HEMA1 - Red Blood Cell Physiology, Metabolism and Membrane Structure

Red blood cell

Also erythrocyte. • Biconcave disc
• 7-8 µm and 1.5-2.5 µm
• Salmon-pink with 1/3 central pallor
• Average life span: 120 days
• The only cell in mature form that contains no nucleus

One true function

Of the RBC: "oxygen delivery from lungs to the tissues".

Secondary function

Of the RBC: transport of carbon dioxide from tissues to the lung (buffering of blood pH).

Erythron

All stages of erythrocytes, in the bone marrow and peripheral blood.

RBC mass

Cells in circulation / peripheral blood.

RBC maturation changes

Five. 1. Decrease in cell diameter
2. Decrease in N:C ratio (decrease in the diameter of the nucleus)
3. Coarser, clumped, and condensed nuclear chromatin pattern ("raspberry-like appearance" / pyknotic)
4. Disappearance of nucleoli
5. Transition to blue to gray-blue to salmon pink color of cytoplasm

Erythroblast / normoblast

Nucleated precursors in the bone marrow ("erythroid islands / erythroblastic islands").

CFU-GEMM

Erythroid progenitor. Colony-Forming Unit, Granulocyte-Erythrocyte-Monocyte-Megakaryocyte.

BFU-E

Burst-Forming Unit-Erythroid. • Earliest committed progenitor
• Multisubunit colonies ("bursts")
• Not actively proliferating (G0/G1 phase)
• 1 week

CFU-E

Colony-Forming Unit-Erythroid. • Actively proliferating (S phase)
• Influenced by EPO (3-5 divisions)
• Has the most EPO receptors and is the most sensitive to EPO rescue

MPP

Multipotent progenitors. In the hematopoietic hierarchy the HSC, carrying a self-renewal loop, gives the MPP, which branches into the CMP and the CLP.

CMP

Common myeloid progenitors. Gives the MEP (then ErP to erythrocytes, and MkP to platelets) and the GMP (then granulocytes, macrophages and dendritic cells).

CLP

Common lymphoid progenitors. Gives Pro-B to B cells, Pro-T to T cells, Pro-NK to NK cells, and dendritic cells.

MEP

Megakaryocyte/erythrocyte progenitors.

GMP

Granulocyte/macrophage progenitors.

ErP and MkP

Erythroid precursors and megakaryocyte precursors. The MEP gives ErP, then erythrocytes; and MkP, then platelets.

Dendritic cells in the hierarchy

They appear on both branches of the figure - under the GMP on the myeloid side and under the CLP on the lymphoid side. That is the figure as printed.

Pronormoblast

Stage 1, also rubriblast. Nucleus: purple red chromatin; 1 or 2 nucleoli. Cytoplasm: dark blue.

Pronormoblast

Stage 1. N:C ratio 8:1. Capable of mitosis: YES. Location: bone marrow. Cellular activity: beginning of globin production. Length of time: > 24 hours.

Basophilic normoblast

Stage 2, also prorubricyte. Nucleus: deep purple red (start of condensation - chromatin); parachromatin larger & sharper. Cytoplasm: deeper richer blue.

Basophilic normoblast

Stage 2. N:C ratio 6:1. Capable of mitosis: YES. Location: bone marrow. Cellular activity: detectable hemoglobin synthesis. Length of time: > 24 hours.

Polychromatic normoblast

Stage 3, also rubricyte. Nucleus: chromatin condensation; no nucleoli present. Cytoplasm: accumulation of hemoglobin pigmentation; "murky gray-blue".

Polychromatic normoblast

Stage 3. N:C ratio 4:1 to 1:1. Capable of mitosis: YES - the last stage capable of mitosis. Location: bone marrow. Cellular activity: increase hemoglobin synthesis. Length of time: 30 hours.

Orthochromic normoblast

Stage 4, also metarubricyte. Nucleus: completely condensed ("pyknotic"). Cytoplasm: increase salmon pink color.

Orthochromic normoblast

Stage 4. N:C ratio 1:2. Capable of mitosis: NO. Location: bone marrow. Cellular activity: nucleus ejection - pyrenocyte; Howell-Jolly bodies for pitting. Length of time: 48 hours.

