Blood plasma and blood cells Biochemistry II Lecture 11 2008 (J.S.) Blood serum proteins- the six main fractions iotai serum proteins bz — oz g /1 Electrophoretic separation on a cellulose acetate strip (pH 8.6) start i globulins y Densitogram A ß2ßi oc2 a, albumin Normal values (mass fraction of total proteins) Albumin 0.50- 0.62 0.55 a,-Globulins 0.03- 0.06 0.05 a2-Globulins 0.07- 0.13 0.10 ß1ß2-Globulins0.09- 0.15 0.12 7-Globulins 0.14- 0.22 0.18 2 Blood plasma proteins About 10 000 proteins were estimated, from which 22 high abundance proteins represent approximately 99 % of total protein in human plasma. Transthyretin (prealbumin) Albumin 0,-Globulins acid ^-glycoprotein ^-antitrypsin antithrombin III apolipoprotein A I, All cc2-Globulins a2-macroglobulin C3, C4-components haptoglobin ceruloplasmin plasminogen P^GIobulins transferrin haemopexin fibronectin apolipoprotein B100 P2-Globulins fibrinogen C1q-component y-Globulins immunoglobulins G, A, M, D Separation effractions in ultracentrifuge Starch-gel electrophoresis (two-dimensional) 5 14 Immunoelectrophoresis 6 Most of the high abundance plasma proteins, except for immunoglobulins, are synthesized in the liver. Albumin is the major plasma protein, normal concentration 35 - 53 g /1; about 10 -12 g albumin are produced daily. Mr« 67 000 (585 amino acid residues). Albumin is essential for the maintaining of oncotic pressure in capillaries. Because of a negative net electric charge (~ 12 mmol/l), it acts as an important buffer base and in binding of Ca2+ (about 50 % of total calcium). Hydrophobic areas of the surface of albumin molecules provides the transport of free fatty acids, bilirubin, and also, weakly and non-specifically of steroid and thyroid hormones, and numerous drugs (e.g., salicylates, penicillins, sulfonamides, and barbiturates). Hypoalbuminaemia occurs in liver diseases, nutritional depletion, due to losses in renal diseases, chronic intestinal inflammations, vast burns, as well as in hyperhydration. A reduction in plasma oncotic pressure results in oedema. 7 Transthyretin (prealbumin) is a tetrameric protein, Mr 50 000. Serum concentration 100 - 400 mg/l. Biological function - binding of thyroxin and retinol-binding protein. Due to its very short biological half-life (2 days) it serves as a marker of malnutrition, impairment or recovery of liver proteosynthesis. Haptoglobin (Hp) is an a2-sialoglycoprotein with haemoglobin-binding capacity that prevents both iron loss and kidney damage during haemolysis; the complexes Hb-Hp are rapidly captured by the reticuloendothelial system. Concentration in serum of adults 0.4 - 2.1 g/l, it falls in haemolysis. Molecular polymorphism of Hp exists: there are three major phenotypes -Hp1 -1, Hp 2-2, and the heterozygous Hp 2-1. Molecular mass of Hp 1 -1 is 86 000, that of Hp 2-2 from 170 000 to 900 000. Hp also protects against free radicals in haemolysis and exhibits an antiinflammatory action by inhibition of prostaglandin synthesis. Haemopexin p^Glycoprotein, Mr« 70 000, binds free haem, if it appears in the plasma, so that it may be captured by the liver cells (receptor-mediated endocytosis). 8 Transferrin pr Glycoprotein, Mr« 79 000, serum concentration 2.5 - 4.0 g /1. It transports Fe3+ ions. Molecule of transferrin can bind two ferric ions, Under normal conditions, about 1/3 of the total iron-binding capacity is saturated. In iron deficiency, the synthesis of transferrin is stimulated. In chronic alcoholism, glycosylation of transferrin is impaired and detection of carbohydrate-deficient transferrin (CDT) may serve as a marker of chronic alcohol abuse. Ceruloplasmin is an a2-globulin, a blue protein, because of firmly bound 8 Cu2+ ions. Mr 132 000. Serum concentration 150 - 600 mg /1. Even though ceruloplasmin contains about 90 % of copper plasma content, it doesn't take part in Cu2+ transport. Biological function of ceruloplasmin is the ferroxidase activity that prevents the occurrence of Fe2+ ions and possible Fenton reaction. Thus it is viewed as one of endogenous antioxidants. ^-Antitrypsin (o,AT, ^-proteinase inhibitor) is an (^-glycoprotein, normal serum concentration about 2 - 4 g /1. Mr« 54 000. This protein inhibits proteinases released from polymorphonuclear leukocytes (namely elastase) and other proteinases, which may occur in blood plasma and attack