemoglobin Chains

• Genes responsible for the alpha (a) and beta (b) chains:
  • a-like chains are located on chromosome 16, while b-like chains are located on chromosome 11.
• Different hemoglobin variants include:
  • Hb Gower 1: $ext{z}<em>2 ext{e}</em>2$ (embryonic)
  • Hb A2: $ext{a}<em>2 ext{d}</em>2$ (minor adult form)
  • Hb A: $ext{a}<em>2 ext{b}</em>2$ (adult)
  • Hb F: $ext{a}<em>2 ext{g}</em>2$ (foetal)
  • Hb Gower 2: $ext{a}<em>2 ext{e}</em>2$ (embryonic)
  • Hb Portland: $ext{z}<em>2 ext{g}</em>2$ (embryonic)

Oxygen Dissociation Curve

• Myoglobin exhibits a higher affinity for oxygen than hemoglobin.
• The relationship can be graphically represented as:
  • Partial Pressure of O2 (pO2) compared to % Saturation with O2 (Y).
• A hyperbolic curve demonstrates the reversible binding of a single O2 to myoglobin, while hemoglobin demonstrates a sigmoid saturation curve indicative of cooperative binding.

O2 Binding Mechanism

• In hemoglobin, the sigmoid saturation curve is indicative of cooperative binding, which is the interaction (or “cross-talk”) between different protein subunits.
• Hemoglobin dissociates oxygen at higher partial pressures than myoglobin, allowing effective oxygen delivery from hemoglobin to myoglobin.

Haem Interactions

• The sigmoidal shape of the O2 binding curve in hemoglobin is due to structural changes initiated when one haem group binds to O2, which are then transmitted to the remaining haem groups.
• The affinity of the fourth O2 bound is 300 times greater than the first O2.

Structural Changes Due to Oxygenation

• Hemoglobin transitions between two forms:
  • T form (tense)
  • R form (relaxed)
• When O2 is bound, the transition from T to R involves breaking ionic bonds and hydrophobic interactions, resulting in a higher affinity for oxygen in the R form.

Allosteric Effects

Haem-Haem Interaction and Cooperativity

• The phenomenon of cooperativity is influenced by haem-haem interactions.

The Bohr Effect

• The Bohr effect describes how O2 is released more readily at low pH or increased pCO2, which results in decreased oxygen affinity and stabilizes the T (deoxy) form of hemoglobin.
• This differential response based on pH gradients (lungs having a higher pH than peripheral tissues) favors oxygen unloading in tissues and loading in the lungs.
• The rightward shift of the oxygen dissociation curve results in an increased P50 value, thus maximizing efficient oxygen handling by hemoglobin.

2,3-BPG (2,3-Bisphosphoglycerate)

• 2,3-BPG is present in erythrocytes at approximately equimolar concentrations to hemoglobin.
• It binds specifically to deoxy-Hb, resulting in decreased oxygen affinity due to stabilization of the taut (T) conformation.

Mechanism of Action

• 2,3-BPG engages in salt bridge formation with positively charged residues on the beta subunits within a central cavity of hemoglobin.
• This action necessitates that these salt bridges be broken during the process of oxygenation as the cavity narrows and causes the 2,3-BPG to be expelled.
• Hemoglobin depleted of 2,3-BPG exhibits an increased oxygen affinity.

Effects of 2,3-BPG Concentration

• Deoxy-Hb combined with 2,3-BPG has a P50 value of 26 Torr, whereas deoxy-Hb alone has a P50 value of 1 Torr.
  • Higher concentrations of 2,3-BPG lead to a rightward shift in the oxygen dissociation curve, facilitating oxygen release in tissues.

Significance

• In conditions of low oxygen (e.g., anaemia or hypoxia) or elevated 2,3-BPG concentrations, hemoglobin enhances oxygen delivery to tissues effectively.

Useful Mnemonic

• The mnemonic “CADET, face Right!” refers to the physiological states (CO2, Acid, 2,3-DPG, Exercise, and Temperature) that increase the tissue requirement for oxygen, causing the oxygen dissociation curve to shift to the right.