PLASMA PROTEINS, IMMUNOGLOBULINS AND BLOOD COAGULATION

ByDr.Hj. Sadiah Achmad,dr

PLASMA PROTEINS, IMMUNOGLOBULINS AND BLOOD COAGULATIONBlood : - Solid elements : - Red & white blood cells - Platelets - Liquid Medium : - Plasma (H2O, electrolytes, metabolites, nutrients, proteins, hormones)

Blood has clotted liquid phase : serum - Lacks of clotting factors - Some degradation products of clotting factors

Functions of Blood :

Transport : - O2 & CO2 Respiration - Nutrients Nutrition - Metabolic waste Excretion - Hormones - MetabolitesHomeostasis : - Maintenance of normal acid-base balance - Regulation of water balance - Regulation of body temperatureDefense : - Immune System - cellular : white blood cells - humoral : circulating anti- bodies - Coagulation

Plasma contains a complex mixture of proteins - single proteins- conjugated proteins Concentration of total plasma protein : 7.0 - 7.5 g/dL Plasma proteins can be separated : - salting out methods - Albumin, Globulin, Fibrinogen - electrophoresis (Cellulose acetate) bands :- albumin- 1, 2, , - globulins Densitometer scanning quantified the amount of bands graph. PLASMA PROTEINS

Albumin 1 2 +Albumin 1 2 a

ba : Separated protein bands are visualized in characteristic positions after being stainedb : Densitometer scanning from cellulose acetate strip converts bands to characteristic peaks of albumin, 1 -globulin, 2 - globulin, - globulin, - globulin,

Concentration of plasma protein determine the distribution of fluid between blood & tissues. - Concentration diminished edema due to : Protein deficiency : - severe protein malnutrition - chronic diarrhea - chronic glomerulo nephritis.

Study of plasma proteins : a. Synthesized in the liver (-globulin in plasma cells) b. Synthesized on membrane bound polyribosomes as preprotein Rer. Ser. Golgi APP secretory vesicles plasma : post translational modifications (proteolysis, glycosylation, phosphorylation) Transit time : 30 - several hours. c. Glycoproteins (except albumin) N/O - oligosaccharide chain various functions e.g. removal of sialic acid (from ceruloplasmin) shorten half life.

Polymorphism.- ABO blood group substances- 1- anti trypsin, haptoglobin, transferin- ceruloplasmin, immunoglobulins Strach gel electrophoresis characteristic migrationCharacteristic half life in circulation - albumin : 20 days- haptoglobin : 5 daysCrohns disease (regional ileitis) : plasma proteins lost into bowl through inflamed intestinal mucosa : protein - losing gastroenteropathy half life albumin : 1 day

Levels of certain plasma proteins increase :1. Acute inflammatory states / secondary to tissue damage acute phase proteins (reactants)- C - Reactive Protein (CRP) : 50% 1000x- 1- anti trypsin, 1- acidglycoprotein- Haptoglobin, fibrinogen2. Chronic Inflammatory states3. Cancer

Response to Inflammation :- CRP : stimulate classical complement pathway- 1 antitrypsin : neutralize proteases released during acute inflammatory states- Interleukin 1 (1L 1) : stimulate synthesis of acute phase reactants by hepatocytes (gene transcription)

Antiproteases : - Antichymotrypsine, Antithrombin - 1- antitrypsin, 2 - macroglobulin Blood clotting : - Coagulation factors, Fibrinogen Enzymes : - function in blood : - coagulation factors - cholinesterase. - leakage from cells/tissue : aminotransferases Immune defense : - Immunoglobulins, complement proteins - 2 - microglobulin. Inflammatory responses : - CRP - 1- acidglycoprotein (orosomucoid) Functions of Plasma Proteins

Transport / binding proteins :- Albumin ( various ligands)- Ceruloplasmin (Cu2+)- Cortico steroid binding globulin / transcortin (cortisol)- Haptoglobin (extracorpuscular Hb)- Lipoproteins ( CM, VLDL, LDL, HDL)- Retinol binding protein (retinol)- Sex hormone binding globulin (testosteron, estradiol)- Thyroid binding globulin (T4, T3)- Transferrin (Fe)

The major plasma protein : 3.4 4.7 g/dL 60% of total plasma protein- 40% in the plasma- 60% in extra cellular space Liver produces 12 g albumin per day 25% of total hepatic protein synthesis Synthesis is depressed in : - liver disease decreased albumin / globulin. ratio - protein malnutrition (kwashiorkor) Consist of : 1 polypeptide chain of 585 amino acids & 17 disulfide bonds (69kDa) Proteases 3 domains : different functionsALBUMIN

Function : - Major determinant of plasma osmotic pressure Analbuminemia : mutation that affect splicing moderate edema ( other plasma proteins increase compensate) - Binds various ligands : FFA, bilirubin, steroid hormones, Ca2+, Cu2+, drugs (sulfonamides, penicillin G, dicumarol, aspirin) Preparations of human albumin treatment of - hemorrhagic shock - burns

HAPTOGLOBIN (Hp) Glycoprotein : 90 kDa Binds extracorpuscular Hb (Hb - Hp) prevents loss of free Hb into kidney and urine - 10% of Hb degraded each day circulation : extracorpuscular - 90% degraded in RES. Hb kidney excreted in urine / precipitates in tubules iron is lost. Hb - Hp kidney : Hb is catabolized by liver cells iron is conserved & reused.

