biochemistry blood

BLOOD BIOCHEMISTRY THE ROLE OF IRON,FOLIC ACID, CYANOCOBALAMIN INTHE FORMATION OF HEMOGLOBINE & RBC BYLiniyanti.D.Oswari, M.D.; MNS.MScForMedical student,Sriwijaya UniversityBlock 8

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Chapt. 44 Biochemistry of ErythrocytesStudent Learning Outcomes:

Describe the structure/ function of blood cell types:

Erythrocytes, leukocytes, thrombocytes Explain the metabolism of the red blood cell Explain basics of hematopoiesis from bone marrow Describe some errors of hemoglobin function, anemias, hemoglobin switching Describe the structure/ function of blood group antigens

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Learning ObjectiveUnderstand of the HematopoesisUnderstand the Metabolism of folic acid

Cyanocobalamine in Erythropoesis.Understand the etiology and the

management of Anemia.

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Blood - FunctionsRespiratory

Transport O2 from lungs to tissuesTransport CO2 from tissues to lungsNutrition

Transport food from gut to tissues (cells)Excretory

Transport waste from tissues to kidney (urea, uric acid, water)Regulatory

Water Content of TissuesWater exchanged through vessel walls to tissue (interstitial fluid)Body Temperature

Water- high heat capacity, thermal conductivity, heat of vaporizationTypical heat generation is 3000 kcal/dayProtective

Antibodies, antitoxins, white blood cells (WBC)

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Blood CompositionBlood composition

5-6 L in an adult70 mL/kg of body weightSuspension of cells in a carrier fluid (plasma)Cells - 45% by volumePlasma - 55% by volumeCells

Red cells (erythrocytes)5x106/mLWhite cells (leukocytes)7x103/mLPlatelets (thrombocytes)3x105/mL

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Blood composition Suspension of cells in plasma (carrier fluid)

45% Cells 55% Plasma Cells

Red cells (erythrocytes) 99%5x106/mLWhite cells (leukocytes)7x103/mL < 1% Platelets (thrombocytes)3x105/mL

Cells of blood

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Blood composition

Composition of BloodThe three main cellular elements in blood are:

1. Erythrocytes: are formed in bone marroware very specialized cells whose main function is to carry O2 to cells and CO2 away from themhave a half-life of about 60-120 days are removed by the liver and spleen and destroyed2. Leukocytes (white blood cells)are formed in the bone marrowmost of the different leukocytes destroy invading bacteria or other foreign substances by phagocytosis

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Normal BloodReticulocyte

Normal Blood

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Blood cellsTable 1 Blood cells (cells/mm3):Erythrocytes5.2 x 106 men carry oxygen 4.6 x 106 womenNeutrophils4300 granules; phagocytic, O2 burst killsLymphocytes2700 immune response, B- and T-cells, NKMonocytes 500 macrophages for bacteria, damageEosinophils 230 granules destroy parasites (worms)Basophils 40 granules hypersensitivity, allergic

histamine, proteases,

Composition of Blood, cont3. Platelets or thrombocytesare formed in the bone marrow and spleencontrol bleeding when there is a cut or injuryPlasma: is 90% water

the fluid remaining after all cellular elements have been removed from whole blood by centrifugationthe dissolved solids are mainly proteins (7%)the remaining 1% contains glucose, lipids, enzymes, vitamins, hormones, and waste products such as urea and CO2

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Composition of Bloodif plasma is allowed to stand, it forms a clot, a gel-like substanceserum: the clear liquid that can be extracted from blood plasmaserum contains all the components of plasma but lacks fibrinogen that makes blood clotMale versus female

Hematocrit (% volume that is red cells)40-50% in males35-45% in females

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Blood Components:Plasma Transports Solutes

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Blood PlasmaStraw colored clear liquid Contains 90% water7% plasma proteinscreated in liverconfined to bloodstreamAlbuminmaintain blood osmotic pressureImmunoglobulinsantibodies bind to foreignsubstances called antigensform antigen-antibody complexesFibrinogenfor clotting 2% other substances Nutrients, electrolytes, gases, hormones, waste products

HematocritsNormal, Hemorrhage, IDA, Leukemia, Hemolysis, B12, P VeraPlasmaWhite cellsRed cells

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Plasma ProteinMore than 200Most abundant

Albumin - 4-5 g/100 mLg-globulins - ~1 g/100 mLfibrinogen - 0.2-0.4g/100 mLOriginal classification by zone electrophoresis at pH 8.6Separation by pI with several molecular weight species within each group

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Blood Components: "Blood Count" % of Each Component

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RBC - Reversible Shape ChangesSurfactants result in cells becoming more sphericalMechanical stress - deformation in capillaries to allow for passage of cellsDisease eg. Sickle Cell AnemiaHemolysis - release of Hb from the cell

Osmotic swellingSurface collisions with artificial organs

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White Blood Cells (Leukocytes)Total count - approximately 7000/mLVarious types

Neutrophils 62% (50-70%)Eosinophils 2.3% (2-4%)Basophils 0.4% (0-1%)Monocytes 5.3% (2-8%)Lymphocytes 30% (25-40%)Plasma cells (mainly in the lymph)Monocytes in tissue become macrophages

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Functions of eosinophilsSecrete lethal substances at the time of exposure to foreign proteins/parasites

1. Function protection from microorganisms, allergic reactions

2. Eosinophill peroxidase detroy worms, bacteria and tumor cells.

3. Eosinophill cationic protein (ECP)- destroys helminths.

4. Eosinophill derived neurotoxin destroys nerve fibres (myelinated nerve fibres)

Functions of basophilsFunction allergic reactions, blood clottingEnergy mainly from oxidative phosphorylationBasophill granules release some important substances like

Histamine Acute hypersensitivity reaction- vascular changes, increase capillary permeability

Heparin prevents intravascular blood clotting

Hyaluronic acid necessary for deposition of ground substances in basement membrane

Proteases exaggerate inflammation

Basophill have IgE receptor hypersensitivity reaction

Functions of neutrophilsFirst line of defence against invading micro-organisms.

