A. Introduction

1. Red blood cells (RBC) are biconcave discs
2. Major role is oxygen transport using its hemoglobin (Hb) content
3. Total RBC content in the body is tightly regulated
   1. RBC destruction must not exceed RBC production
   2. Therefore, RBC must maintain a normal (~120 day) lifespan
   3. Cells must survive capillaries (deformation), oxygen stress, ostmotic stress
4. Three critical features of RBC structure and function
   1. Deformable membrane - allows survival in microcirculation
   2. Maintenance of intact Hb and modulation of Hb oxygen affinity
   3. Generate energy to maintain constancy of osmotic environment of cell
5. RBC Metabolism
   1. RBC do not have mitochondria or nuclei
   2. They use glycolysis only, creating 2 moles each ATP and NADH from each glucose
   3. They can also use hexose monophosphate shunt, creating 2 moles NADPH from glucose
   4. 2,3-diphosphoglycerate (DPG) is created in the Embden-Meyerhof pathway
   5. DPG is a critical modulator of oxygen-Hb dissociation function

B. Normal Red Blood Cells (RBC) Values

1. Hematocrit (HCT): M 38-54% F 36-47%
2. Hemoglobin (Hb): M 14-18g% F 12-16g% Child: 12-14g% Newborn: 14-24g%
3. Erythrocytes (per mm3): M 4.5-6 million F 4.3-5.5 million
4. Reticulocytes (per mm3): 0-1.5%
5. Diameter: Adult: 5.5-8.8 µm Newborn: 8.6 µm
6. Mean Corpuscular Volume (MCV): 80-90 cµm (fL) Newborn: 100-110 cµm
7. Mean Corpuscular Hemoglobin (MCH): 27-32 pg
8. Sedimentation Rate (ESR): M 0-9mm/hr F 0-20mm/hr (age dependent)
9. RDW (red cell distribution width): Normal <14.5%; RDW >14.5% called anisocytosis

C. RBC Membrane

1. Biconcave disc shape maximizes area for respiratory exchange
2. Shape maintained by highly evolved underlying protein skeleton
3. Major scaffold is composed of tetrameric (alpha2-beta2) spectrin
   1. Spectrin multimers are cross-linked by protein 4.1 and short actin filaments
   2. ThisSkeletin is attached to membrane by binding of spectrin to ankryn
4. Ankryn binds to another protein (band 3) which is the anion exchanger
5. Protein skeleton maintains shape and plays major role in transport across membrane
6. Blood group antigens are found on the surface of the RBC

H. Hemoglobin [2]
[Figure] "Oxygen-Hemoglobin Dissociation Curve"

1. Structure
   1. Adult Hb consists of two alpha and two beta (or delta) polypeptides
   2. Each globin contains one heme molecule: porphyrin ring with ferrous (Fe2+ iron) atom
   3. Molecular weight is 64.5K
   4. Deoxyhemoglobin is in a tense (T) conformation with low O2 affinity
   5. Normal oxy-Hb is more flexible
   6. The affinity of Hb for O2 is dependent on many factors, but mainly oxygen tension
   7. Thus, O2 is taken up at high pO2 (lung) and is released easily at low pO2 (tissues)
2. Hb Polypeptide Chains
   1. Structure of human Hb changes during development
   2. All Hb consists of two different pairs of peptide chains: alpha-like and beta-like (a2b2)
   3. The alpha-like genes are found in clusters on chromosome 16
   4. The beta-like genes are found in clusters on chromosome 11
   5. Initially, embryonic alpha-like and beta-like genes are expressed
   6. Fetal Hb (HbF) has an a2g2 (gamma peptides instead of usual beta)
   7. Fetal Hb replaces embryonic Hb by 12 weeks after conception
   8. Adult Hb (HbA) is mainly a2b2, with some HbA2 consisting of a2d2 (delta peptides)
3. There are multiple oxygen-binding sites on Hb
   1. One O2 molecule can bind to ferrous atom
   2. This induces changes in conformation of Hb leading to changes in conformation
   3. These changes lead to relaxed conformation and ~500 fold increase in Hb affinity for O2
   4. In addition, there is a cooperativity among O2 binding sites
   5. Thus, occupancy of O2 binding sites leads to increased affinity
   6. These properties lead to the sigmoid-shaped Hb-oxygen dissociation curve
4. Modulators of Hb Affinity for O2
   1. Oxygen, the primary ligand, induces increased affinity for itself (homotropic effector)
   2. There are three major heterotropic effectors
   3. These are hydrogen ion (pH), carbon dioxide (CO2), red-cell 2,3-diphosphoglycerate (DPG)
   4. Hydrogen ions (decreased pH) and CO2 reduces O2 binding of Hb (Boehr Effect)
   5. O2 binding to Hb reduces its affinity for CO2 (Haldane Effect)
   6. Reduction in Hb affinity for O2 shifts O2-Hb Dissociation Curve to the right
   7. In addition, temperature increases reduce O2 affinity (similar to acid)
   8. Chloride concentrations can also affect Hb-O2 affinity
   9. These concepts explain much of O2 delivery physiology between lung and tissue
5. Red Cell DPG
   1. DPG is sequestered by deoxyhemoglobin and acumulates at high levels
   2. DPG binds to Hb, stabilizes the T conformation, and reduces O2 affinity
   3. In addition, DPG binding also lowers intracellular pH
   4. In chronic acidosis, reduced RBC DPG levels compensate partially for drop in pH
6. Nitric oxide, acid-base disturbances, and other factors affect Hb-O2 interactions