Human Hp : 3 polymorphic forms : Hp1-1, Hp2-1,Hp2-2 (no significant functional differences) Starch gel electrophoresis Hp1-1 : single band Hp2-1 Hp2-2 2 genes direct the 3 phenotypes : Hp1 & Hp2 Hp2-1 : heterozygous phenotype. complex band patterns

Concentration in plasma : 300 mg/dL bind 300 g/dL Fe total iron binding capacity of plasma Normal : 1/3 saturated with iron Iron def. Anemia : less saturated ( < 1/3) Hemochromatosis : > 1/3 saturated

Fe deficiency anemia : occurs frequently.- Inadequate intake of Fe (pregnancy, older people )- Inadequate utilization of Fe- Excessive loss of Fe

* Stores Fe Intracellular* Contains 23% Fe Apoferritin : - 440 kDa (24 subunits of 18,5 kDa) - surround 3000 - 4500 Fe 3+ in micellar form * Normal : a little ferritin in plasma In excess Fe : ferritin in plasma Hemochromatosis : ferritin in tissue (liver, spleen) Ferritin in plasma can be measured by Ria (sensitive & specific) index of body iron stores

FERRITIN

* Synthesis of ferritin & transferrin receptor (TfR) : reciprocally linked to cellular iron content. Iron Response Elements (specific untranslated sequences of mRNAs for both proteins) interact with IRE- binding protein . Fe levels - ferritin mRNA synthesize ferritin- tfR mRNA : degraded Fe level - synthesis TfR - ferritin mRNA inactive

HEMOSIDERIN* A partly degraded form of ferritin, still containing Fe Excessive storage of Fe detected histologically by prussian blue staining ( for Fe)

HEMOCHROMATOSIS* Primary CHR : Genetic disorder Excessive storage of Fe in tissue damage* Secondary CHR : - repeated transfusions - Excessive oral intake of Fe.

Lab tests for assessing disorder of Fe metabolism :- RBC count & estimation of Hb- determination of TIBC & % transferrin saturation- determination of ferritin in plasma (RIA)- prussian blue stain of tissue sections- determination of amount of Fe (g/g) in tissue biopsy.

CERULOPLASMIN( 160 kDa)* 2 - globulin* Carries 90% Cu in plasma : 1 mol binds tightly 6 atoms Cu (albumin carries 10 % plasma Cu & binds less tightly donates Cu to tissues)* Ceruloplasmin - liver disease - wilson disease (hepatolenticular degeneration), abnormal metabolism of Cu)

1-ANTIPROTEINASE (1-ANTITRYPSIN)* Single chain (52 kDa) : 394 amino acids + 3 oligo - saccharide chains* Major component (> 90%) of 1 fraction of plasma* Synthesized by hepatocytes & macrophage* Serin protease inhibitor : inhibiting elastase of neutrophils* 75 polymorphic forms ( can be separated by electrophoresis)* Deficiency 1 - A.T. emphysema (5%)

Def. 1 - A.TWBC PMN in the lung (pneunomia) lacks acounter- check to proteolytic damage by protease (elastase) :- active elastase + 1 - A.T inactive El : 1 - A.T complex (residu 358,methionine is involved) no proteolysis of lung no tissue damage - active elastase + or no 1 - A.T active El. proteolysis tissue damageSmoking : oxidizes met met. sulfoxide inactive emphysemaTreatment of patients with emphysema due to 1 - A.T Def. : i.v. 1 - A.T (augmentation therapy)

2 MACROGLOBULIN* Large plasma glycoprotein : 720 kDa ( 4 identical subunits of 180 kDa)* 8 - 10% of total plasma protein * Transport 10% of Zn (the rest : by albumin)* Synthesized by ; monocytes, hepatocytes & astrocytes* Thiol ester plasma protein family (contain internal cyclic thiol ester bond)* - Binds & neutralizes many proteinase panproteinase inhibitor - Targets certain cytokines (PDGF, TGF-) toward particular tissues / cells.

IMMUNOGLOBULINS (Ig)* - globulin , glycoprotein* Synthesis : plasma cells (B lymphocytes)* Function : key role in defense mechanism (humoral anti - bodies) Two components of immune system : 1. B lymphocytes (bone marrow cells) humoral antibodies (Ig)2. T lymphocytes (thymic) cell - mediated immunologic processes.