2. Main source of energy: Glucose in glycolysis (small amount of mitochondria)Hundreds of granules (contain hydrolyses, peroxidases, phosphatases, lysocim....)3. Powerful and effective killer machine contains enzymes like protease, elastase, metalloproteinase, NADPH oxidase; antibody like substances called defensins. Defensins antimicrobial peptides active against bacteria and fungi.

4.Secrete Platelet Aggregation Factor (PAF) accelerates the aggregation of platelet during injury to the blood vessels

Function

Defense against foreign invadersbacteriavirusesforeign materials (including biomaterials)Phagocytosis

Neutrophils, macrophagesMove to foreign particle by chemtaxisChemicals induce migrationToxins, products of inflamed tissues, complement reaction products, blot clotting productsResponse is extremely rapid (approx 1 h)

*Macrophages - much larger particles

Lymphocytes

B cells - responsible for humoral immunityT cells - responsible for cell mediated immunityB cells responsible for production of antibodies

Receptor matches antigenCells multiplyAntibodiesAbs are just immunoglobulins discussed earlier

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T cells

Cytotoxic T cells (Killer T cells)Bind to cytotoxic cells (eg infected by virus)SwellRelease toxins into cytoplasmHelper T cellsMost numerousActivate B cells, killer T cellsStimulate activity by secretion of IL2Stimulate macrophagesSuppressor T cellsRegulate activities of other cell types

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AIDSHIV - attacks many cell types

epithelial cellsmacrophagesneuronslymphocytes (helper T)Infected helper T cells when stimulated, produces viral proteins which kill the cellHelper T cell population disappears

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MonocytesFunction phagocytosis, exit into tissues tissue macrophages

4-8 % of all leucocytesAre accumulated in the place of inflammation

A lot of lysosomal hydrolases

Aerobic pathway of energy obtaining prevails

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Functions of PlateletsBlood clotting

Clot retraction

Defence mechanism

Homeostasis

Repair and rupture of blood vessel

PlateletsNon-nucleated disk shaped cells3-4 mm diameterVolume 10 x 10-9 mm3250 000 cells/mL10 day circulation timeSurface contains membrane bound receptors (GP Ib and IIb/IIIa)

mediate surface adhesion reactions, aggregation reactionsinteract with coagulation proteins

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Blood Components: PlateletsFigure 16-10c: Megakaryocytes and platelets Coagulate, form plug, prevent blood lossFormed by fragmentation from megakaryoctyes

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Contain muscle proteins actin and myosin which contract when platelet is activatedAlso a granules, dense granules, lysosomal granulesPlatelets activated by minimal stimulation

Become stickyShape changeRelease of cell contentsStimulate other plateletsFunction

Initially arrest bleeding through formation of platelet plugsStabilize platelet plugs by catalyzing coagulation reactions leading to formation of fibrin

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Overview of Hemostasis: Clot Formation & Vessel RepairFigure 16-11: Overview of hemostasis and tissue repair

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Hemostasis: Vasoconstriction & Plug Formation

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Platelet Adhesion

Site of injury - exposure of connective tissue elements (eg collagen)Artificial surfaces through forming thrombi (clots)Platelet Aggregation

Caused by ADP, collagen, thrombin, epinephrine, PAF, TXA2Release of cell contents

Induced by ADP, collagen, thrombin, TXA2 and epinephrine

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*Platelet function Adhesion to endothelial cells is mediated by glycoprotein Ib which binds vWF on endothelial cells. Aggregation is mediated by GPIIb/IIIa on another platelet by fibrinogen. Various agonists ADP, PAF, are shown as interacting with specific receptors and activating phospholipase C. Coagulant activity is generated by coagulation factors on the platelet external membrane to convert prothrombin (II) to thrombin (IIa).

CoagulationMaintenance of hemostasis (prevention of blood loss)At least 12 plasma proteins interact in series of reactionsCascade of reactionsInactive factors become enzymatically active following surface contact, proteolytic cleavage by other enzymesAmplification is rapidReactions are localized

*Reactions are amplified quickly because one enzyme can activate a number of moleculesLocalized - diluted due to blood flow, actions of inhibitors, reactions only occur at a sufficient rate when at surface of activated platelets or at sites in injury

Coagulation Cascade *

Zone Electrophoresis of Plasma Proteins

-+

pI6.05.65.14.7globulinsalbumingba1a2

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Functions of Plasma ProteinsMaintenance of:

Colloid osmotic pressure(COP) (p)pHelectrolyte balanceCOP relates to blood volume

ProteinsolnWaterDP = p

*pH maintained by buffering action of proteins

If membrane present p importantIsotonic - same osmotic pressureHuman blood - 300 milliOsmoles /LNormal saline - 0.9% NaCl by weight

0.15 mol/L0.30 mol/L of particlesAt physiological temperature, two solutions differing in pressure by 1 mOsm have an osmotic pressure of 19.3 mm Hg between them.

