RBC Membrane Lecture Notes PDF

BLOOD BANKING MT TERM LECTURE \ BAISAC 06...

BLOOD BANKING MT TERM LECTURE \ BAISAC 06 MODULE 4: RBC MEMBRANE & METABOLISM Adducin OUTLINE Band 4.9 protein I RBC Membrane Tropomyosin A Integral Proteins B Peripheral Proteins C RBC Deformability D RBC Permeability II Metabolic Pathways A RBC Metabolism III O2-Hgb Dissociation Curve [italicized à senior trans] RBC MEMBRANE Essentially, this is the covering of the red blood cell and it’s not plainly a covering but it is a highly complex INTEGRAL MEMBRANE PROTEINS structure that helps the red blood cell perform a lot of Proteins that are quite large and extend from the outer its functions especially oxygen delivery. surface and span the entire membrane to the inner We have to remember that the red blood cell membrane cytoplasmic side of the red cell is semipermeable, meaning it allows passage of o Span the entire thickness of the RBC membrane certain substances and molecules in and out of the red (phospholipid bilayer) blood cell. o Because of this, it has an extracellular domain and The membrane is supported by a meshlike protein an intracellular domain cytoskeleton structure. o It communicates with both the extracellular and In the illustration, the membrane, intracellular compartments made up of a phospholipid Extracellular domains are often glycosylated, meaning bilayer, has some meshwork most of the time they have associated carbohydrates or below which is like a net or sieve certain sugars and thus are able to express several that gives the RBC its specific blood group antigens. characteristic biconcave shape. o Many of the RBC antigens are where they are on Phospholipids, the main lipid components of the the RBC membranes because of these glycosylated membrane, are arranged in a bilayer structure extracellular domains of integral proteins. comprising the framework in which globular proteins 2 major families: Glycophorins (A, B, C) and Band 3 traverse and move. proteins o Underneath the bilayer within the cytoplasmic Glycophorin C compartment are the proteins that constitute the o Plays an important role in attaching the underlying cytoskeletal framework. cytoskeletal protein network to the cell membrane This is the basic structure of the o Communicates with peripheral proteins, which are RBC membrane; it is a purely intracellular, found within the RBC phospholipid bilayer. o Glycoproteins are special in a way that it is able to Phospholipids have a specific anchor the peripheral proteins to the inner surface shape in that they have of the RBC membrane hydrophilic/polar heads § This is not unique to Glycophorin C as Band arranged towards the 3 is capable of doing this as well. extracellular domains where they can interact with water Band 3 without any problems. Whereas, o Binds hemoglobin and acts as additional their hydrophobic/non-polar anchoring site for peripheral proteins (cytoskeletal (afraid of water) tails are directed proteins) more inwardly due to their o Can anchor peripheral proteins to the inner surface properties in terms of interacting of the RBC membrane via a big peripheral protein with water. called ankyrin o Hydrophilic heads = outside o In association with special globular proteins like ankyrin o Hydrophobic tails = inside and band 4.2, they are also able to anchor the cytoskeletal meshwork to the RBC phospholipid bilayer membrane. The RBC membrane does not only comprise of phospholipids, but there are other molecules present. The membrane is actually composed approximately of o 52% protein o 40% lipid o 8% carbohydrate We are also able to categorize the proteins into: o Integral o Peripheral INTEGRAL PROTEINS PERIPHERAL PROTEINS Glycophorins A, B, C Spectrin Band 3 proteins Actin Band 4.1 protein TRANS BY: ABELLA :3 | 3B-MT 1  TRANS: RBC Membrane & Metabolism PERIPHERAL PROTEINS There are specific sugar Located and limited to the linkages/groups that attach inner (cytoplasmic) surface to those glycophorin proteins of the membrane, forming and depending on the the RBC cytoskeleton arrangement of the sugar (meshwork/net-like sieve). molecules, they will dictate o Entirely intracellular; only found inside the RBC whether this RBC will express o This arrangement contributes greatly to the the A antigen thus making the maintenance of the RBC’s shape and the stability of person a Type A blood type, the membrane. etc. o It gives it that durability in the scaffolding to maintain Other examples include Rh the RBC’s shape. and miscellaneous blood groups like Duffy, Kidd, Kell, Organized into a hexagonal lattice network that is positioned parallel to the membrane. MNS and Diego. Cytoskeletal proteins