* Structure ; Consist of : - 2 identical light (L) chains (23 kDa) - 2 identical heavy (H) chains (53 75 kDa) Held together as tetramer (L2H2) by disulfide bonds (intra & interchain) Y - shaped. L-chain : 2 domain : - constant region (CL) - variable region (VL) H-chain : 4 domain : - CH1, CH2, CH3 & VH CH2 : complement binding site CH3 : attaches to receptor on neutrophils & macrophages Antigen binding site : hypervariable regions of L & H chains ( located on VL & VH)

S S S S S S S S L chainH chain VL CLVHCH1+H3N

+H3N

-S - S--S - S-SSSS +H3N

+H3N

Hinge regionS S S S S S S S H chainH chainCOO-

COO-CH2 CH3F.cPepsinPapainCleavage sitesStructure of IgGS S S S S S S S H chainL chain FabFab

* Digestion of Ig by papain (on hinge region) - 2 antigen binding fragments (Fab) : bind 2 mol antigen (on antigenic determinant = epitope) divalent / bivalent- 1 crystallizable fragment (Fc). * L chains : , , ( differences in CL region) Mol. Ig always contains : 2 or 2 ( chains are more frequent) * H chains : , , , & (differences in CH region) & : 4 CH domains 5 Ig class : IgG, IgA, IgM, IgD, IgE

IgM: - produced in the primary response to an antigen - fixes complement - does not cross placenta - antigen receptor on the surface of B cellsIgD: - uncertain - found in serum & on the surface of many B-cellsIgE: - mediates immediate hypersensitivity (releasing mediators from mast - cells & basophils upon exposure to allergen) - defends against helminthic infections (releasing enzymes from eosinophils) - does not fix complement.

Interchain disulfide bondsIntrachaindisulfidebondsHeavy chainhypervariableregionsLight chainhypervariableregionsVL CL VH CHSchematic model of an IgG molecule showing approximate positions of the hypervariable regions in heavy and light chains

* Variable Regions of Ig mol - consist of VL & VH, heterogenous - comprise of invariable regions & hypervariable regions - L chains : 3 hypervar. region H chains : 4 hypervar. region - hypervar. r : antigen binding site & dictate specificity of antibodies complementarity - determining regions (CDRs) : 5-10 amino acids in each CDR - CDRs : located on small loops of V domains surrounding peptide regions between CDRs : frame work regions CDRs from VH & VL : form a single hypervar. surface : antigen binding site

* Various combinations of H & L chains CDRs (combinatorial diversity) different specificities of antibody diversity of antibody molecules. - large antigens : interact with all of CDRs - small ligands : interact with only 1 or a few CDRs that form a pocket or groove in the molecule* Constant regions C- region : CH2, CH3 (CH4 of IgM & IgE) : Fc fragment responsible for class-specific effector functions of different Ig mol. e.g. - complement fixation - transplacental passage

Dimer

J.chainL H

H LMonomerA. Serum IgASchematic representation of serum IgA, secretory IgAL H

H LB. Secretory IgA (dimer)Secretory component LH

H LJ. chain

* IgG : basic tetrameric structure IgA : polymers of 2 3 tetrameric units IgM : 5 tetrameric units* Both L & H chains : products of multiple genes L - chain : product of 3 structural genes : - VL gene - Joining region (J) gene - CL gene H - chain : product of 4 different genes : - VH gene - Diversity region (D) gene - J gene - CH gene

* Disorders of Ig - Increased production of specific classes of Ig / specific Ig mol Multiple myloma : tumor of plasma cells Electrophoresis of serum /urine increased of 1 particular Ig / 1 particular L chain (Bence Jones Protein) - Decreased production : single class of Ig mol ( e.g. IgA or IgG) a gamma globulinemia : genetic abnormality of IgG impairment of the bodys defense against microorganism (immunodeficiency) all classes of Ig

The Complement System * Involved in : - cell lysis, inflammation, other process* - comprises 20 plasma proteins - labile when heated at 56oC - major protein components : C1-9, C9 associated with C5 8 complex membrane attack complex : generating a lipid-soluble pore in cell membrane osmotic lysis* Detail of the system : complex Basic concept : Inactive protein of the system stimulated activated (by proteolysis) interact with other proteins of the system result : - cell lysis - generation of peptide fragments various aspects of inflammation : chemotaxis, phagocytosis etc.

Other function of the system : - clearance of antigen - antibody complexes from circulation

* Activation of the system :- classic pathway : interaction of C1 with antigen antibody complexes- alternative pathway : direct interaction of bacterial cell surfaces (polysaccharides) with C3b ( not involving antibody)