Pyrenocyte

The ejected nucleus of the orthochromic normoblast.

Polychromatic erythrocyte

Stage 5, also the reticulocyte. Nucleus: no nucleus. Cytoplasm: salmon pink. N:C ratio 0. Capable of mitosis: NO.

Polychromatic erythrocyte

Stage 5. Location: bone marrow (1-2 days); peripheral blood (1 day); pitting and membrane polishing - splenic macrophages. Cellular activity: completes the hemoglobin production from the remaining ribosomes. Length of time: 72 hours.

Mature erythrocyte

Stage 6. Nucleus: no nucleus. Cytoplasm: salmon-pink with central pale area or central pallor (1/3 of the cell). N:C ratio 0. Capable of mitosis: NO.

Mature erythrocyte

Stage 6. Location: peripheral blood. Cellular activity: oxygen delivery (lungs to tissues); carbon dioxide delivery (tissues to lungs). Length of time: 120 days.

The N:C ratio across the stages

8:1, then 6:1, then 4:1 to 1:1, then 1:2, then 0 and 0 for the last two stages.

Last stage capable of mitosis

The polychromatic (polychromatophilic) normoblast / rubricyte, stage 3.

Hypoxia

Also tissue hypoxia. "Decrease in oxygen content within the tissues". • Impaired oxygen transport to the tissues (i.e., anemia)
• Low oxygen tension (i.e., high altitude)

Peritubular fibroblasts

In the kidneys. The primary oxygen-sensing system.

2,3-diphosphoglycerate (2,3-DPG)

Oxygen affinity is modulated by the concentration of phosphates, of which this is the one the lecture names.

The HIF-1 chain

Five boxes as the lecture draws them: hypoxia (peritubular fibroblasts) » production of HIF-1 (hypoxia-inducible factor-1) » proteasomal degradation of HIF-1α » EPO formation » stimulates proliferation and differentiation of BFU-E & CFU-E.

Erythropoietin

• Produced primarily by the kidneys (mediated by GATA1)
• Thermostable, nondialyzable, glycoprotein hormone - carbohydrate unit + terminal sialic acid unit
• 34,000 kD

EPO as a growth factor

Signal transduction in developing erythroid cells. EPO-EPOR (EPO-Receptor gene) » JAK2 » STAT5 pathway. Promotes transcription of specific genes from the RBC nucleus.

Major effects of EPO

Three. • Early release of reticulocytes
• Inhibition of apoptosis
• Reduced marrow transit time

Measurement of EPO

• Immunologic methods (i.e., chemiluminescence) - serum, plasma, other body fluids (i.e., urine)
• 10-30 U/L

Increased EPO

In most patients with anemia - except in patients with anemia caused by renal disease.

Decreased EPO

After transfusion and with primary polycythemia (polycythemia vera).

Early release of reticulocytes

EPO effect 1. • Increase width of the spaces - changes in adventitial cell layer
• Decreased expression of fibronectin receptor
• "Shift / stress reticulocytes"

Inhibition of apoptosis

EPO effect 2. • "Direct EPO rescue from apoptosis"
• Reduced production of Fas ligand
• Increased production of the antiapoptotic molecule Bcl-XL (now called Bcl-2-like protein 1)

Reduced marrow transit time

EPO effect 3. • Increased rate of cellular processes and decreased cell cycle times - early cell cycle arrest (cessation of cell division)
• Secretion of erythroferrone - decreases hepcidin production
• "True shift reticulocytes"
• Normoblastic hyperplasia

Testosterone

Directly stimulates erythropoiesis. Higher hemoglobin concentration in men than in women.

Pituitary and thyroid hormones

Indirectly affect erythropoiesis, and also affect EPO production.

Extravascular hemolysis

Also macrophage-mediated. • The major physiological pathway for removal of senescent ("aged") or abnormal cells - splenic ("culling") or hepatic macrophages
• Lack of ATP » oxidation of membrane lipids and proteins (globin)
• Loss of discoid shape (spheroid) - difficulty squeezing through the splenic sieve

Erythrophagocytosis

Removal of red cells by the mononuclear phagocyte system, in extravascular hemolysis.