*Driving force developed by difference in water concentration between solution in question and pure water. Can be measured by apparatus similar to that on the previous slide.

Solutions with same concentration of solute particles will have same osmotic pressure even if solute particles are different.Solution with higher concentration of solute particles is hyperosmoticSolution with lower concentration of solute particles is hyposmotic

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Colloid - large particle that cannot easily cross a membrane

Stays in the compartmentIn blood pprotein = 20-30 mmHgTotal ~ 5000 mmHgProtein stays in the blood as p is maintained in the bloodWater content is therefore maintained

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Hypotonic - lower p than normal

Hemolysis of RBC

Hypertonic1.5% NaClHbH2OGhost Cells

Crenated CellsHypertonic higher p than normalCreation of cells

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Functions of Plasma Proteins (contd)Transport of ions, fatty acids, steroids, hormones etc.

Albumin (fatty acids), ceruloplasmin (Cu2+), transferrin (Fe), lipoproteins (LDL, HDL)Nutritional source of amino acids for tissuesHemostasis (coagulation proteins)Prevention of thrombosis (anticoagulant proteins)Defense against infection (antibodies, complement proteins)

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Function and Properties of Selected Plasma ProteinsConsider three abundant plasma proteins

Structure, function

Coagulation, fibrinolysis, complement

*Objective - illustrate previous discussion, lead in to coagulation, fibrinolysis, complement

State of knowledge is currently very sophisticated- many proteins purified, MW, amino acid sequence, 2y, 3y, 4y structure determined

Consider three abundant plasma proteins

Recall - analogous to synthetic polymers - macromolecules but not polymers perse

MWD - monodisperseShape in solution is fixed - not random coil as other polymers

Some trace proteins exist whose biological function has not yet been determined

AlbuminMW 66 000Single chain, 580 amino acids, sequence is knownDimensions - Heart shaped molecule50% a helix [He and Carter, Nature, 358 209 (1992)]Modeled as:

30 80

*Dimensions - 3yMW - 1y50% a helix - 2y

Any quaternary structure? - only one chain

Synthesis

Mainly liver cells then exportedAssembly time on ribosome ~ 1-2 mint0.5 in circulation - 19 days14 g lost per day0.4 mg synthesized per hour per g of liverNeed liver of approximately 1.5 kg in weight to maintain

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Functions

Colloid osmotic pressure of blood is 80% due to albuminrelatively low molecular weightregulates water distributionTransport of fatty acids Liver to tissues, bindingSource of amino acids for tissue cells (pinocytosis)60% albumin in tissue (interstitial) fluid

*Recall from high school biologypinocytosis is engulfment of liquid matter by the cell membranephago (eat) involves solid

g-Globulins20% of plasma proteinsg refers to electrophoretic mobilityRepresents a group of proteins of variable structure

immunoglobulinsMain functional task is immunochemical

Antibodies - combine with specific antigens

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Basic 4 chain structural unit

MW = 2x55000 +2x27000 = 160000

*Constant - same amino acid sequence for all of the moleculesVariable - varies between molecules- results in specificity of moleculesTwo heavy and two light chains joined by disulfide bonds. Variable region - red - binds the antigen Constant region (black) can activate complement pathway or attach the Ab to cells such as macrophages

Variable region varies with respect to primary, secondary and tertiary structuresBasis of specificity of antigen binding (106 average number)5 classes of immunoglobulins

IgG, IgA, IgM, IgD, IgEDifferent structures of constant regions of heavy chainsSome are polymers (multiples of 4 chain unit - IgA - dimer - MW 350 000, IgM - pentamer - MW 900 000See any immunology book for more details

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*The basic antibody unit is a monomer, IgA is a dimer and IgM is a pentamer

Classes of ImmunoglobulinsIgG Identifies microorganisms for engulfment or lysisIgE Inhibits parasite invasion; involved in allergic reactionsIgD UnknownIgA Basis for passive immunity provided by breast milk, agglutinates infectious agents in secretions outside the body, present in tears, mucousIgM Identifies microorganisms for engulfment or lysis

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Functions

Primary function is antigen binding (immune response)Secondary function is complement binding (after antigen)Synthesis

In lymphocytes (T and B)Made in response to presence of antigen (foreign macromolecule, virus particle etc.)

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FibrinogenCoagulationStructure

MW 340 000Sequence of amino acids is known (3000)4y, 3y structure6 polypeptide chains, 2a (67,000), 2b (56,000), 2g (47,000)

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abgdisulfideTriple dumbell model (EM)DDE90 450 as, bs and gs are intertwined

*N terminals in E and C terminals in Da chain in D is more like a random coil synthetic polymer

Clotting - thrombin breaks off FPA and FPB

Function

Blood coagulation (clotting)Plasmin is end product of fibrinolytic systemClot needs to be removedNot needed foreverCould embolize to lungs, brain

*Coagulation - very important consideration when processing blood outside of the bodyStructure must incorporate the ability to be easily broken down after clotting

What is a clot and how does it form from the polymerization of fibrinogen?

