Abdul Salam M. SofroDept.of Biochemistry, Fac. of MedicineYARSI University

Teaching aimsBy the end of the lecture, students would be able to understand & describe various biochemical aspects of blood

Reference:Murray K et al. 2000. Harpers Biochemistry, 25th ed & other lecture sources

Core topicsIntroductionComposition and main functions of bloodPlasma and its proteinsHemostasis and thrombosis

IntroductionBlood is a liquid tissue circulates in what is virtually a closed system of blood vesselsBlood consists of solid elements (RBC, WBC & platelets) suspended in a liquid medium called plasma critical for the maintenance of health

Composition and main functions of blood

FunctionsRespirationNutritionExcretionMaintenance of normal acid-base balanceRegulation of water balanceRegulation of body temperature

Defense against infection by WBC & circulating antibodiesTransport of hormones & regulation of metabolismTransport of metabolitesCoagulation

CompositionSolid elements : RBC, WBC, PlateletsLiquid medium : plasma consisting of water, electrolytes, metabolites, nutrients, proteins, hormones, etc. Water & electrolyte composition of plasma is practically the same as that of all extracellular fluids Once the blood has clotted (coagulated), the remaining liquid phase (called serum) lacks of the clotting factors (including fibrinogen)

19-*Composition of Blood

Red blood cells (erythrocytes)Delivering Oxygen to the tissues & helping in the disposal of carbon dioxide & protons formed by tissue metabolismMuch simpler structure than most human cells membrane surrounding a solution of Hb (about 95% of intracellular protein of the RBC)Contain cytoskeletal components important in determining their shape (Spectrin, ankyrin & other peripheral membrane protein)

Red blood cells (cont.)Possess many blood group systems (eg. ABO, Rh & MN systems)The ABO system is crucial in blood transfusionThe ABO substances are glycosphingolipids & glycoproteins sharing common oligosaccharide chains

Red blood cells (cont.)Life span : 120 daysTheir production is regulated by erythropoietin (synthesized in kidney & is released to the blood stream and travels to bone marrow in response to hypoxia)

Red blood cells (cont.)About 2 million RBC enter the circulation per secondMetabolically active (but unique & relatively simple) (facilitated diffusion involving specific protein, i.e. glucose transporter/permease, but not insulin dependent like in muscle & adipose cells)

Red blood cells (cont.)SOD, Catalase & Glutathione protect cells from oxidative stress & damage linked to Hexose Monophosphate Shunt (HMS =Pentose Phosphate Pathway)

Leukocyte (WBC)There are 3 groups : granulocytes (polymorphonuclear leukocytes = PMNs):NeutrophilsBasophilseosinophilsmonocyteslymphocytes

Neutrophils phagocytose bacteria and play a major role in acute inflammationBasophils resemble mast cells, contain histamin & heparin and play a role in some types of immunologic hypersensitivity reactionsEosinophils are involved in certain allergic reactions & parasitic infections

Monocytes are precursors of macrophages which, like neutrophils are involved in phagocytosisLymphocytes B lymphocytes synthesize antibodies, T lymphocytes play major roles in various cellular immune mechanisms, such as killing virally infected cells & some cancer cells

Platelets (Thrombocytes)cell-like particles smaller than RBCs and WBCs.Help with clotting process by gathering at bleeding site and clumping together to form a plug that helps seal the blood vessel.

Blood group systemVery important in blood transfusionDetermined by antigens in blood cell membrane and antibody in plasmaABO blood group system:Blood group A : antigen A, antibody Anti BBlood group B : antigen B, antibody Anti ABlood group AB : antigen A & B, antibody nonBlood group O: antigen non, antibody anti A & anti B

PrecursorsubstanceGALNAcGALGNAcGALFUC A

BA GalNAcB Gal

Genes & their product in ABO blood group systemGene H : fucosyltransferaseGene A : N-acetylgalactosamine glycosyltransferaseGene B : galactosyltransferaseGene O : inactive enzyme

Gene product Antigen Gene product AntigenH & AH & B HH P s r u e b c s u t r a s n o c r eTr-ATr-BOPrecursorsubstanceTr-Hhh