E. Development of Erythrocytes
[Figure] "Blood Cell Development"

1. Generated in bone marrow from hematopoietic precursor cells
2. Precursors are usually considered CD34+ stem cells (CFU-S, express SCL/tal-1)
3. These differentiate to CFU-GEMM
   1. CFU-GEMM is colony forming unit - granulocyte-erythroid-megakaryocyte-macrophage
   2. CFU-GEMM expresses transcription factors GATA-2, GATA-1 and c-myb
4. CFU-GEMM differentiate into BFU-E, which are committed/restricted to RBC lineage
   1. BFU is burst forming unit
   2. BFU-E differentiates into CFU-E
5. CFU-E differentiate into proerythroblasts
   1. CFU-E and proerythroblasts are increasingly sensitive to erythropoietin (EPO)
   2. Express erythroid Kruppel-like factor and Nuclear factor erythroid 2
6. Proerythroblast Development
   1. Several stages of development occur between proerythroblast and nucleated RBC
   2. Nucleated RBC develop into reticulocytes
   3. Reticulocytes are the first non-nuceated stage of RBC development
   4. Reticulocytes differentiate into final stage erythrocytes
7. EPO [5,6]
   1. Major growth for regulation of erythropoiesis
   2. Expression regulated by oxygen sensing (distributed in many tissues)
   3. In adults, production of EPO is mainly the kidney
   4. EPO drives bone marrow production of RBC
   5. EPO binds dimerised EPO-receptor on surface of erythroid progenitor cells
   6. This leads to receptor conformational change and activation of JAK-2 kinase
   7. JAK-2 kinase phosphorylates tyrosine residues on various proteins including STAT-5
   8. STAT-5 causes gene activation, stimulates erythrocyte development, blocks apoptosis
   9. Therapeutic uses for various types of anemia (see below)
8. N-acetyl-Seryl-Aspartyl-Lysyl-Proline (NASDKP) [4]
   1. NASDKP is a natural inhibitor of hematopoietic stem cell growth
   2. Angiotensin converting enzyme (ACE) degrades NASDKP
   3. ACE Inhibitors may lead to increased NASDKP
   4. This may be beneficial in patients with polycythemia
   5. Anemia or other bone marrow effect is not a side effect of ACE I

F. Erythrocyte Stimulation [6]

1. Various treatments stimulating erythropoiesis have been approved
2. Recombinant EPO (epoetin, Procrit®, Eprex®, NeoRecormon®)
   1. Mainstay of therapy anemia of renal failure
   2. EPO and iron supplementation before surgery reduced need for transfusion [3]
   3. Also used for chemotherapy-associated and other forms of anemia
   4. EPO has neuronal anti-apoptotic effects, may be useful in some forms of stroke [5]
   5. Recominant EPO is a 30.4K glycoprotein with elimination half-life ~8.5 hours
   6. Typically given 2-3 times weekly intravenously
3. Darbepoetin Alpha (Aranesp®)
   1. EPO with additional N-linked glycosylation changes (37.1K glycoprotein)
   2. Elimination half-life 25.3 hours, usually given once weekly or biweekly
4. Methoxy-Polyethylene Glycol Epoetin ß (mPEG-epoetin) [7]
   1. Long acting, pegylated epoetin designed for infrequent administration
   2. Similar efficacy when given IV q2-4 weeks as standard epoetin given three times weekly
   3. Tolerability similar to standard epoetin
5. Continuous EPO-receptor Activator (CERA)
   1. Investigational, having completed Phase III clinical development
   2. CERA created by integrating single 30K polymer chain into EPO to yield ~60K molecule
   3. Can be given once every 3-4 weeks
6. Synthetic Erythropoiesis Protein: 51K protein-polymer construct, activates EPO receptor
7. EPO fusion proteins
8. EPO Mimetic Peitides that activate JAK-2/STAT-5 Pathway
9. Hypoxia inducible factor alpha stabilizers
10. Gene therapy

References

1. Weatherall DJ and Provan AB. 2000. Lancet. 355(9120):1169
2. Hsia CCW. 1998. NEJM. 338(4):239
3. Feagan BG, Wong CJ, Kirkley A, et al. 2000. Ann Intern Med. 133(11):845
4. Plata R, Cornejo A, Arratia C, et al. 2002. Lancet. 359(9307):663
5. Kaushansky K. 2006. NEJM. 354(19):2034
6. Macdougall IC and Eckardt KU. 2006. Lancet. 368(9539):947
7. Levin NW, Fishbane S, Canedo FV, et al. 2007. Lancet. 370(9596):1415