Rbc membrane

HI I AM DR.GAYATHRI. JUNIOR RESIDENT MD TRANSFUSION MEDICINE, SCTIMST TRIVANDRUM, KERALA, INDIA

1. RBC MEMBRANE & METABOLISM Dr.Gayathri.A.M . Presentation-3

2. Historical Aspect Discovery: 17 TH century by ANTONJ VAN LEEUWENHOEK Presence of iron in blood: LEMERY in 17 TH century Functional significance of RBC as oxygen transporter:FELIX HOPPE-SEYLER IN 1877 Late 20 TH century: Mechanism of oxygen, carbon dioxide & nitrous oxide exchange Lipid bilayer hypothesis: first proposed in 1925 and refined by Danielli and Davson in 1935 Uniquely anuclear ,highly specialized of cells Lacks cytoplasmic organelles as nucleus, mitochondria, or ribosomes , hence unable to synthesize new protein, carry out the oxidative reactions associated with mitochondria, or undergo mitosis More than 95% of the cytoplasmic protein is hemoglobin ERYTHROCYTES

3. EM VIEW OF: A) RBC B) WBC C) PLATELET

4. At rest when suspended in isotonic solution: flattened, bilaterally indented sphere ( biconcave disc ) In fixed, stained blood smears: appears circular, with a diameter of about 7 to 8 μm and an area of central pallor corresponding to the indented regions disc shape is well suited to erythrocyte function; (surface to volume ratio maximum) thereby facilitating gas transfer and cascading in the microcirculation SHAPE & DIMENSION

5. DIMENSIONS Volume: 94 +/-14 fl

6. Changes its shape with size of the vessel & under shearing stress without remodelling Due to cytoplasm(very low viscosity) & plasma membrane(elasticity & viscocity ) Spherical: hypotonicity , discocyte-echinocyte transformation, discocyte-stomacyte transformation. DEFORMABILITY

7. DISCOCYTE-ECHINOCYTE TRANSFORMATION Decrease in IC ATP Increase in IC Calcium & pH Cell exposed to stored plasma Anionic detergents Lysolecithin /Fatty acid Washed cell when kept between glass slide & coverslip Stages of echinocyte transformation STAGE I STAGE II STAGE III STAGE IV IR REGULARLY CONTOURED DISC CRENATION OVER FLAT SURFACE SPICULES OVER SURFACE SPHERO-ECHINOCYTE

8. DISCOCYTE-STOMACYTE TRANSFORMATION Low pH Cationic detergent Stages of stomacyte transformation STAGE I STAGE II

9. STAGES OF SHAPE CHANGE TYPICAL OF PROLONGED STORAGE

10. 1935: Danielli & Davson gave lipid bilayer model MEMBRANE STRUCTURE

11. Two electron-dense ( osmophilic ) layers approximately 2.5 nm thick separated by an electron-penetrable layer about 2.0 nm thick, for a total thickness of some 7.0 nm. ULTRA STRUCTURE

12. Material following hemolysis due to membrane rupture: stroma & if membrane intact: cell ghost CHEMICAL COMPOSITION

13. Phospholipids Phosphatidylcholine (lecithin) Phosphatidylethanolamine ( cephalin ) Sphingomyelin Phosphatidylserine Lysolecithin Cholesterol Glycolipid Lipid components

14. Helps in regulating the SHAPE of RBC & mechanical membrane stability in case of shear stress Certain provide bilipid layer attachment to cytoskeleton ( spectrin – phosphatidylserine interaction) Mobility of lipids: Outer layer > inner layer sphingomyelin , fluidity Phospholipid composition: phosphatidylcholine (PC) and sphingomyelin in outer leaflet and phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in inner leaflet PHOSPHOLIDIPS

15. Loss of phospholipid asymmetry results in exposure of PS ( apoptopic marker) on the red cell surface promotes red cell removal from the circulation Phospholipid asymmetry is maintained by the ATP-dependent flippase ( aminophospholipid translocase ) activity which counteracts phospholipid scrambling in which PS moves from the inner to the outer cell surface Flippase activity decreases during storage, but can be corrected by rejuvenation of the red cells Phospholipid scrambling is normally low during storage, but can be enhanced by photodynamic treatment for pathogen inactivation

16. FATTY ACID COMPOSITION SATURATED Palmitic Stearic Others UNSATURATED Oleic Linoleic Arachidonic Others

17. CHOLESTEROL Increases in membrane cholesterol content decrease membrane deformability Abnormally high levels of cholesterol lead to distortions in red cell shape; bizarre spicules form (“spur cells”), deformability of the cells is reduced, and they are destroyed in the spleen.