RBC Precursor StructureGlucose Galactose N-acetylglucosamineGalactose Precursor Substance (stays the same) RBCSource: cls.umc.edu/COURSES/.../ABOsystem.ppt

Formation of the H antigenGlucose Galactose N-acetylglucosamineGalactose H antigen RBCFucosecls.umc.edu/COURSES/.../ABOsystem.ppt

Formation of the A antigenGlucose Galactose N-acetylglucosamineGalactose RBCFucoseN-acetylgalactosaminecls.umc.edu/COURSES/.../ABOsystem.ppt

Formation of the B antigenGlucose Galactose N-acetylglucosamineGalactose RBCFucoseGalactosecls.umc.edu/COURSES/.../ABOsystem.ppt

GeneticsThe H antigen is found on the RBC with the Hh or HH genotype, but NOT from the hh genotypeThe A antigen is found on the RBC with the Hh, HH, and A/A, A/O, or A/B genotypesThe B antigen is found on the RBC with the Hh, HH, and B/B, B/O, or A/B genotypes

H antigenCertain blood types possess more H antigen than others:

O>A2>B>A2B>A1>A1BGreatest amount of HLeast amount of Hcls.umc.edu/COURSES/.../ABOsystem.ppt

Group OGroup AMany H antigen sites Fewer H antigen sitesAAAAAMost of the H antigen sites in a Group A individual have been converted to the A antigencls.umc.edu/COURSES/.../ABOsystem.ppt

Plasma and its proteins

Plasma proteinsTotal plasma protein approx. 7.0-7.5 g/dlA complex mixture of simple & conjugated proteins such as glycoproteins & various types of lipoproteins, thousands of antibodiesCan be separated by:sodium or amm. sulfate into three major groups fibrinogen, albumin & globulins electrophoresis using cellulose acetate into five bands albumin, 1, 2, & globulin

Cont.Concentration of plasma protein is important in determining the distribution of fluid between blood & tissuesOsmotic pressure (oncotic pressure) exerted by plasma protein is approx. 25 mm Hg. Hydrostatic pressure in arterioles is approx. 37 mm Hg a net outward force of about 11 mm Hg drives fluid out into interstitial spaces.Hydrostatic pressure in venules is approx. 17 mm Hg a net force of about 9 mm Hg attracts water back into circulation

Cont.The above pressures are often referred to as the Starling forces.If plasma protein concentration is markedly diminished (eg. due to severe protein malnutrition fluid is not attracted back into the intravascular compartment and accumulates in extravascular tissue spaces oedema

Cont.Most plasma proteins are synthesized in the liverPlasma proteins are generally synthesized on membrane-bound polyribosomesAlmost all plasma proteins are glycoproteinsMany plasma proteins exhibit polymorphism

Some functions of plasma proteinsAntiprotease (antichymotrypsin, a1 antitrypsin, a2 macroglobulin, antithrombin)Blood clotting (various coagulation factors, fibrinogen)Hormones

Immune defence (Ig, complement proteins, b2-microgloblin)Involvement in inflammatory responses (acute phase response protein eg. C-reactive protein, a1-acid glycoproteinOncofetal (a1-fetoprotein = AFP)Transport or binding proteins such as:

Cont.albumin for bilirubin, FFA, ions, metals, metheme, steroids, other hormones, variety of drugsCeruloplasmin contains Cu but albumin is more important in physiological transport of CuCorticosteroid-binding globulin (transcortin)Haptoglobin binds extracorpuscular HbLiproproteins (chylomicron, VLDL, LDL, HDL)

Cont.Hemopexin Retinol-binding proteinSex hormone-binding globulinThyroid-bindingTransferrinTransthyretin (formerly pre albumin, binds T4 & forms a complex with Retinol-binding protein)

Detail functions of some plasma proteinAlbumin:Major protein of human plasma (3.4-4.7 g/dL)Some 40% in plasma, 60% in extracellular spaceSynthesized in liver as preproprotein, depressed in a variety of diseases, particularly those of liver (decreases albumin/globulin ratio)Responsible for 75-80% of osmotic pressure of human plasmaAbility to bind various ligands (include FFA, Ca, certain steroid hormones, bilirubin etc.Play an important role in transport of Cu, drugs