Overhead from Brashs notes next

Importance of Protein Structure - Sickle Cell AnemiaOccurs because of a minor variation in one amino acid in the b chain of HbResults in Hb that, when exposed to low O2 concentrations precipitates into long crystalsElongate cellDamage cell membraneDecrease in amount of RBC

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Cellular Elements of BloodRed cells

40 - 50% of blood volume5 x 106 cells /mLbag of hemoglobinnon-nucleatedno proliferationcell membrane in excess so that deformation does not ruptureShapeBiconcave disc8 mm in diameter, 2.7 mm thick, volume ~ 90 mm3, area ~ 160 mm2

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Scanning Electron Micrograph of Red Blood Cells

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Why this shape?

Area to volume ratio is high (maximal?)Facilitates diffusion of O2 and CO2minimal distance of contents from surfaceOriginates in bone marrow (hematopoiesis)

Molecular explanation based on the properties of the proteins in the cell membrane is found in Elgsaeter et al. Science, 234, 1217 (1986)

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Oxygen Binding of HbBlood must carry 600 L of O2 from lungs to tissues each day

Very little carried in plasma since O2 only sparingly solubleNearly all bound and transported by Hb of RBCPossible for Hb to carry four O2 molecules, one on each a chain, one on each b chain

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O2 depleted Hb solution placed in contact with O2(g)Equilibrium reactionFraction (s) of Hb converted to oxyhemoglobin

*Measure of % saturation of hemoglobin as a function of O2 partial pressureDepends on temperature, pH (CO2 content) of blood - we will come back to thisNB in the lungs PO2 is approximately 100 mm Hgin the tissues PO2 is approximately 45 mm Hg

In the lungs we want HbO2In the tissues we want Hb (O2 delivered to the tissues)

Hb cycles between 95% saturated in the lungs, 65% saturated in the tissuesMust also consider CO2 - high pH in the lungs and low pH in the tissues

Binding of O2 to 4 heme sites given by:

Equilibrium constants for different reactions differentBinding of first O2 relatively low affinity2nd, 3rd and 4th - much higher affinityCooperative effect

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Compare with binding curve for myoglobin

*Oxygen binding curves of Mb and Hb. Mb has a much greater affinity for O2 than Hb. It is 50% saturated at partial pressures of 0.15 - 0.30 kPa whereas Hb requires a pO2 of about 3.5 kPa for 50% saturation. Note that both are 95% saturated at the pO2 of arterial blood leaving the lungs (13 kPa) Sigmoid O2 saturation is an adaptation for transport function of RBC assuring binding and release of O2 in appropriate tissues.

Myoglobin - oxygen reaction

At equilibrium

*Simple hyperbolic curve - monomer with no positive cooperativity

Acid Effect - O2 DissociationO2 binding causes release of H+pH decreases, [H+] increases then the equilibrium moves to left% saturation decreases, more dissociation for a given pO2Tissues are at a lower pH than the lungs due to CO2 which facilitates release of O2 to tissues

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Hb versus MbHb carry O2 to tissues where it is released

Releases quickly in tissues where pO2 is lowerMb store O2 in the muscle, make available to cells

Releases very little in tissuesReference: Science 255 54 (1992)

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Extrinsic system

Blood comes in contact with traumatized vascular wall or extravascular tissuesIntrinsic system

Initiated by surface contact (often negatively charged surface)Most reactions are Ca++ dependentChelaters of Ca++ effective anticoagulants

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*Contact activation - the initial event is the adsorption of factor XII to a negatively charged surface where it undergoes a conformational change to expose its active site. Factor XII converts prekallikrein to kallikrein. Additional factor XIIa and kallikrein are then generated by reciprocal activation. Factor XIIa also activates factor XI. Both kallikrein and factor XI bind to a cofactor, HMWK which serves to anchor them to the charged surface.

FibrinolysisResults in dissolution of fibrin clot

Conversion of plasminogen to plasminPlasminogen activators synthesized by and released from endothelial cellsTPA - tissue plasminogen activator

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Erythrocyte metabolismErythrocyte metabolism: Only glycolysisATP for Na+/K+, Ca2+HMP shunt makes NADPH G6PD is 1st enzyme Lifetime rbc by G6PD activity2,3-BPG modulates O2 bindingNeed Fe2+ Hb bind O2; If ROS made Fe3+, NADH can reduce

Fig. 1

HematopoiesisFig. 15Hematopoiesis:Stem cells in bone marrow (1/105)Proliferate, differentiate, mature by growth factors, hormones signal transduction pathsMyeloid, lymphoid linesLeukemias: immature cells keep proliferating; defined by cell type

Hematopoiesis Factors affecting erythropoiesis:-A)-Oxygen supply of tissues:Decreased oxygen supply (hypoxia) to tissues stimulates secretion of erythropoietin (EP) hormone.Hypoxia stimulates kidney to release renal erythropoietic factor (REF).Hypoxia stimulates liver to produce a special type of globulin.Both REF & globulin unite in plasma and form EP.EP then stimulates bone marrow to produce RBCs.Erythropoietin accelerates nearly all stages of RBCs formation, i.e. it stimulates proliferation & differentiation of progenitor stem cells to produce mature RBCs.