18. GLYCOLIPIDS Ceramide glycolipids : with one glucose(GL-1: 5%), one glucose & one galactose (GL-2: 14%), and one glucose & two galactose (GL-3: 12%), one glucose & 3 galactose (GL-4: 69%) molecules attached Fucose -containing ceramide glycolipids , in trace amounts: surface antigenic activity corresponding to the A, B, H, and Lewis blood groups

19. Solubilization of membrane proteins done by adding detergents to cell ghost Separation & analysis: high resolution by means of electrophoresis in polyacrylamide gel Main proteins are: Peripheral:- SPECTRIN, ANKYRIN, PROTEIN KINASE, ACTIN & G-3PD Integral:- GLYCOPHORINS ( Sialoglycoproteins ), ANION CHANNELS & GLUCOSE TRANSPORTERS Surface glycoprotein CD47 inhibits phagocytosis (decreased in old RBCs & during storage) PROTEIN COMPOSITION

20. TRANSMEMBRANE PROTEINS Two prominent ones-: Glycophorin A (GPA) and Anion channels Sialic acid residues attached to glycophorins : 60% negative charge of membrane; GPA also bears blood group antigens & also binding site for many pathogens AE1(Anion Exchange Protein)/ Band3 : traverses the membrane 12 times; the extracellular domain is highly glycosylated - bears CHO blood group antigens & ABO system antigens ; Within the membrane, AE1 exists predominantly as a dimer ;its function appears to be Cl–HCO 3 exchange AE1 also interacts with the erythroid cytoskeleton by binding ankyrin and binds NO , possibly facilitating its transit across the erythrocyte plasma membrane Role of band 3 in anion and CO 2 transport

21. Rh PROTIENS only 100,000 copies per cell RhD protein is the most immunodominant determinant of red cells outside the ABO antigens. Complete absence results in multiple erythrocyte defects and mild hemolytic anemia . The proteins that carry the D, C (or c), and E (or e) (Rh antigens are highly homologous to one another) traverse the membrane multiple times Minor role in CO 2 transport Interaction between integral proteins & cytoskeletal proteins

22. CYTOSKELETAL PROTEINS most abundant of the peripheral proteins: spectrin-actin cytoskeletal complex The complex includes large α and β spectrin polypeptide chains and the smaller actin chain It preserve erythrocyte integrity in the face of the shear stresses of the circulatory system and spleen Protein 4.1 : promote spectrin-actin interaction Protein 2.1/ Ankyrin : serves as a mode of attachment of the cytoskeleton to the membrane Other proteins include protein (band) 4.9, tropomyosin , tropomodulin , and adducin : play vital roles in formation and stability of the cytoskeleton

23. In Stored blood : spectrin oxidation occur with time, leading to loss of membrane surface area by formation of lipid vesicles comprising membrane detachment from cytoskeleton. Other cytoskeletal proteins may also be affected by oxidative processes that alter their ability to interact with members of the spectrin-actin meshwork underpinning the erythroid membrane.

24. MEMBRANE TRANSPORT PROTEINS Nonpolar substances diffuse through the membrane Polar solutes cross the membrane at specialized transport proteins, including the anion transporter (AE1/ band 3), glucose transporter, urea transporter,Ca 2+ -ATPase, Na- K- ATPase , the GSSG (oxidized glutathione) transport ATPases , Amino Acid transporters and a water channel aquaporin-1 ( 85% of the osmotic water permeability)

25. ION EXCHANGE Rapid exchange : mediated by the band 3 anion-exchange protein and plays an important role in the chloride–bicarbonate exchanges that occur as the red cell moves between the lungs and tissues Slower ionic diffusion : accounting for net loss or gain of anions d isomers of glucose, mannose, galactose , xylose & arabinose are transported easily whereas fructose is not transported under physiological conditions

26. Glucose enters the erythrocyte by facilitated diffusion , mediated by a transmembrane protein GLUT1 (5% of erythrocyte membrane protein) Potassium is the predominant cation and sodium is a relatively minor constituent, whereas the relationship is reversed in plasma; preservation of these gradients is the result of the cation transport process(passive diffusion & active transport) Active Na + and K + transport depends on the activity of the membrane protein Na-K ATPase 3Na i + + 2K o + + ATP i → 3Na o + + 2K i + + ADP i + P i Urea transporter : transports urea rapidly across the membrane and helps preserve red cell osmotic stability and deformability

27. The Na-K- ATPase is highly sensitive to changes in temperature and scarcely functions at 4ºC During cold storage , Na diffuses into the cells and K leaks out until a new equilibrium is reached The leakage of K is further increased by irradiation Increased K content in plasma or in the additive solution of stored RBC units presents a potential hazard to neonates Citrate in plasma increases K toxicity Reducing RBC supernatant volume results in less K leakage before equilibrium is reached