Cont.Haptoglobin:A plasma glycoprotein that binds extracorpuscular Hb in a tight noncovalent Hb-Hp complexPrevent loss of free Hb into kidneyIts plasma levels are of some diagnostic use low level in hemolytic anemias

Cont.Transferrin:a 1-globulin, a glycoprotein, synthesized in liverPlays an important role in the bodys metabolism of iron (two mol of Fe3+ per mole of transferrin) diminishes potential toxicity of free ironPlasma concentration is approx. 300 mg/dL can bind 300 g of iron per dL (Total Iron Binding Capacity of plasma)

Ceruloplasmin (Cp)2-globulinBinds copper (Cu)Exhibits a copper-dependent oxidase activityLow levels of Cp are associated with Wilson diseaseTissue levels of Cu & certain other metals are regulated in part by metallomethionins (small protein found in the cytosol of cells particularly liver, kidney & intestine)

1-Antiproteinase (1-antitrypsin)Synthesized by hepatocytes & macrophages Principal serine protease inhibitor of human plasma inhibits trypsin, elastase & certain other proteasesDeficiency of this protein has a role in certain cases (approx. 5%) of emphysema

2-Macroglobulin A large plasma glycoproteinComprises 8-10% of the total plasma protein in humanSynthesized by a variety of cell types, including monocytes, hepatocytes & astrocytes.Binds many proteinases (an important panproteinase inhibitor)Binds many cytokines

ImmunoglobulinPlay a major role in the bodys defence mechanismSynthesized by B lymphocytes

Immunoglobulin (Ig)A group of proteins involved in mediating immune response in higher organismsIn gamma globulin fraction of serumVery heterogeneousSimilar in different species106 different antibodies may be produced in human adult

Source: http://pathmicro.med.sc.edu/mayer/IgStruct2000.htm

Ig structureTetramer : * a pair of light chains (two identical =kappa or =lambda chains)* a pair of heavy chains (two identical =alpha, =gamma, =delta, =epsilon or =mu chains)Light chain has one variable region (VL) & one constant region (CL)Heavy chain has one variable region (VH) and three (, , ) or four (, ) constant regions

Ig class Mol. Struct Carbohydr IgG 22 22 4 %

IgA 22 22 10 %

IgM 22 22 15 %

IgD 22 22 18 %

IgE 22 22 18 %

Ig functional groupsN terminal of H & L chains (VL/VH & CL /CH1) => antigen binding fragmentC terminal of L chain (CL) => interchain disulphide bondC terminal of H chain (CH) particularly C 2 & C 3 * and C 4 of IgM & IgE) constitute the Fc fragment responsible for class specific effector function => complement fixation or placental transfer, cell surface binding etc

Hemostasis and thrombosis

Hemostasis is the cessation of bleeding from a cut or severed vessel, whereas thrombosis occurs when the endothelium lining blood vessels is damaged or removed (eg. upon rupture of an atherosclerotic plaque)Hemostasis & thrombosis share three phases:Formation of a loose & temporary platelet aggregate at the site of injuryFormation of fibrin mesh that binds to the platelet aggregate, forming a more stable hemostatic plug or thrombusPartial or complete dissolution of the hemostatic plug or thrombus by plasmin

ThrombiThree types of thrombi:White thrombusRed thrombusDisseminated fibrin deposit in very small blood vessels or capillaries

Intrinsic and Extrinsic pathway of blood coagulationTwo pathways lead to fibrin clot formationThese pathways are not independentInitiation of fibrin clot in response to tissue injury is carried out by extrinsic pathway, but how intrinsic pathway is activated in vivo is unclear (but it involves a negatively charged surface)Intrinsic & extrinsic pathways converge in a final common pathway

Involves many different proteins can be classified into 5 types:zymogens of serine dependent proteases which become activated during the process of coagulationcofactorsfibrinogena transglutaminase, which stabilizes fibrin clotregulatory & other proteins

Blood clotting factorsF I: FibrinogenF II: ProthrombinF III: Tissue factorF IV: Ca2+F V: Proaccelerin, labile factor, accelerator (Ac-) globulinF VII: Proconvertin, serum prothrombin conversion accelerator (SPCA), cothromboplastin

