2. biokimia darah

BIOCHEMICALASPECTOFBLOOD

AbdulSalamM.SofroDept.ofBiochemistry,Fac.ofMedicine

YARSIUniversity

IntroducHon

AnaksayawaktudisunatdarahnyasusahberhenH,kenapayadok?

Tiapkalisayasikatgigikeluardarah,kenapayadok? Hidungadiksayaseringmimisen.Kenapayadok? Tekanandarahsayarendah.Apasayaharusmakansatekambingterusdok?

KadarHemoglobin(Hb)pamansayakurang.Katadokterharusditransfusi.Apakalaukurangdarahharusditransfusidok?

Anaksayakurangdarah,apaharusdiberilaukhaH&ampelaterusdok?

QsrelatedtoBlood

Iqra bismi Rabbikalladzii kholaq

(Bacalah, dengan nama Rabb/Tuhan mu yang telah menciptakan)

Kholaqal insaana min alaq ........ (Yang menciptakan manusia dari alaq -

segumpal darah, sesuatu yang menempel) - QS. AlAlaq

Venice, Italy Dec 2012

Theverybeginningofhumandevelopment

Fer$lizedovum

Zygote

cogito ergo sum = saya berpikir maka saya ada (Rene Descartes Filsuf Prancis)

Cesky Crumlov, Cezk Dec 2013

Paris, France, Dec 2012

Teaching aims

By 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

Coretopics

Introduction Composition and main functions of blood Plasma and its proteins Hemostasis and thrombosis Hemoglobin synthesis and degradation

IntroducHon

Blood is a liquid tissue circulates in what is virtually a closed system of blood vessels

Blood consists of solid elements (RBC, WBC & platelets) suspended in a liquid medium called plasma critical for the maintenance of health

ComposiHonandmainfuncHonsofblood

FuncHons

Respiration Nutrition Excretion Maintenance of normal acid-base balance Regulation of water balance Regulation of body temperature

Defense against infection by WBC & circulating antibodies

Transport of hormones & regulation of metabolism

Transport of metabolites Coagulation

Composition

Solid elements : RBC, WBC, Platelets Liquid 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)

Composition of Blood

19-13

Red blood cells (erythrocytes)

DeliveringOxygentotheHssues&helpinginthedisposalofcarbondioxide&protonsformedbyHssuemetabolism

MuchsimplerstructurethanmosthumancellsmembranesurroundingasoluHonofHb(about95%ofintracellularproteinoftheRBC)

Containcytoskeletalcomponentsimportantindeterminingtheirshape(Spectrin,ankyrin&otherperipheralmembraneprotein)

Red blood cells (cont.)

Possessmanybloodgroupsystems(eg.ABO,Rh&MNsystems)TheABOsystemiscrucialinbloodtransfusion

TheABOsubstancesareglycosphingolipids&glycoproteinssharingcommonoligosaccharidechains

Redbloodcells(cont.)

Lifespan:120days TheirproducHonisregulatedbyerythropoieHn(synthesizedinkidney&isreleasedtothebloodstreamandtravelstobonemarrowinresponsetohypoxia)


About2millionRBCenterthecirculaHonpersecond

MetabolicallyacHve(butunique&relaHvelysimple)(facilitateddiusioninvolvingspecicprotein,i.e.glucosetransporter/permease,butnotinsulindependentlikeinmuscle&adiposecells)


SOD,Catalase&GlutathioneprotectcellsfromoxidaHvestress&damagelinkedtoHexoseMonophosphateShunt(HMS=PentosePhosphatePathway)

Leukocyte(WBC)

Thereare3groups:granulocytes(polymorphonuclearleukocytes=PMNs):NeutrophilsBasophilseosinophils

monocyteslymphocytes

NeutrophilsphagocytosebacteriaandplayamajorroleinacuteinammaHon

Basophilsresemblemastcells,containhistamin&heparinandplayaroleinsometypesofimmunologichypersensiHvityreacHons

EosinophilsareinvolvedincertainallergicreacHons&parasiHcinfecHons

Monocytesareprecursorsofmacrophageswhich,likeneutrophilsareinvolvedinphagocytosis

LymphocytesBlymphocytessynthesizeanHbodies,Tlymphocytesplaymajorrolesinvariouscellularimmunemechanisms,suchaskillingvirallyinfectedcells&somecancercells

Platelets(Thrombocytes)

cell-likeparHclessmallerthanRBCsandWBCs.

Helpwithcloingprocessbygatheringatbleedingsiteandclumpingtogethertoformaplugthathelpssealthebloodvessel.