Eryptosis

Nonnucleated cell death.

Intravascular hemolysis

Also mechanical or fragmentation hemolysis. • Rupture intravascularly - within the lumen of blood vessels
• Purely mechanical / traumatic stress - "fragmentation"
• Small contributor to RBC destruction under normal circumstances

Chuvash polycythemia

Also hereditary polycythemia. Autosomal recessive; increased cellular expression of HIF-1α due to a 598C>T mutation (von Hippel-Lindau gene).

Familial polycythemia

Autosomal dominant; defect in the regulation of EPO.

Other disorders of erythropoietin

• Renal neoplasms or disorders
• Smoking - secondary erythropoiesis

Red cell increases

Increase in packed cell volume (hematocrit) or total erythrocyte count - decreased plasma volume (i.e., dehydration).

Megaloblastic anemia

Defective nuclear maturation - nuclear maturation lags behind cytoplasmic maturation (vitamin B12 / folate deficiencies); impaired ability of the cells to synthesize DNA.

Membrane composition

8 % carbohydrates, 52 % proteins, 40 % lipids (cholesterol and phospholipids).

Cholesterol in the membrane

Tensile strength. The ratio of cholesterol to phospholipids remains constant, which is the balance of deformability and strength.

Membrane phospholipids

Phosphatidylcholine and sphingomyelin in the outer layer; phosphatidylserine and phosphatidylethanolamine in the inner layer.

Glycolipids

Clumps/rafts; support the carbohydrate side chains that anchor the glycocalyx; bear copies of carbohydrate-based blood group antigens (i.e., ABH, Lewis).

Transmembrane proteins

Function as transport sites, adhesion sites, and signaling receptors.

Two major macromolecular complexes

The ankyrin complex and the actin junctional complex (protein 4.1 complex). They provide membrane structural integrity and vertical membrane structure.

Phospholipid asymmetry

Outer leaflet mostly phosphatidylcholine and sphingomyelin; inner leaflet mostly phosphatidylethanolamine and phosphatidylserine; phosphatidylinositol and cholesterol on both faces.

Protein classes in the bilayer

• Transmembrane proteins crossing the bilayer
• Extracellular protein
• Glycosylphosphatidylinositol (GPI) linked protein
• Lipid-linked protein
• Cytosolic protein

Aquaporin 1

Transmembrane protein. Gene AQP1; 28 kD; 120-160 copies/cell. Water transporter, Colton antigen.

Band 3

Anion exchanger, AE1. Gene SLC4A1, band 3; 90-102 kD; 1200 copies/cell, 27 % of total protein. Anion transporter, location of ABH antigens.

Glut-1

Transmembrane protein. Gene SLC2A1, band 4.5; 45-75 kD; 5 % of total protein. Glucose transporter, location of ABH blood group antigens.

Glycophorin A

Transmembrane protein. Gene GYPA, PAS-1; 36 kD; 85 % of glycophorin. Sialic acid transporter, location of MN blood group antigens.

Glycophorin B

Transmembrane protein. Gene GYPB, PAS-4; 20 kD; 10 % of glycophorin. Sialic acid transporter, location of Ss blood group antigens.

Glycophorin C

Transmembrane protein. Gene GYPC, PAS-2; 14-32 kD; 4 % of glycophorin. Sialic acid transporter, location of Gerbich system antigens.

Duffy

Transmembrane protein. Genes FY, DARC, ACKR1; 35-43 kD. G protein-coupled receptor, chemokine receptor, Duffy antigens, receptor for malarial parasites.

Kell

Transmembrane protein. Gene KEL; 93 kD. Zn²⁺-binding endopeptidase, Kell antigens.

Kidd

Transmembrane protein. Gene SLC14A1; 43 kD. Urea transporter, Kidd (Jk) antigens.

Rh

Transmembrane protein. Genes RHCE, RHD; 30-45 kD. D and CcEe antigens; stabilizes band 3 and Rh macrocomplexes.

RhAG

Transmembrane protein. Gene RHAG; 45-100 kD. D and CcEe antigen component; CO₂, cation, and ammonium transporter.