28. MEMBRANE ASSOCIATED ENZYMES Externally Oriented : Hydrolytic enzymes ( glycosidases and acid phosphatases ), AChE Membrane Bound : Production of ATP – 3 enzymes:- aldolase , glyceraldehyde 3-phosphate dehydrogenase (G3PD), and phosphoglycerate kinase These three enzymes convert fructose diphosphate to 3-phosphoglycerate with the production of ATP Use of ATP : protein kinases , and adenosine triphosphatases ( ATPases ) ATP cAMP Protein kinases : phosphorylated structural proteins have lower-affinity for their target proteins Eg : dephosphorylation due of ATP depletion, stress develops; more rigid spectrin-actin network and reduced membrane deformability ATPases (Na-K ATPase , Ca-Mg ATPase , and Mg ATPase ) : Na-K ATPase maintains the high internal K and low internal Na concentration ADENYLYL CYCLASE

29. ERYTHROCYTE METABOLISM

30. GLUCOSE HEXOKINASE ATP ADP GLU-6-PHOSPHATE FRUCTOSE-6-PHOSPHATE PHOSPHOGLUCOSE ISOMERASE FRU 1,6 BIS PHOSPHATE PHOSPHO FRUCTO KINASE ATP ADP ALDOLASE DIHYDROXY ACETONE-P GLYCERALDEHYDE 3 P TPI NAD+ NADH G-3 P DEHYDROGENASE 1,3 BISPHOSPHO GLYCERATE GLYCOLYTIC PATHWAY

31. 1,3 BISPHOSPHO GLYCERATE 3- PHOSPHO GLYCERATE 2- PHOSPHO GLYCERATE 2- PHOSPHOENOL PYRUVATE PYRUVATE LACTATE PHOSPHOGLYCEROKINASE ADP ATP MONO PHOSPHOGLYCERATE MUTASE ENOLASE PYRUVATE KINASE ADP ATP LACTATE DEHYDROGENASE NAD+ NADH

32. GLUCOSE GLU-6-PHOSPHATE FRUCTOSE-6-PHOSPHATE FRU 1,6 BIS PHOSPHATE DIHYDROXY ACETONE-P 1,3 BISPHOSPHO GLYCERATE PENTOSE PATHWAY/SHUNT GLYCERALDEHYDE 3 P 6-PHOSPHOGLUCONATE G-6-P DEHYDROGENASE NADP+ NADPH RIBULOSE-5-P NADP+ NADPH 6PG DEHYDROGENASE

33. PENTOSE PATHWAY Source of NADPH NADPH maintains glutathione (GSH) in its reduced form (eliminate peroxide, protection of protein sulfhydryl groups and detoxification processes) Ribulose-5-phosphate needed for production of phosphoribosyl pyrophosphate ( PRPP )essential for the synthesis of adenine nucleotides required for ATP synthesis Pentose pathway accelerated when NADPH is oxidized to NADP (oxidative stress) and as well as when Hb in R state(O 2 bound)

34. 1,3 BISPHOSPHO GLYCERATE 3- PG 2- PG 2- P E P PYRUVATE 2,3 DPG SHUNT OR RAPOPORT-LUEBERING SHUNT DIPHOSPHOGLYCERATE MUTASE 2,3 BISPHOSPHO GLYCERATE DIPHOSPHOGLYCERATE PHOSPHATASE phosphate

35. Production of large quantities of 2,3-DPG is a unique feature of glycolysis in the red cell This is to stabilise low affinity state of Hb ( T state ) Both reactions are catalyzed by the same enzyme and are balanced at physiologic pH . At higher pH , the enzyme acts only as a mutase At low pH , the enzyme acts as phosphatase 2,3-DPG is made at the expense of ATP (shunt bypasses 1 ATP-making steps) In storage systems, a high pH can shut down ATP production , while a lower pH leads to a burst of ATP production driven by breakdown of 2,3-DPG.

36. 2,3 DPG Storage source of phosphate Membrane shape and deformability are controlled by the ATP-driven cytoskeleton Free 2,3-DPG increases cell flexibility by weakening the links between the membrane and the cytoskeleton, and facilitates gas exchange by allowing the red cell to slip into narrow capillaries and splenic sinusoids Because some capillaries in the microcirculation have a diameter of only half that of a RBC, loss of flexibility and deformability is a serious storage lesion responsible for removal of rigid cells

37. 2,3 DPG + Hb T state of Hb R state of Hb Hb FREE 2,3 DPG BAND 3 PROTEIN 4.1 PROTEIN 4.2 ANKYRIN