Bloodgroupsystem

Very important in blood transfusion Determined by antigens in blood cell

membrane and antibody in plasma ABO blood group system:

Blood group A : antigen A, antibody Anti B Blood group B : antigen B, antibody Anti A Blood group AB : antigen A & B, antibody non Blood group O: antigen non, antibody anti A &

anti B

Genes&theirproductinABObloodgroupsystem

GeneH:fucosyltransferaseGeneA:N-acetylgalactosamineglycosyltransferaseGeneB:galactosyltransferaseGeneO:inacHveenzyme

Gene product Antigen Gene product Antigen

H & A

H & B

H

H P s r u e b c s u t r a s n o c r e

Tr-A

Tr-B

O

Precursor substance

Tr-H

hh

RBCPrecursorStructure

Glucose

Galactose

N-acetylglucosamine

Galactose

Precursor Substance (stays the

same)

RBC

Source: cls.umc.edu/COURSES/.../ABOsystem.ppt

FormaHonoftheHanHgen

Glucose

Galactose

N-acetylglucosamine

Galactose

H antigen

RBC

Fucose cls.umc.edu/COURSES/.../ABOsystem.ppt

FormaHonoftheAanHgen

Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

Fucose N-acetylgalactosamine

cls.umc.edu/COURSES/.../ABOsystem.ppt

FormaHonoftheBanHgen

Glucose

Galactose

N-acetylglucosamine

Galactose

RBC

Fucose Galactose


Group O Group A

Many H antigen sites

Fewer H antigen

sites

A

A A

A A

Most of the H antigen sites in a Group A individual have been

converted to the A antigen


GeneHcs

The H antigen is found on the RBC with the Hh or HH genotype, but NOT from the hh genotype

The A antigen is found on the RBC with the Hh, HH, and A/A, A/O, or A/B genotypes

The B antigen is found on the RBC with the Hh, HH, and B/B, B/O, or A/B genotypes

HanHgen

Certain blood types possess more H antigen than others:

O>A2>B>A2B>A1>A1B Greatest

amount of H Least

amount of H


Plasmaanditsproteins

Plasmaproteins

Total plasma protein approx. 7.0-7.5 g/dl A complex mixture of simple & conjugated

proteins such as glycoproteins & various types of lipoproteins, thousands of antibodies

Can 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 & tissues

Osmotic 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 liver

Plasma proteins are generally synthesized on membrane-bound polyribosomes

Almost all plasma proteins are glycoproteins

Many plasma proteins exhibit polymorphism

SomefuncHonsofplasmaproteins

Antiprotease (antichymotrypsin, 1 antitrypsin, 2 macroglobulin, antithrombin)

Blood clotting (various coagulation factors, fibrinogen)

Hormones

Immune defence (Ig, complement proteins, 2-microgloblin)

Involvement in inflammatory responses (acute phase response protein eg. C-reactive protein, 1-acid glycoprotein

Oncofetal (a1-fetoprotein = AFP) Transport or binding proteins such as:

Cont. albumin for bilirubin, FFA, ions, metals,

metheme, steroids, other hormones, variety of drugs

Ceruloplasmin contains Cu but albumin is more important in physiological transport of Cu

Corticosteroid-binding globulin (transcortin) Haptoglobin binds extracorpuscular Hb Liproproteins (chylomicron, VLDL, LDL,

HDL)

Cont. Hemopexin Retinol-binding protein Sex hormone-binding globulin Thyroid-binding Transferrin Transthyretin (formerly pre albumin, binds

T4 & forms a complex with Retinol-binding protein)

Detail functions of some plasma protein

Albumin: Major protein of human plasma (3.4-4.7 g/dL) Some 40% in plasma, 60% in extracellular space Synthesized 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 plasma

Ability 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 complex

Prevent loss of free Hb into kidney Its plasma levels are of some

diagnostic use low level in hemolytic anemias

Cont. Transferrin:

a 1-globulin, a glycoprotein, synthesized in liver

Plays an important role in the bodys metabolism of iron (two mol of Fe3+ per mole of transferrin) diminishes potential toxicity of free iron

Plasma concentration is approx. 300 mg/dL can bind 300 g of iron per dL (Total Iron Binding Capacity of plasma)

Ceruloplasmin (Cp) 2-globulin Binds copper (Cu) Exhibits a copper-dependent oxidase activity Low levels of Cp are associated with Wilson

disease Tissue 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 proteases

Deficiency of this protein has a role in certain cases (approx. 5%) of emphysema

2-Macroglobulin A large plasma glycoprotein Comprises 8-10% of the total plasma

protein in human Synthesized by a variety of cell types,

including monocytes, hepatocytes & astrocytes.