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HematopoiesisFactors affecting erythropoiesis:-B) Dietary factors:i-Proteins: Proteins of high biological value are needed in the formation of RBCs.ii-Metal ions:Iron Fe: is essential for RBCs formation because it enters in the formation of the hem part.Copper Cu: It is carried & transported by plasma protein ceruloplasmin. It catalyses the oxidation of Fe++ to Fe+++, a reaction that must occur before transferrin can combine and transport iron.Cobalt Co: It stimulates EP release from kidney. So, excess Co may produce polycythaemia.

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Factors affecting erythropoiesis:-B) Dietary factors:iii-Vitamins:Both vitamins B12 & folic acid are essential for final maturation of RBCs because they are needed in DNA synthesis.Deficiency of either B12 or folic acid results in failure of nuclear maturation and causing maturation failure anemia.Vitamin C is a strong reducing agent which is important in reducing the ferric form of iron to ferrous to facilitate its absorption and transport.

Hematopoiesis

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Factors affecting erythropoiesis:-C) Hormonal factors:i-Androgens: increase erythropoiesis by stimulating the production of erythropoietin from kidney.ii-Thyroid hormones: Stimulate the metabolism of all body cells including the bone marrow cells, thus, increasing erythropoiesis.Hypothyroidism is associated with anemia while hyperthyroidism is associated with polycythaemia.Hematopoiesis

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HematopoiesisFactors affecting erythropoiesis:-C) Hormonal factors:iii-Glucocorticoids: Stimulate the general metabolism and also stimulate the bone marrow to produce more RBCs. In Addisons disease (hypofunction of adrenal cortex) anemia present, while in Cushings disease (hyperfunction of adrenal cortex) polycythaemia present.

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Factors affecting erythropoiesis:-C) Hormonal factors:iv-Pituitary gland: Affects erythropoiesis both directly and indirectly through the action of several hormones.v- Haematopoietic growth factors: Are secreted by lymphocytes, monocytes & macrophages to regulate the proliferation and differentiation of proginator stem cells to produce blood cells.Hematopoiesis

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Factors affecting erythropoiesis:-D)-State of liver & bone marrow:i-Liver: Healthy liver is essential for normal erythropoiesis because the liver is the main site for storage of vitamin B12 , folic acid, iron & copper. In chronic liver disease anemia occurs.ii-Bone marrow: When bone marrow is destroyed by ionizing irradiation or drugs, aplastic anemia occurs.Hematopoiesis

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AnemiaAnemia means a decrease in hemoglobin content,or RBCs count, or both of them below the normal range.Anemia leads to a decrease in blood ability to transport oxygen to tissue cells.

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AnemiaAnemias: hemoglobin concentration is low:Normal Hb g/dL: men 13.5-17.5; women 11.5-15.5

Anemias classified by red blood cell morphology:

Rbc morphology functional deficitpossible causeMicrocytic, impaired Hb thalassemia, lead, hypochromic synthesis iron deficiency

Macrocytic impaired DNA vit B12 or folic acidnormochromic synthesis deficient, erythroleukemia

Normocyticred cell lossacute bleeding, normochromic sickle cell defects

Types & causes of anemia:

I-Blood loss anemia:A-Acute blood loss anemia:Due to severe hemorrhage.Plasma volume is replaced rapidly by the fluids present in tissue spaces.This leads to marked dilution of the blood. RBCs are replaced within 2-3 weeks.Sufficient iron gives normocytic cells but insufficient iron will produce microcytic RBCs.Anemia

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Types & causes of anemia:

I-Blood loss anemia:B-Chronic blood loss anemia:Due to repeated loss of small amounts of blood over a long period e.g.:-Gastrointestinal bleeding (peptic ulcer)-Excessive menstruation.-Hemorrhagic diseases.Due to depletion in iron stores the newly formed RBCS are microcytic.Anemia

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Types & causes of anemia:II-Aplastic anemia: It results from destructione of bone marrow.It may result from: 1-Excessive exposure to x-rays or gamma rays.2-Chemical toxins e.g. cancer therapy & prolonged exposure to insecticides or benzene. 3-Invasion of bone marrow by cancer cells.4-Following infection by hepatitis. Damaged bone marrow dont produce any RBCs, so in aplastic anemia RBCS are normocytic. It is associated with decrease in WBCs & platelets.Anemia

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AnemiaTypes & causes of anemia:III-Hemolytic anemia:It results from increased rate of destruction of RBCs inside the cardiovascular system.Causes of hemolytic anemia:A-Hereditary:1-Membrane abnormalities.2-Enzyme deficiency e.g. G-6-P Dehydrogenase.3-Hemoglobin abnormalities.B-Acquired:1-Incompatible blood transfusion.2-Parasitic infection e.g. malaria.3-Toxic agents e.g. snake venom & insect poisons.4-Thermal e.g. several burns.

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Bone Marrow (BM) BiopsyNormalAplastic

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AnemiaTypes & causes of anemia:IV-Dyshemopoietic anemia: Which may be due to:1-Iron deficiency anemia.2-Maturation failure (megaloblastic) anemia:-a-Vitamin B12 deficiency.b-Folic acid deficiency.3-Anemia of endocrine disorders.4-Nutritional anemia.5-Anemia of renal failure.