the elastin between alveoli in the lung. ^-Antitrypsin deficiency is one of the common inborn error. Individuals with the genotype ZZ produce less than 15 % on usual amounts of o^AT and they are exposed to a high risk of pulmonary emphysema due to enzymatic degradation of elastin in the lungs, with consequent reduction of the surface area available for gas exchange. Smokers also risk an insufficient effectivity of cc, AT: Components of tobacco smoke oxidize the sulfide group of methionyl residue in position 358 of o^AT (which takes part in interactions with proteinases) to sulfinyl group that disables the interactions. In addition, the smoke irritates the tissue and increased occurrence of leukocytes results in a higher local activity of proteinases. 10 The acute phase proteins (APP) Positive acute phase proteins Their hepatic synthesis is induced by numerous cytokines that enter the circulation as products of, e.g., macrophages, epithelial cells, and fibrocytes. C-reactive protein, CRP Serum amyloid A protein Acid (^-glycoprotein (^-Antitrypsin Haptoglobins Fibrinogen Ceruloplasmin C3 and C4 components response time 6 - 8 h increase 10-100 times response time 24 h increase 2-4 times response time 48 h increase by 50 % APP type I APP type II stimulated by TNF-oc, IL-1, and IL-6 stimulated by IL-6 and glucocorticoids CRP fibrinogen acid ^-glycoprotein a2-macroglobulin haptoglobins ^-antitrypsin and other serpins haemopexin ceruloplasmin, hepcidin 11 Negative acute phase proteins Their synthesis in the liver is decreased in the catabolic state Transthyretin (prealbumin) response time < 24 h Transferrin 24-48 h Albumin >48h Biological half-lives of some plasma proteins in davs Albumin 17- 19- 23 Immunoglobulins G 15- -18- 26 Transferrin 7- -8.5- -10 Immunoglobulins A 5.5 Acid o^-glycoprotein 5.2 Fibrinogen 4- -4.5- -5.5 Haptoglobins 4 Immunoglobulins M 4 Transthyretin (prealbumin) 2 Haemostasis If the smaller vessels are injured by traumas, the leakage of blood is discontinued normally in few minutes due to a series of interactions between the vessel wall, blood platelets, coagulation factors, and the fibrinolytic system. The initial step in haemostasis is arteriolar vasoconstriction, which temporarily reduces local blood flow. Blood platelets adhere then to the vessel wall at the site of injury, aggregate to each other, forming so the initial, unstable primary platelet plug ("white thrombus"). Vascular injury also activates coagulation factors that form thrombin, which converts plasma fibrinogen to insoluble, crosslinked fibrin and relatively resistant - the secondary, platelet-fibrin plug ("red thrombus"). The blood cells are caught in a network of fibrin. Local formation of fibrin activates local generation of plasmin, an enzyme of the fibrinolytic system, which digests fibrin plugs (in parallel with tissue repair processes). Vasoconstriction is either a reflex to an injury or the result of stimulation by serotonin, thromboxane TXA2, and platelet derived growth factor (PDGF), which are released from activated platelets. Injury of endothelial cells enables the contact blood with subendothelial collagen fibres and endothelial cells begin to secrete von Willebrand factor (vWF), a large protein, which is the carrier for coagulation factor VIII and promotes platelet adhesion to collagen. Blood platelets adhering to collagen are activated - they change their shape to spherical and form pseudopodia. http://www. platelet-research .org/ 14 Activated platelets release from their granules compounds that stimulate aggregation of platelets and thus formation of the primary platelet plug serotonin (5-hydroxytryptophan), ADP, thromboxane TXA2, fibronectin, platelet derived growth factor (PDGF), and platelet activating factor (PAF, an 1-O-alkylglycerophospholipid). Aggregation of platelets is supported also by von Willebrand factor (vWF produced by endothelial cells), thrombin, and fibrin which originate as products of coagulation cascade from plasma proteins prothrombin and fibrinogen. 