include spectrin, actin, band 4.1 Duffy is a blood group antigen and attaches to a special protein, adducin, band 4.9 protein and tropomyosin integral protein called a multi-pass protein since it that form a network or mesh. passes through the red cell membrane 7 times (it differs for every blood group antigen). The lattice is anchored to the lipid bilayer by the globular protein ankyrin, which interacts with band 4.2 protein as well as with band 3 integral membrane protein; and IMPORTANT FUNCTIONS OF RBCS Glycophorin C is also able to interact with some The normal chemical composition and structural peripheral proteins. arrangement and molecular interactions of the RBC membrane are crucial to RBC survival in the circulation; This unique cytoskeletal arrangement contributes to erythrocyte shape and imparts stability to the also, they maintain two important roles – deformability membrane. and permeability. The cytoskeleton is not static; it undergoes continuous RBC DEFORMABILITY rearrangement in response to RBCs are carried through our blood vessels and various physical factors and sometimes our blood vessels can get really tiny, so the chemical stimuli as the cell moves RBCs should be able to deform in a such a way that through the vascular network. they can still pass through those very small diameter o Peripheral proteins adjust to the shape that they have to sinusoids or blood vessels. take RBCs need to be easily deformable to navigate small o Example: One has to pass through the blood vessel, the sinusoids which may be only a few micrometers in RBC has to conform to the shape of the blood vessel. The diameter. cytoskeleton has to rearrange themselves in such a way to Deformability means flexibility and pliability. allow the contortion to happen. We mentioned that peripheral proteins are vital in Proteins and lipids, which maintaining the stability of the membrane, but besides differ in composition, are that, it also allows for a certain degree of deformability organized asymmetrically of the RBC membrane without disrupting it. within the RBC membrane; The red cell membrane, especially the cytoskeleton is lipids are not equally responsible for facilitating this phenomenon à distributed in the two layers requires energy of the membrane. o Spectrin phosphorylation (addition of a o It isn’t like the phospholipid bilayer where it’s just the phosphate group which needs energy) – must be hydrophilic heads and hydrophobic tails arranged done in order for RBCs to deform without rupturing. side-by-side. The loss of ATP (energy) levels leads to a decrease in o The proteins and lipids can be scattered or irregularly the phosphorylation of spectrin and in turn, a loss of distributed throughout the membrane and can membrane deformability. undergo constant rearrangement. o External layer: rich in glycolipids and choline Increased deposition of calcium within the membrane and intracellularly causes increased rigidity of the phospholipids membrane and thus less deformability/pliability. o Internal layer: rich in amino phospholipids o If calcium (cation) builds up in our RBCs and isn’t Note: § In a resting RBC, the network pumped out actively, it will accumulate in the RBC should somewhat look like a membrane and will cause the membrane to become lattice. When it is stretched (e.g. more rigid rather than pliable. when RBC is markedly swollen), o RBC is more rigid = less deformable (can’t go it stretches accordingly. through or blocks the small-bore blood vessels or it § The function of the cytoskeleton is to maintain the integrity ruptures) of the red cell membrane while accommodating to shape RBCs which are less deformable are at a disadvantage change. when they pass through small sinusoidal orifices of the spleen and thus are sequestered and removed from the IMPORTANCE OF RBC STRUCTURE IN RELATION circulation much more readily. TO BLOOD BANKING o Less deformable RBCs can block the sinusoids or get A lot of RBC antigens destroyed resulting to decreased red blood cell survival. (e.g. ABO, A antigens and B antigens) are RBC PERMEABILITY found on the Semi-permeable glycosylated o It doesn’t mean that anything and everything can extracellular domains of just go in and out anytime (selective) glycophorins, which are o There are certain conditions that the RBC integral proteins. membrane has to adjust to in order to maintain its shape. TRANS BY: ABELLA :3 | 3B-MT 2  TRANS: RBC Membrane & Metabolism Permeability means penetrability; It is how much it allows certain molecules in and out of the membrane. The volume of any given RBC is controlled by several cation transport systems which often require energy in