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Classification of AnemiaBased on cell size (MCV)Macrocytic (large) MCV 100+ fl (femtoliters)Normocytic (normal) MCV 80-99 flMicrocytic (small) MCV

MEGALOBLASTIC (Macrocytic) ANEMIA

High MCVHigh MCHNormal MCHC

Macrocytic AnemiaMegaloblastic : defective DNA synthesisNon-megaloblastic : numerous mechanisms

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Nutritional Requirements forHematopoiesis

Metals : iron copper cobaltB12 and FolateOther vitamins: B6, A, E, CRiboflavin, Niacin

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Megaloblastic AnemiasA form of anemia characterized by the presence of large, immature, abnormal red blood cell progenitors in the bone marrow95% of cases are attributable to folic acid or vitamin B12 deficiency

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corin nucleusbenzylimidazolecobalt coordinated

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Vitamin B12Source : food of animal origin

- liver- muscle- eggs- cheese and milk- Not in plants- Made by bacteria

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B12 Absorption

1. Release from food sources gastric proteases and acids2. Binding by salivary cobalophilins3. Digestion of cobalophilin-B12

complex by pancreatic enzymes4. Binding to intrinsic factor (IF)IF is secreted by gastric parietal cells5. Attachment of B12-IF to receptors6. Endocytosis and binding to

transcobalamin II

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B12 Dependant Reactions

1. Synthesis of methionine fromhomocysteine requires : B12 and folate

2. Synthesis of succinyl CoA frommethyonyl CoA requires :methylmalonyl CoA mutase

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Macrocytic Anemias Macrocytic Anemias

Macrocytic AnemiaMacrocytic Anemia

High MCV

High MCH

Normal MCHC


Megaloblastic : defective DNA synthesis

Non-megaloblastic : numerous mechanisms

Nutritional Requirements for Nutritional Requirements for HematopoiesisHematopoiesis

Metals : iron copper cobalt

BB1212 and Folateand Folate

Other vitamins: B6, A, E, CRiboflavin, Niacin

Vitamin BVitamin B1212 (Cyanocobalamin)(Cyanocobalamin)

Vitamin BVitamin B1212Source : food of animal origin

livermuscleeggscheese and milk

Not in plants

Made by bacteria

BB12 12 Absorption Absorption

1. Release from food sources gastric proteases and acids

2. Binding by salivary cobalophilins

3. Digestion of cobalophilin-B12complex by pancreatic enzymes

BB12 12 AbsorptionAbsorption4. Binding to intrinsic factor (IF)

IF is secreted by gastric parietal cells

5. Attachment of B12-IF to receptors

6. Endocytosis and binding to transcobalamin II

BB12 12 Dependant ReactionsDependant Reactions

1. Synthesis of methionine from homocysteine requires : B12 and folate

2. Synthesis of succinyl CoA from methyonyl CoA requires : methylmalonyl CoA mutase

Folic AcidFolic Acid


Sources : synthesized by plants and microorganisms

Vegetables, fruits, dairy products

PolyglutamatedThermo labile

Folic AcidFolic AcidAbsorptionAbsorption--TransportTransport

1. Polyglutamates converted to monoglutamatesIntestinal carboxypeptidase

2. Binding of monoglutamates to brush border receptor

3. Conversion to methyltetrahydrofolate during absorption


4. Bind to serum protein

5. Receptor mediated cell uptake

6. Polyglutamated in cytoplasm

Folic Folic AcidAcid-- BB1212 InteractionInteraction

Folic Acid and DNAFolic Acid and DNA

Pernicious AnemiaPernicious AnemiaAn Autoimmune DiseaseAn Autoimmune Disease

Antibodies against :parietal cellsintrinsic factor (IF)

Thyroid - myxedemaMelanosomes - vitiligo

Pernicious AnemiaPernicious Anemia

Hematologic features : anemiapancytopeniamegaloblastic hematopoiesiscellular bone marrowineffective hematopoiesis

Folate DeficiencyFolate Deficiency

Hematologic features : same as P.A.

Clinical Picture : no neurologic findings

Folate Deficiency Folate Deficiency DiagnosisDiagnosis

Dietary historyClinical conditions

pregnancymalabsorption (sprue)hemolytic anemiadrugs

Laboratoryserum or red cell folate levels

Pernicious Anemia Pernicious Anemia Presenting ComplaintPresenting Complaint

Symptoms of anemia 58%Sensory paresthesis 13%GI complaints 11%Sore tongue or mouth 7%Weight loss 5%Difficulty walking 3%Other 3%

Pernicious Anemia Pernicious Anemia -- DiagnosisDiagnosisHistory and Physical

glossitispallorneurologic exam

Laboratoryblood smearantibody assaysB12 level

OtherSchilling test

Schilling TestSchilling TestFirst stage :

1. Inject B12 IM (1,000 ug) to saturate transcobalamin II

2. Administer oral B12 - radiolabeled

3. Collect 24 h urine

4. Measure radioactivity in urine

Schilling TestSchilling TestSecond stage :

1. Inject B12 IM (1,000 ug) to saturate transcobalamin II, (Same as 1st stage)

2. Administer oral B12 - radiolabeled plus intrinsic factor (HOG)

3. Collect 24 h urine, (Same as 1st stage)

4. Measure radioactivity in urine, (Same as 1st stage)

Pernicious Anemia Pernicious Anemia TreatmentTreatment

B12 by IM injection

Frequent at first

Monthly thereafter life long

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FOLIC ACIDSources : synthesized by plantsand microorganismsVegetables, fruits, dairy productsPolyglutamatedThermo labile

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Folic Acid Absorption-Transport