Cytoskeletal proteins

Also peripheral proteins. • Assemble to form an antiparallel heterodimer - filamentous α-spectrin and β-spectrin
• Provide lateral or horizontal membrane stability
• Spectrin dimer bonds disassociate and reassociate (open and close) during deformation

α-spectrin

Gene SPTA1, band 1; 240-280 kD; 242 copies/cell, 16 % of total protein. With β-spectrin it forms the filamentous antiparallel heterodimer, the primary cytoskeletal proteins.

β-spectrin

Gene SPTB, band 2; 220-246 kD; 242 copies/cell, 14 % of total protein. With α-spectrin it forms the filamentous antiparallel heterodimer, the primary cytoskeletal proteins.

Ankyrin

Cytoskeletal protein. Gene ANK1, band 2.1; 206-210 kD. Anchors band 3, protein 4.2, and other proteins in the ankyrin complex to spectrin.

Adducin

Cytoskeletal protein. Genes ADD1, ADD2, band 2.9; 80-103 kD. Caps actin filament, binds Ca²⁺/calmodulin.

Dematin

Cytoskeletal protein. Gene EPB49, band 4.9; 43-52 kD. Actin bundling protein.

β-actin

Cytoskeletal protein. Gene ACTB, band 5; 42-43 kD. Binds β-spectrin.

G3PD

Cytoskeletal protein. Gene GAPD, band 6; 35-37 kD. Carbohydrate metabolism, phosphorylates G3P.

Protein 4.1

Cytoskeletal protein. Gene EPB41, band 4.1; 66-80 kD. Anchors the actin junctional complex to spectrin tetramers, RBC cytoskeleton shape.

Protein 4.2

Protein kinase. Gene EPB42, band 4.2; 72-77 kD. Part of ankyrin complex, ATP binding protein.

Tropomodulin

Cytoskeletal protein. Gene TMOD1, band 5; 41-43 kD. Caps actin filament.

Tropomyosin

Cytoskeletal protein. Gene TPM3, band 7; 27-38 kD. Stabilizes and regulates actin polymerization.

Unbound band 3

One of the three regions the membrane diagram labels. Band 3 with GPA and Prx2.

Ankyrin complex

Band 3, GPA, GPB, Rh, RhAG, LW, CD47, protein 4.2 and GEC, all anchored by ankyrin down onto spectrin. It anchors vertically into the bilayer.

Actin junctional complex

GPC/D, Kell, Duffy, Kx, GPA, Glut1, protein 4.2, adducin, stomatin, p55, GEC, and protein 4.1 with its EF and CH1/CH2 domains, running onto F-actin with tropomyosin, tropomodulin and dematin.

Self-association sites

Marked on the spectrin braid beneath the bilayer, where tetramers join.

Membrane permeability

• Permeable to water and anions (chloride and bicarbonate)
• Impermeable to cations (sodium, potassium, calcium)

Aquaporin 1 in osmotic balance

Formation of pores/channels to create inward flow of water.

Calmodulin

Controls the Ca²⁺ATPase pump. Cations are held out by ATP-dependent cation pumps.

Acanthocytes & codocytes

Deficiency in enzymes maintaining the cholesterol concentration - exchanging membrane and plasma cholesterol.

Paroxysmal nocturnal hemoglobinuria (PNH)

Acquired mutation in the PIGA gene - deficient CD55 and CD59.

Hereditary elliptocytosis

Autosomal dominant mutations affecting spectrin dimer-to-dimer lateral bonds or the protein 4.1 junction - horizontal interaction defects, failing to rebound from deformation.

Hereditary spherocytosis

1. Autosomal dominant mutations affecting the integrity of band 3, ankyrin, protein 4.2 or α-/β-spectrin - too few vertical anchorages to maintain membrane stability (lipid membrane peels off in small fragments / blebs).
   2. Decrease in Aquaporin 1 expression

Horizontal against vertical defect

Hereditary elliptocytosis is the horizontal defect - spectrin dimer-to-dimer lateral bonds. Hereditary spherocytosis is the vertical one - too few vertical anchorages.

Overhydrated stomatocytosis

Defect in ion channels - increase intracellular sodium.