the form of adenosine triphosphate (ATP). Any abnormality that alters cationic transport may decrease RBC survival. o Abnormalities causes aberrations to the RBC METABOLIC PATHWAYS shape, which causes it to be destructed faster in our Where does the red cell get energy (ATP)? bodies. RBC is freely permeable to water and anions (chloride & bicarbonate) RBC METABOLISM o Chloride and bicarbonate can also traverse the RBCs produce energy in the form of ATP (adenosine membrane rapidly trisphosphate) mainly through anaerobic metabolic pathways o These don’t require energy to be able to go in and o The function of the RBC is to DELIVER oxygen, not out of the RBC membrane. to consume it (no organelles to consume it) Relatively impermeable to cations (sodium, potassium, and calcium) Because the mature erythrocyte has no cytoplasmic organelles (no nucleus and no mitochondrial apparatus; o Can allow cations to enter or exit through cation only hemoglobin) or no equipment to use oxygen to transport systems and that requires energy generate energy, oxidative metabolism is not possible By controlling the intracellular concentrations of thus energy must be generated almost exclusively sodium, potassium and water, RBCs are able to through the anaerobic (does not require oxygen) maintain their distinct shape. breakdown of GLUCOSE. o RBC volume and water homeostasis are maintained There are four metabolic pathways maintaining the Erythrocyte intracellular-to-extracellular ratios for Na+ normal RBC; all are essential for effective oxygen and K+ are 1:12 and 25:1 respectively transport: o Normally, sodium is predominantly extracellular (1:12) Glycolytic Converts glucose into a modest o Potassium is predominantly intracellular (25:1) pathway amount of energy (2 ATP per (anaerobic) – glucose molecule) There are about 300 cationic pumps which actively “Glycolysis” Main pathway through which the transport sodium out of the cell and potassium into the RBC is able to produce energy for cell – these require energy in the form of ATP its pumps and to maintain its o Active transport = requires energy shape and deformability o It tries to maintain the arrangement that potassium stays capabilities. within and sodium stays outside. Need to specify (anaerobic) because Ca2+ is also actively pumped out from the RBC interior there is an aerobic glycolytic pathway which utilizes mitochondria which RBC regularly through energy-dependent calcium-ATPase does not have. pumps; these pumps are probably controlled by a Pentose Helps prevent oxidative cytoplasmic calcium-binding protein called phosphate damage to the red blood cell Calmodulin, preventing excessive intracellular Ca2+ pathway Glycolysis introduces certain buildup (this changes the shape of the RBC and makes reactive oxygen molecules as by- it more rigid). products and they are capable of When RBCs are ATP-depleted, Ca2+ and Na+ are damaging the RBC. allowed to accumulate intracellularly, while K+ and This pathway helps avoid injury that is potentially caused by those water are lost, resulting in a dehydrated, rigid cell which free radicals or reactive oxygen is readily sequestered in the spleen à decreased RBC species from the Glycolytic survival. process. We need to maintain that delicate balance of the Produces ATP but in a lesser degree pumps, energy, electrolytes and water. than anaerobic. Methemoglobin Helps retain hemoglobin iron in its reductase reduced state / ferrous state HYPERTONIC HYPOTONIC pathway (Fe2+) Water usually follows If the cationic pumps are not Iron in its ferric state (oxidized) is where sodium goes, so functioning properly and the not very effective in transporting with the abundance of extracellular fluid gets too oxygen, so it’s important to keep hypotonic, the cell becomes sodium in the iron in its reduced state. swollen and will lyse (because extracellular domain, all of the water is trying to move Luebering- Permits the water inside the RBC will Rapaport shunt accumulation/production of 2,3- inside). try and go out and be diphosphoglycerate (2,3-DPG) / with sodium. 