1. Polyglutamates converted to mono-glutamates Intestinal carboxypeptidase2. Binding of monoglutamates to brush border receptor3. Conversion to methyltetrahydrofolate during absorption4. Bind to serum protein5. Receptor mediated cell uptake6. Polyglutamated in cytoplasm

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High MCV

High MCH

Normal MCHC











Not in plants

Made by bacteria












































OtherSchilling test












B12 by IM injection

Frequent at first


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8.pdf



High MCV

High MCH

Normal MCHC











Not in plants

Made by bacteria












































OtherSchilling test












B12 by IM injection

Frequent at first


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Pernicious AnemiaAn Autoimmune Disease

Antibodies against :parietal cells intrinsic factor (IF)Thyroid - myxedemaMelanosomes - vitiligo

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Pernicious AnemiaHematologic features :

anemiapancytopeniamegaloblastic hematopoiesiscellular bone marrowineffective hematopoiesis

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High MCV

High MCH

Normal MCHC











Not in plants

Made by bacteria












































OtherSchilling test












B12 by IM injection

Frequent at first


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High MCV

High MCH

Normal MCHC











Not in plants

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OtherSchilling test












B12 by IM injection

Frequent at first


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Folate Deficiency

Hematologic features : same as Pernicious Anemia.Clinical Picture : no neurologic findings

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Folate Deficiency :Diagnosis

Dietary historyClinical conditions:

pregnancymalabsorption (sprue)hemolytic anemiadrugsLaboratory:

serum or red cell folate levels

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Pernicious AnemiaPresenting Complaint

Symptoms of anemia : 58%Sensory paresthesis :13%GI complaints :11%Sore tongue or mouth : 7%Weight loss : 5%Difficulty walking : 3%Other :3%

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Pernicious Anemia - Diagnosis

History and Physicalglossitispallorneurologic examLaboratoryblood smearantibody assaysB12 levelOtherSchilling test

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Schilling Test

First stage :1. Inject B12 IM (1,000 ug) to saturatetranscobalamin II2. Administer oral B12 - radiolabeled3. Collect 24 h urine4. Measure radioactivity in urine

Second stage :1. Inject B12 IM (1,000 ug) to saturate transcobalamin II (Same as 1st stage)2. Administer oral B12 radiolabeled plus intrinsic

factor (HOG)3. Collect 24 h urine, (Same as 1st stage)4. Measure radioactivity in urine,(Same as 1st stage)

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Static Test for Folate/B12 StatusFolateMeasured in whole blood (plasma and cells) and then in the serum aloneDifference is used to calculate the red blood cell folate concentration (may better reflect the whole folate pool)Can also test serum in fasting patient

B12Measured in serum

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Functional Tests for Macrocytic AnemiasHomocysteine: Folate and B12 are needed to convert homocysteine to methionine; high homocysteine may mean deficiencies of folate, B12 or B6Methylmalonic acid measurements can be used along with homocysteine to distinguish between B12 and folate deficiencies ( in B12 deficiency)Schilling test: radiolabeled cobalamin is used to test for B12 malabsorption

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Pernicious AnemiaA macrocytic, megaloblastic anemia caused by a deficiency of vitamin B12. Usually secondary to lack of intrinsic factor (IF)May be caused by strict vegan diet Also can be caused by gastric acid secretion, gastric atrophy, H-pylori, gastrectomy, disorders of the small intestine (celiac disease, regional enteritis, resections), drugs that inhibit B12 absorption including neomycin, alcohol, colchicine, metformin, pancreatic disease

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Symptoms of Pernicious AnemiaParesthesia (especially numbness and tingling in hands and feet)Poor muscular coordinationImpaired memory and hallucinationsDamage can be permanent

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Vitamin B12 DepletionStage Iearly negative vitamin B12 balanceStage IIvitamin B12 depletionStage IIIdamaged metabolism: vitamin B12 deficient erythropoiesisStage IVclinical damage including vitamin B12 anemiaPernicious anemianumbness in hands and feet; poor muscular coordination; poor memory; hallucinations

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Causes of Vitamin B12 DeficiencyInadequate ingestionInadequate absorptionInadequate utilizationIncreased requirementIncreased excretionIncreased destruction by antioxidants

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Treatment of B12 DeficiencyBefore 1926 was incurable; until 1948 was treated with liver extractNow treatment consists of injection of 100 mcg of vitamin B12 once per week until resolved, then as often as necessaryAlso can use very large oral doses or nasal gelMNT: high protein diet (1.5 g/kg) with meat, liver, eggs, milk, milk products, green leafy vegetables B12 by IM injection; Frequent at firstMonthly thereafter life long

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Folic Acid DeficiencyTropical sprue; pregnancy; infants born to deficient mothersAlcoholicsPeople taking medications chronically that affect folic acid absorptionMalabsorption syndromes

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Causes of Folate DeficiencyInadequate ingestionInadequate absorptionInadequate utilizationIncreased requirementIncreased excretionIncreased destructionVitamin B12 deficiency can cause folate deficiency due to the methylfolate trap

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Methylfolate TrapIn the absence of B12, folate in the body exists as 5-methyltetrahydro-folate (an inactive form)B12 allows the removal of the 5-methyl group to form THFA

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Stages of Folate Depletion and DeficiencyStage Iearly negative folate balance (serum depletion)Stage IInegative folate balance (cell depletion)Stage IIIdamaged folate metabolism with folate-deficient erythropoiesisStage IVclinical folate deficiency anemia

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Diagnosis of Folate DeficiencyFolate stores are depleted after 2-4 months on deficient dietMegaloblastic anemia, low leukocytes and plateletsTo differentiate from B12, measure serum folate, RBC folate (more reflective of body stores) serum B12High formiminoglutamic acid (FIGLU) in the urine also diagnostic

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Hemolytic AnemiaOxidative damage to cellslysis occursVitamin E is an antioxidant that seems to be protective.This anemia can occur in newborns, especially preemies.

