Abdul Salam M. Sofro Dept.of Biochemistry, Fac.

of Medicine YARSI University

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

Core topics
Introduction Composition

and main functions of blood Plasma and its proteins Hemostasis and thrombosis

Introduction
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

Composition and main functions of blood

Functions
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

19-9

Red blood cells (erythrocytes)

Delivering

Oxygen to the tissues & helping in the disposal of carbon dioxide & protons formed by tissue metabolism Much 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 transfusion The ABO substances are glycosphingolipids & glycoproteins sharing common oligosaccharide chains

Red blood cells (cont.)

Life

span : 120 days Their 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 second Metabolically 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): Neutrophils Basophils eosinophils monocytes lymphocytes

 Neutrophils

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

 Monocytes

are precursors of macrophages which, like neutrophils are involved in phagocytosis Lymphocytes 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 system

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

A GalNAc GAL GNAc

GALNAc

A
B
B Gal

GAL

Precursor substance
FUC

Genes & their product in ABO blood group system

Gene

H : fucosyltransferase Gene A : N-acetylgalactosamine glycosyltransferase Gene B : galactosyltransferase Gene O : inactive enzyme

Gene product Antigen Gene product

Antigen

Tr-A Ps r u e b c s u t r a s n o c r e H Tr-B O hh Precursor substance

H&A

Tr-H

H&B

RBC Precursor Structure

RBC

Glucose
Galactose N-acetylglucosamine Galactose

Precursor Substance (stays the same)

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

Formation of the H antigen

RBC

Glucose

H antigen

Galactose N-acetylglucosamine Galactose

Fucose

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

Formation of the A antigen

RBC

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

Glucose
Galactose N-acetylglucosamine Galactose N-acetylgalactosamine

Fucose

Formation of the B antigen

RBC

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

Glucose
Galactose N-acetylglucosamine Galactose Galactose

Fucose

Genetics
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

H antigen
Certain

blood types possess more H antigen than others:

Greatest amount of H

O>A2>B>A2B>A1>A1B

Least amount of H

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

A A Group O

A A Group A

Many H antigen sites

Fewer H antigen sites

Most of the H antigen sites in a Group A individual have been converted to the A antigen
cls.umc.edu/COURSES/.../ABOsystem.ppt

Plasma and its proteins

Plasma proteins
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

Some functions of plasma proteins

(antichymotrypsin, a1 antitrypsin, a2 macroglobulin, antithrombin) Blood clotting (various coagulation factors, fibrinogen) Hormones
Antiprotease

 Immune

defence (Ig, complement proteins, b2-microgloblin) Involvement in inflammatory responses (acute phase response protein eg. C-reactive protein, a1acid 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

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

Ig class
IgG
IgA

Mol. Struct 22
22

Carbohydr
4%
10 %

22
22

IgM
IgD IgE

22
22 22

22
22 22

15 %
18 % 18 %

Ig functional groups
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

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 injury Formation of fibrin mesh that binds to the platelet aggregate, forming a more stable hemostatic plug or thrombus Partial or complete dissolution of the hemostatic plug or thrombus by plasmin

Thrombi
Three

types of thrombi: White thrombus Red thrombus Disseminated fibrin deposit in very small blood vessels or capillaries

Intrinsic and Extrinsic pathway of blood coagulation

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

Blood clotting factors

F

I F II F III F IV FV
F

VII

: Fibrinogen : Prothrombin : Tissue factor : Ca2+ : Proaccelerin, labile factor, accelerator (Ac-) globulin : Proconvertin, serum prothrombin conversion accelerator (SPCA), cothromboplastin

Blood clotting factors (cont.)

F F

VIII IX

X F XI
F

XII F XIII

: Antihemophilic factor A, antihemophilic globulin (AHG) : Antihemophilic factor B, Christmas factor, plasma thromboplastin component (PTC) : Stuart Prower Factor : Plasma thromboplastin antecedent (PTA) : Hageman factor : Fibrin stabilizing factor (FSF), fibrinoligase

Intrinsic pathway
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.

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 activation F 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 pathway PK HK XII XIIa HK Extrinsic pathway VII XIa VIIa/Tissue factor IX VIII Ca 2+ VIIIa IXa Ca 2+ PL Xa Va Ca 2+ PL Thrombin X

Ca 2+
XI

X V

Prothrombin

Prothrombin

Thrombin XIII

Fibrinogen XIIIa Fibrin monomer

Fibrin polymer

Cross-linked Fibrin polymer

Extrinsic pathway
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

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

Final common pathway

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

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 notes
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)

Some notes(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)

Some notes(cont.)
Activators

of plasminogen are found in most body tissues e.g. tissue plasminogen activator (alteplse, tPA) 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 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