Binds many proteinases (an important panproteinase inhibitor)

Binds many cytokines

Immunoglobulin Play a major role in the bodys defence

mechanism Synthesized by B lymphocytes

Immunoglobulin(Ig)

A group of proteins involved in mediating immune response in higher organisms

In gamma globulin fraction of serum Very heterogeneous Similar in different species 106 different antibodies may be produced

in human adult

Basic structure of immunoglobulins

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

Ig structure Tetramer :

* 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

Igclass Mol.Struct Carbohydr

IgG 2222 4%IgA 2222 10%IgM 2222 15%IgD 2222 18%IgE 2222 18%

IgfuncHonalgroups

N terminal of H & L chains (VL/VH & CL /CH1) => antigen binding fragment

C terminal of L chain (CL) => interchain disulphide bond

C 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

Hemostasisandthrombosis

HemostasisisthecessaHonofbleedingfromacutorseveredvessel,whereasthrombosisoccurswhentheendotheliumliningbloodvesselsisdamagedorremoved(eg.uponruptureofanatheroscleroHcplaque)

Hemostasis&thrombosissharethreephases:FormaHonofaloose&temporaryplateletaggregateatthesiteofinjury

FormaHonofbrinmeshthatbindstotheplateletaggregate,formingamorestablehemostaHcplugorthrombus

ParHalorcompletedissoluHonofthehemostaHcplugorthrombusbyplasmin

Thrombi

Three types of thrombi: White thrombus Red thrombus Disseminated fibrin deposit in very

small blood vessels or capillaries

IntrinsicandExtrinsicpathwayofbloodcoagulaHon

Two pathways lead to fibrin clot formation These pathways are not independent Initiation 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 coagulation

cofactors fibrinogen a transglutaminase, which stabilizes

fibrin clot regulatory & other proteins

Bloodcloingfactors

F I : Fibrinogen F II : Prothrombin F III : Tissue factor F IV : Ca2+ F V : Proaccelerin, labile factor,

accelerator (Ac-) globulin F VII : Proconvertin, serum prothrombin

conversion accelerator (SPCA), cothromboplastin

Bloodcloingfactors(cont.) F VIII : Antihemophilic factor A,

antihemophilic globulin (AHG) F IX : Antihemophilic factor B, Christmas

factor, plasma thromboplastin component (PTC)

F X : Stuart Prower Factor F XI : Plasma thromboplastin antecedent

(PTA) F XII : Hageman factor F XIII : Fibrin stabilizing factor (FSF),

fibrinoligase

Intrinsicpathway

Involves 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.

Intrinsicpathway(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 activation

F XIIa once formed, activates F XI to F XIa and also release bradykinin from HMW kininogen

Intrinsicpathway(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 pathway PK HK

XII XIIa

IX IXa

X Xa

Prothrombin Thrombin

XI XIa

HK

X

VIIa/Tissue factor

Extrinsic pathway VII

Ca 2+

Ca 2+

Ca 2+ PL

Ca 2+

PL VIII VIIIa

V Va

Prothrombin Thrombin

Fibrinogen

Fibrin monomer

Fibrin polymer

Cross-linked Fibrin polymer

XIII

XIIIa

Extrinsicpathway

Also 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 Xa

It is initiated at the site of tissue injury with the expression of tissue factor on endothelial cells

Extrinsicpathway(cont.)

Tissue factor interacts with & activates F VII. Tissue factor acts as a cofactor for F VIIa, enhancing its enzymatic activity to activate F X

Activation of F X provides an important link between those two pathways

Finalcommonpathway

Involves activation of prothrombin to thrombin

F 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

Finalcommonpathway(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

Somenotes

Levels 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 thrombin

Inactivation of any thrombin formed by circulating inhibitors (the most important of which is antithrombin III)

Somenotes(cont.)

Endogenous activity of antithrombin III is greatly potentiated by the presence of heparin

Coumarin anticoagulants (eg. Warfarin) inhibit vit.K-dependent carboxylation of F II, VII. IX & X

Fibrin clots are dissolved by plasmin (circulates in plasma in the form of its inactive zymogen, plasminogen)

Somenotes(cont.)

Activators of plasminogen are found in most body tissues e.g. tissue plasminogen activator (alteplase,

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)

Somenotes(cont.)

Hemophilia A is due to deficiency of F VIII Hemophilia B is due to deficiency of F IX Endothelial cells synthesize prostacyclin

(potent inhibitor of platelet aggregation)& other compounds that affect clotting & thrombosis

Aspirin is an effective antiplatelet drug Some laboratory tests measure coagulation

& thrombolysis

TERIMAKASIHTHANKYOU

International Society of Blood Transfusion (ISBT) Meeting

2. biokimia darah

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