38. GLYCOPHORIN C PROTEIN 4.1 ACTIN SPECTRIN

39. ALTERNATE SUBSTRATES FOR RBC METABOLISM RBCs are capable of metabolizing other sugars like fructose, mannose, galactose , and the three-carbon sugar dihydroxyacetone but none proven to be useful in the design of blood preservatives. Nucleosides: such as inosine to support ATP synthesis by action of nucleoside phosphorylase Inosine + Pi → Ribose-1-P + Hypoxanthine R-1-P (without ATP expenditure) is then readily converted to F-6-P by the pentose shunt:- generation of ATP through Glycolysis

40. REGULATION OF GLYCOLYSIS I) Feedback mechanisms Negative feedback mechanisms between glycolytic pathway and 2,3-DPG & ATP II) pH HK and PFK inhibited by hydrogen ions Accumulation of lactic acid & acidity of first-generation additive solutions in blood storage slows glycolysis III) Pentose Shunt Activity : facilitatory effect

41. IV) States of Hb N-terminal cytoplasmic domain of Band 3 binds Hb , cytoskeletal proteins, and glycolytic enzymes Hb in T state & Band 3 interaction causes release of glycolytic enzymes Glycolysis Hb in R state pentose shunt (enzymes activity reduced in bound state with Band 3)

42. ADENOSINE NUCLEOTIDES Equilibrium exist between ATP , ADP & AMP As ADP increases, some is converted to AMP& it inturn is deaminated in the AMP- deaminase reaction Total adenine pool decreases during storage leading to depletion of ATP (so adenine is added to anticoagulant and/or the additive solution ) Adenine in storage system enters the cells & purine nucleotides are synthesized through adenine phospho - ribosyl transferase reaction Adenine + PRPP → AMP + PP PRPP synthesised from pentose pathway : for AMP production

43. GUANINE NUCLEOTIDES Guanine nucleotides : formed by action of hypoxanthine-guanine phosphoribosyl transferase . Main functions: 1 ) signal transduction of membrane shear into secretion of local vasodilators cyclic AMP and ATP 2 ) high concen . of GTP inhibit RBC trans – glutaminase (which has Factor XIII like activity that interacts with the cytoplasmic domain of band 3 and with protein 4.1) GTP concentration & transglutaminase is reduced when RBCs age, which facilitate the removal of old RBCs by binding them to fibrin clots

44. IN NUTSHELL……. metabolize GLUCOSE by the glycolytic pathway with its pentose and 2,3-DPG shunts ATP maintain ion and glucose concentration gradients between the plasma and erythrocyte secure red cell deformability For optimal dissociation of oxygen from Hb & as a phophate depot rise and fall of non- Hb -bound 2,3-DPG induces repetitive changes in the membrane-cytoskeleton architecture (implications for red cell flexibility and gas transport) 2,3DPG Adequate levels of ATP, NADH, NADPH , and 2,3-DPG for these metabolic functions are secured by the glycolytic pathway with its pentose and 2,3-DPG shunts

45. GASEOUS EXCHANGE OXYGEN TRANSPORT CO 2 TRANSPORT

46. Maintaining the Iron of Hb in a reduced state is a prerequisite for effective oxygen transport(by NADH) Fe 3 Fe 2 NADH NAD+ GSH is synthesised from precursor AA s & nucleotides through salvage pathway SH groups of Hb and membrane proteins is protected from oxidation by maintaining adequate amounts of reduced GSH by oxidation of NADPH to NADP GSH GSSG MetHb reductase PEROXIDASE H 2 O 2 H 2 Fe 3 Fe 2

47. Citrate dextrose : nutrient for red cells Acid-citrate-dextrose : Shelf life of 21 days; Acid pH(pH 5) does not help in maintaining 2,3-DPG levels Citrate-phosphate-dextrose : Alkaline pH and PO 4 ( phoshate ) help in maintaining 2,3-DPG & shelf life of 28 days Citrate-phosphate-dextrose-adenine (CPDA-1) : Improved ATP synthesis & longer shelf life (35 days) Citrate Phosphate Dextrose Adenine 2 (CPDA 2) : more glucose content than CPDA-1 DIFFERENT PRESERVATIVES & THEIR IMPACTS ON RBCs

48. Citrate phosphate Double Dextrose (CP2D) :100% more glucose than CPD and 60% more than CPDA-1;used with an additive solution ( AS3)which doesnot contain glucose Added nucleotides : I) ADENOSINE : Restores ATP II) INOSINE : generates ATP III) GUANOSINE : Used in PAGGS-M which provides 7 weeks of RBC storage with recovery of 74 %

49. RBC STORAGE LESIONS CHARACTERISTICS CHANGE % VIABLE CELLS GLUCOSE ATP pH 2,3 DPG LACTIC ACID PLASMA K + PLASMA Hb O 2 DISSOCIATION CURVE SHIFT TO LEFT

50. REFERENCE Rossi’s Principles of Transfusion Medicine, Fourth Edition Wintrobe’s Clinical Hematology 12th Edition

51. THANK YOU