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Nonnutritional AnemiasSports anemia (hypochromic microcytic transient anemia)Anemia of pregnancy: dilutionalAnemia of inflammation, infection, or malignancy (anemia of chronic disease)Sickle cell anemiaThalassemias

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Sports AnemiaTransientusually in athletes who are runners; from compression of RBCs in feet until they burst, releasing hemoglobinCheck lab valuesCounsel about a proper diet

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ReferencesFirst Known Heart Attack Associated With Beta- thalassemia Major Reported." Heart Disease Weekly February 22, 2004: 10.

Bowden, Vicky R., Susan B. Dickey, and Cindy Smith Greenberg. Children and Their Families: The continuum of care . Philadelphia: W.B. Saunders Company, 1998.

"Thalassemias." In Principles and Practice of Medical Genetics , Volume 2, edited by Alan E.H. Emery, MD, PhD, and David L. Rimoin, MD, PhD. New York: Churchill Livingstone, 1983.

Thompson, M.W., R. R. McInnus, and H. F. Willard. Thompson and Thompson Genetics in Medicine , Fifth Edition. Philadelphia: W.B. Saunders Company, 1991.

Olivieri, N. F. "The Beta Thalassemias." The New England Journal of Medicine 341 (1999): 99-109.

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*Macrophages - much larger particles*

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*Platelet function Adhesion to endothelial cells is mediated by glycoprotein Ib which binds vWF on endothelial cells. Aggregation is mediated by GPIIb/IIIa on another platelet by fibrinogen. Various agonists ADP, PAF, are shown as interacting with specific receptors and activating phospholipase C. Coagulant activity is generated by coagulation factors on the platelet external membrane to convert prothrombin (II) to thrombin (IIa).*

*Reactions are amplified quickly because one enzyme can activate a number of moleculesLocalized - diluted due to blood flow, actions of inhibitors, reactions only occur at a sufficient rate when at surface of activated platelets or at sites in injury*

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*pH maintained by buffering action of proteins*Driving force developed by difference in water concentration between solution in question and pure water. Can be measured by apparatus similar to that on the previous slide. *

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*Objective - illustrate previous discussion, lead in to coagulation, fibrinolysis, complement

State of knowledge is currently very sophisticated- many proteins purified, MW, amino acid sequence, 2y, 3y, 4y structure determined

Consider three abundant plasma proteins

Recall - analogous to synthetic polymers - macromolecules but not polymers perse

MWD - monodisperseShape in solution is fixed - not random coil as other polymers

Some trace proteins exist whose biological function has not yet been determined*Dimensions - 3yMW - 1y50% a helix - 2y

Any quaternary structure? - only one chain*

*Recall from high school biologypinocytosis is engulfment of liquid matter by the cell membranephago (eat) involves solid*

*Constant - same amino acid sequence for all of the moleculesVariable - varies between molecules- results in specificity of moleculesTwo heavy and two light chains joined by disulfide bonds. Variable region - red - binds the antigen Constant region (black) can activate complement pathway or attach the Ab to cells such as macrophages*

*The basic antibody unit is a monomer, IgA is a dimer and IgM is a pentamer*

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*N terminals in E and C terminals in Da chain in D is more like a random coil synthetic polymer

Clotting - thrombin breaks off FPA and FPB*Coagulation - very important consideration when processing blood outside of the bodyStructure must incorporate the ability to be easily broken down after clotting

What is a clot and how does it form from the polymerization of fibrinogen?

Overhead from Brashs notes next*

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*Measure of % saturation of hemoglobin as a function of O2 partial pressureDepends on temperature, pH (CO2 content) of blood - we will come back to thisNB in the lungs PO2 is approximately 100 mm Hgin the tissues PO2 is approximately 45 mm Hg

In the lungs we want HbO2In the tissues we want Hb (O2 delivered to the tissues)

Hb cycles between 95% saturated in the lungs, 65% saturated in the tissuesMust also consider CO2 - high pH in the lungs and low pH in the tissues*

*Oxygen binding curves of Mb and Hb. Mb has a much greater affinity for O2 than Hb. It is 50% saturated at partial pressures of 0.15 - 0.30 kPa whereas Hb requires a pO2 of about 3.5 kPa for 50% saturation. Note that both are 95% saturated at the pO2 of arterial blood leaving the lungs (13 kPa) Sigmoid O2 saturation is an adaptation for transport function of RBC assuring binding and release of O2 in appropriate tissues.*Simple hyperbolic curve - monomer with no positive cooperativity*

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*Contact activation - the initial event is the adsorption of factor XII to a negatively charged surface where it undergoes a conformational change to expose its active site. Factor XII converts prekallikrein to kallikrein. Additional factor XIIa and kallikrein are then generated by reciprocal activation. Factor XIIa also activates factor XI. Both kallikrein and factor XI bind to a cofactor, HMWK which serves to anchor them to the charged surface.*

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