Blood clotting factors (cont.)F VIII: Antihemophilic factor A, antihemophilic globulin (AHG)F IX: Antihemophilic factor B, Christmas factor, plasma thromboplastin component (PTC)F X: Stuart Prower FactorF XI: Plasma thromboplastin antecedent (PTA) F XII: Hageman factorF XIII: Fibrin stabilizing factor (FSF), fibrinoligase

Intrinsic pathwayInvolves factors XII, XI, IX, VIII, & X as well as prekallikrein, HMW kininogen, Ca2+ & platelet phospholipids results in the production of factor Xa.Commences with the contact phase in which prekallikrein, HMW kininogen, F XII & F XI are exposed to a negatively charged activating surface.

Intrinsic pathway (cont.)When the components of the contact phase assemble on the activating surface, F XII is activated to F XIIa upon proteolysis by kallikrein. This F XIIa attacks prekallikrein to generate more kallikrein, setting up a reciprocal activationF XIIa once formed, activates F XI to F XIa and also release bradykinin from HMW kininogen

Intrinsic pathway (cont.)F XIa in the presence of Ca2+ activates F IX. This in turn cleaves an Arg-Ile bond in F X to produce F Xa

Intrinsic pathwayPKHKXII XIIaIX IXaX XaProthrombin ThrombinXI XIaHKXVIIa/Tissue factorExtrinsic pathwayVIICa 2+Ca 2+Ca 2+PLCa 2+PLVIII VIIIa V Va

Prothrombin ThrombinFibrinogenFibrin monomerFibrin polymerCross-linkedFibrin polymerXIIIXIIIa

Extrinsic pathwayAlso leads to activation of F X but by different mechanism. Involves tissue factor, F VII, F X & Ca2+ and results in the production of F XaIt is initiated at the site of tissue injury with the expression of tissue factor on endothelial cells

Extrinsic pathway (cont.)Tissue factor interacts with & activates F VII. Tissue factor acts as a cofactor for F VIIa, enhancing its enzymatic activity to activate F XActivation of F X provides an important link between those two pathways

Final common pathwayInvolves activation of prothrombin to thrombinF Xa produced by either intrinsic or extrinsic pathway, activates prothrombin (F II) to thrombin (F IIa)Activation of prothrombin, like that of factor X, occur on the surface of activated platelets & requires the assembly of a prothrombinase complex, consisting of platelet anionic phospholipid, Ca2+, F Va, F Xa, & prothrombin

Final common pathway (cont.)Conversion of fibrinogen to fibrin is catalyzed by thrombin (thrombin also converts F XIII to F XIIIa, a factor highly specific transglutaminase that covalently cross-links fibrin molecules by forming peptide bonds between the amide groups of glutamine & the e-amino groups of lysine recidues, yielding a more stable fibrin clot with increased resistance to proteolysis

Some notesLevels of circulating thrombin must be carefully controlled achieved in 2 ways:Feedback mechanism through a cascade of enzymatic reactions for the conversion of prothrombin to thrombinInactivation of any thrombin formed by circulating inhibitors (the most important of which is antithrombin III)

Some notes(cont.)Endogenous activity of antithrombin III is greatly potentiated by the presence of heparinCoumarin anticoagulants (eg. Warfarin) inhibit vit.K-dependent carboxylation of F II, VII. IX & XFibrin clots are dissolved by plasmin (circulates in plasma in the form of its inactive zymogen, plasminogen)

Some notes(cont.)Activators of plasminogen are found in most body tissues e.g. tissue plasminogen activator (alteplse, t-PA) is a serine protease that is released into circulation from vascular endothelium under condition of injury or stress & is catalytically inactive unless bound to fibrin (recombinant t-PA is used therapeutically as a fibrinolytic agent as is Streptokinase Urokinase (precursor: prourokinase)

Some notes (cont.) Hemophilia A is due to deficiency of F VIIIHemophilia B is due to deficiency of F IXEndothelial cells synthesize prostacyclin (potent inhibitor of platelet aggregation)& other compounds that affect clotting & thrombosisAspirin is an effective antiplatelet drugSome laboratory tests measure coagulation & thrombolysis

*H enzyme is fucosyltransferase