2,3-biphosphoglycerate (2,3- BPG), the amount of which has a Result: Shrunken, Result: Plumper, swollen significant effect on the affinity of shriveled-up RBC RBC hemoglobin for oxygen and Since the water has gone If there is less sodium therefore affects how well RBCs out, the cell becomes extracellularly, water will try function post-transfusion. dehydrated. to go where there is 2,3-DPG has a special role in the relatively more sodium and Oxygen-Hemoglobin Dissociation will go inside the cell Curve. Note: o Glycolysis generates about 90% of the ATP needed o Water follows sodium movement, i.e. if sodium by the RBC; approximately 10% is provided by the accumulates intracellularly, water goes with it leading to pentose phosphate pathway. swelling and eventual lysis. o Important for pumps to be maintained and that there is constant source of energy to keep the pumps working to maintain the normal and ideal biconcave shape. TRANS BY: ABELLA :3 | 3B-MT 3  TRANS: RBC Membrane & Metabolism OXYGEN-HEMOGLOBIN DISSOCIATION CURVE Hemoglobin’s primary function is gas transport; oxygen delivery to the tissues and carbon dioxide excretion from the tissues and back to the lungs for exhalation. 2,3-DPG (from the Luebering-Rapaport shunt) is an organic RBC phosphate which is important in controlling the affinity of hemoglobin for oxygen; influences how attracted oxygen is to hemoglobin: o The presence of 2,3-DPG (the so-called tense or T form) accompanied by the formation of anionic salt Conditions that increase the Conditions that decrease the bridges between the beta chains encourages the affinity of O2 to Hgb: affinity of O2 to Hgb and unloading of oxygen from hemoglobin to the tissues release of O2 to the tissues: (ergo, it has a lower affinity for oxygen). o Low/Absent 2,3-DPG o Presence of 2,3-DPG o When hemoglobin loads oxygen (becomes o Low temperature (i.e. o High Temperature (i.e. oxyhemoglobin), the established salt bridges are places with high altitude) fever) broken and 2,3-DPG is expelled (becomes the o Low PCO2 (i.e. patients who o Increased PCO2 (i.e. patient are hyperventilating) with asthmas or breathing relaxed or R form) and this has a higher affinity for problems) oxygen. o High pH (i.e. alkalotic o Decreased pH (i.e. acidotic The relaxed form is more common in areas of the body patients) patients) wherein there is a high partial pressure of oxygen (i.e. lungs). o Hemoglobin F: has many R forms of hemoglobin because SHIFT TO THE LEFT SHIFT TO THE RIGHT it facilitates oxygen delivery from the mother to the baby. OF THE CURVE OF THE CURVE The allosteric changes that occur as the hemoglobin loads and Results conversely, in Occurs in situations of unloads oxygen are referred to as the respiratory movement. an increase hypoxia to alleviate the tissue When we talk about the curve, there are certain hemoglobin-oxygen oxygen deficit. conditions that can increase or decrease the partial affinity and thus, a pressure of oxygen in our bodies that will influence the decrease in tissue affinity of oxygen for hemoglobin. oxygen delivery. The hemoglobin-oxygen dissociation curve is Increased affinity of This shift is mediated by characteristically sigmoid-shaped (S-shaped) and this oxygen for hemoglobin increased levels of 2,3-DPG, is important to permit a considerable amount of oxygen thus hemoglobin decreasing hemoglobin’s to be delivered to the tissues with a small drop in oxygen holds on to the oxygen affinity to oxygen and tension. and will not release it. increasing its delivery to the It illustrates the relation between the partial pressure of tissues. oxygen (x-axis) and the oxygen saturation (y-axis). Oxygen will be released to the o Neither linear nor static, but can change or shift tissues easily depending on several factors: Temperature, PCO2, Note: 2,3-DPG, and pH (regulated by the amount of H+ o These concepts are important in the field of blood ions). banking or transfusion medicine because multiple transfusions of 2,3-DPG depleted blood units can EXAMPLE actually shift the hemoglobin-oxygen dissociation In the lungs, the oxygen tension (pO2) is normally curve to the left, preventing adequate tissue nearly 100 mmHg thus in this environment, the oxygenation. hemoglobin molecule is almost completely saturated o Happens frequently with old blood units or those that have with oxygen; as the erythrocytes leave the lungs and been sitting in the refrigerator for quite some time or are reaching their expiration dates. travel to the different tissues, the oxygen tension drops to an average of about 40 mmHg thus hemoglobin releases its oxygen supply to the tissues, about 25% REFERENCES released at basal metabolic rate. Notes from the discussion and PPT by Isabelle Baisac, It’s ideal if the hemoglobin in the red blood cell goes to RMT, MD the lungs where there is high O2 perfusion and there is no 2,3-DPG so that oxygen has higher affinity for hemoglobin. Once, it gets to the tissues, 2,3-DPG will then be inserted into the hemoglobin molecule so that the oxygen will be released. NORMAL CURVATURE DEPENDS ON SEVERAL LIGANDS: 1. H+ ions (and thus, pH) 2. Carbon dioxide 3. Organic phosphates – of which, 2,3-DPG is the most important 4. Temperature TRANS BY: ABELLA :3 | 3B-MT 4