Introduction

Pyruvate Kinase Deficiency (PKD) is one of the most common causes of chronic hereditary haemolytic anaemia, an inherited disorder characterised by premature destruction of red blood cells (haemolysis), leading to anaemia. Pyruvate kinase is an enzyme involved in the production of adenosine triphosphate (ATP) during glycolysis. Red blood cells rely on glycolysis to obtain the energy needed to maintain their structure and function; therefore, a defect in this pathway leads to the body producing red blood cells that don’t develop properly and break down too quickly.^1,2

The prevalence of PKD in Caucasian populations is between 1:20,000 and 1:300,000, with genetic backgrounds and differences in the function of the spleen resulting in a broad spectrum of symptoms from person to person with varying levels of severity. A wide range of complications can also arise as a result of PKD, one of which includes iron overload. Manifestations of the disease can occur at any point between the newborn period to adult life. This article will explore the underlying pathophysiology of PKD as well as some of the associated complications that commonly arise alongside the deficiency.^1,2,3

What is glycolysis and the role of pyruvate kinase? 

Glycolysis is a metabolic pathway that occurs in the cytoplasm of a cell. Without the need for oxygen, it converts glucose into pyruvate through a series of steps that require different enzymes. Through the process of glycolysis, 2 molecules of ATP are generated and can be used as sources of energy for the cell. Pyruvate kinase is an enzyme involved in the final step of glycolysis, converting phosphoenolpyruvate to pyruvate, generating ATP as a by-product.⁴

Unlike other cells that can generate ATP through a range of different pathways, including oxidative phosphorylation, red blood cells lack mitochondria and a nucleus; hence, they rely solely on glycolysis to generate ATP. Therefore, the lifespan of red cells is dependent on the ATP produced during glycolysis. 50% of the total ATP is produced during the final step with pyruvate kinase, and a deficiency not only reduces ATP output but also leads to the accumulation of intermediates of the previous steps of the pathway, more specifically, 2,3-bisphosphoglycerate. Interestingly, a study identified that an increase in this intermediate leads to better anaemia tolerance in a small group of people.^1,3,4

During PKD, there is a loss of red cell structure and function, with red cell dehydration and disturbed cell membranes commonly observed. In addition, the red cells are variably damaged, with reticulocytes (immature red cells) most susceptible to destruction as they require increased levels of ATP, while mature red cells are less affected.^2,4

Genetic basis of pyruvate kinase deficiency 

Pyruvate kinase is regulated by the expression of 4 key isoenzymes, with the red cell reliant on the R-type. Both red blood cell and liver pyruvate kinase are expressed by the control of a specific gene, PKLR, located on chromosome 1q21. Over 200 different mutations have been detected in different people with PKD within the same gene. The most common type of mutations in PKLR are missense mutations, characterised by a genetic substitution of a single base pair in DNA, leading to a different amino acid and thereby a different protein with altered function.^2,3,4,5

PKD is inherited in an autosomal recessive manner, where the condition only manifests if a person inherits two faulty copies of the PKLR gene (one from each parent). This could either be compound heterozygous, where 2 different mutations that cause the disease are inherited from each parent, or homozygous, where the person has 2 identical faulty copies of the gene. In both cases, there is defective pyruvate kinase activity leading to haemolysis. Studies have shown that PKLR variants not only lead to decreased stability of the enzyme, but also alter their affinity to phosphoenolpyruvate (their substrate).^3,4

Pathophysiology of haemolysis in PKD

PKD leads to chronic hqemolysis due to red blood cells not being able to generate appropriate amounts of ATP to maintain sufficient energy levels in cells. There are several mechanisms that contribute to the resulting red cell instability, deformability, and premature destruction. These include changes in ATP levels, membrane integrity, ion pumps, and oxidative stress.^2,6,7

ATP depletion

Pyruvate kinase is an essential enzyme in the glycolytic pathway directly involved in ATP generation. Mutations in this enzyme lead to impaired enzyme function and depleted ATP stores. Since ATP is required for membrane stability, deformability, and activity of ion transport, its depletion directly promotes red blood cell dysfunction.^1,3,6,7

Loss of membrane integrity 

Red blood cells’ deformability (ability to change shape) allows them to travel through the smallest of capillaries, transporting oxygen to cells. Many people with haemolytic anaemia have aberrant deformability, which affects the strength of red cells and shortens their lifespan. Deformability relies on the interactions of cytoskeletal proteins and integral transmembrane complexes as well as the processes controlling intracellular ion handling. ATP depletion weakens these processes and impairs deformability, leading to red blood cells’ failure to navigate vessels and their consequent removal.^6,7

Failure of ion pumps: dehydration and rigidity 

Deformability is influenced by the volume and ion content of red blood cells, both of which are regulated by pumps and channels. These processes are regulated to withstand osmotic changes to maintain the volume of red blood cells. ATP depletion causes some transport mechanisms to be dysregulated, leading to several complications:

• Na⁺/K⁺ ATPase dysfunction leads to red blood cells accumulating Na⁺ ions and leaking K⁺ ions more rapidly, consuming ATP at an accelerated rate. This disrupts the osmotic balance of the cell
• Ca²⁺ ATPase dysfunction leads to Ca²⁺ ions overload, as the red blood cells are less able to remove it from the cell. Abnormally high levels of Ca²⁺ lead to decreased ATP levels and degraded cytoskeletal proteins

These ionic imbalances lead to cellular dehydration and membrane rigidity that affect red blood cell survival.^6,7

Increased oxidative stress

Red blood cells are constantly exposed to oxidative stress, and ATP is required to maintain antioxidant defences. In people with PKD, oxidative damage to membrane lipids and proteins affects the survival of red blood cells. Under high oxidative stress conditions, more glucose is consumed, while red blood cells are unable to generate new ATP themselves. Studies have identified destabilisation in membranes and a dramatic reduction in deformability.⁶

Splenic clearance

The spleen is a quality control organ that filters out deformed and rigid red blood cells. Those with reduced deformability fail to pass through the spleen, leading to their destruction by splenic macrophages. Increased haemolysis contributes to both anaemia and splenomegaly (enlargement of the spleen).^2,3,7

Clinical implications of PKD

Symptoms and manifestations of both PKD and chronic haemolysis vary from person to person and may even impact quality of life. Some of the symptoms that could arise include:^2,3,4

• Chronic haemolytic anaemia, which can worsen with acute infections, stress or pregnancy, leading to progressive fatigue, poor growth and low energy levels
• Iron overload from haemolysis and transfusions, which can lead to organ-related complications
• Splenomegaly due to increased red blood cell destruction
• Pulmonary hypertension by the release of toxic haemoglobin to the plasma during haemolysis
• Jaundice and hyperbilirubinemia by increased breakdown of red blood cells, leading to yellowing of the skin and eyes as well as potential cognitive defects such as poor concentration
• Gallstones, a complication of chronic bilirubin accumulation

Summary 

Pyruvate kinase deficiency (PKD) is a type of chronic hereditary haemolytic anaemia characterised by the destruction of red blood cells. The enzyme pyruvate kinase is involved in glycolysis, a crucial metabolic pathway used by red blood cells to produce ATP. A strong genetic component has been linked to the condition, with a mutation in the PKLR gene leading to decreased enzyme function and stability. Chronic haemolysis results in red blood cell instability, deformability and premature destruction by a range of mechanisms. These include ATP depletion, loss of membrane integrity, oxidative stress and failure of ion pumps, which promote dehydration and rigidity. In addition, premature removal of red blood cells in the spleen contributes to anaemia and splenomegaly. Other clinical signs of PKD include iron overload, pulmonary hypertension and jaundice.