15 The blood clotting cascade Intrinsic pathway Contact system Damaged surface - contact with subendothelial collagen (negatively-charged) (ph ospholipids) activated platelet surface High MW kininogen Kallikrein I Prekallikrein Factor XII (Hageman) Factor XI (PTA) Xlla I Ca2+, PL Xla I Factor VIII (AH PA) Factor IX (AHP B) Villa Thrombin Common pathway Factor V i f\ f*/r\ fZ) f** q I f lpi vOUUCICl Factor X/^ Ca2+, PL (Stuart-Prower) Va ........... Xa I Extrinsic pathway Cellular injury Tissue factor (tissue thromboplastin, t-TP) I ■ PL Factor VII (plasma protein) PL Factor X Prothrombin i Din Vitamin K metabolic cycle in the liver cells Glu -HH-CH-CO- OH K hydroquinone -I IH-CH-Cu- lt 2,3-epQxide CO, + H,0 Gla y-Carboxylation of Glu residues that forms Gla Ca2+-bindinq centres is an essential step of posttranslational processing of blood coagulation factors VII, IX, X, and prothrombin. The two stages of reduction of vitamin K epoxide to the hydroquinone are inhibited by coumarin anticoagulants warfarin ordicoumarol (analogues of vitamin K) used as inhibitors of blood clotting in the treatment of thrombosis. 17 Fibrinogen central domain terminal D-domain N-ends C-ends fibrinopeptide B jfiy. fibrinopeptide A Glycoprotein, 330 kDa 6 chains - (Aa B(3 y)2 1.5-4 g/l (plasma (32-globulin fraction) C-ends disulfide bridges 40 40 Fibrin monomer (a ß y)2 a Fibrin "soft" clot (electrostatic interactions) + 2 fibrinopeptides A (16 AA) + 2 fibrinopeptides B (14 AA) Fibrin "hard" clot - factor Xllla (transglutaminase, fibrin ligase) catalyses formation ; of covalent cross-links (isopeptide bonds) between side chains of glutaminyl and lysyl residues 18 The cascade of the clotting system permits enormous amplification of its triggering signals. Factors limiting clot growth: PROTEIN C (a proteinase) 1 Factor Xlla Heparin (mast cells) ANTITHROMBIN III, a2-macroglobulin, heparin cofactor II, (^-antitrypsin Activation of platelet surface Fibrin clot THROMBOMODULIN '(complex with thrombin) PROTEIN S ENDOTHELIUM \ Fibrin degradation 19 The fibrinolytic system HMW kininogen Prekallikrein Surface-activated factor XII T (Streptokinase) Urinary-type plasminogen activator (urokinase) Tissue-type plasminogen activator (tPA) I' Plasminogen-activator inhibitor 1 __a2-Anti plasm in Plasminogen ^ PLASMIN (a proteinase) Fibrinogen......► Fibrin Clot Fibrinogen and fibrin degradation products (FDP) (soluble fragments D, E, D dinners, etc.) Thrombolytic treatment in myocardial infarction or embolism are effective, if administered early enough, before irreversible damage of the tissue occurs. Urokinase is an proteinase that activates plasminogen directly. It is secreted by epithelial cells of renal tubules. Streptokinase is a plasminogen activator produced by ß-haemolytic streptococci. Tissue-type plasminogen activator (t-PA, alteplase) and other thrombolytic drugs (streplase, saruplase) are produced by recombinant gene technology. 21 Red blood cells - erythrocytes (RBC, Eres) Biconcave shape, diameter 8 jam, deformations are possible. High surface-to-volume ratio facilitates gas exchange. Nonnucleated, no cellular organelles, cytoskeletal components. Concentration of haemoglobin in RBC is about 330 g /1 (~ 95 % of all proteins). Production of erythrocytes from red cell progenitors is located in the bone marrow and regulated by erythropoietin synthesized mainly by the kidney. Reticulocytes still containing ribosomes and elements of ER are released into the circulation where they transform into adult red blood cells. Colored scanning electron micrograph (SEM) of red blood calls flowing through a blood vessel (Photo by 'Dr. Philippa Uwins, WhistiBr Research). 22 Erythrocyte membrane 52 % proteins, ^E^mcy* 40 % lipids, and |. 8 % saccharides V 1 Erythrocyte Aquaporins, glucose transporters as well as other membrane proteins are not shown. Glycophorins are transmembrane single-passing glycoproteins. The saccharidic component (60 % by mass) consists of numerous oligosaccharides. It is highly sialylated and represents the major part of the glycocalyx on the outer surface. The negative electric charges prevent agglutination of RBC. Polymorphism of glycophorin A in its amino acid sequence denotes the MN blood groups of individuals' erythrocytes. Cytoskeletal proteins are fixed to the inner surface of the membrane and help determine the shape and flexibility of the RBC. Spectrin is the major cytoskeletal protein. It consists of two long polypeptide chains that form a loosely coiled dimer; two dimer form a tetramer, on which are binding sites for other cytoskeletal and membrane proteins (ankyrin, actin, protein 4.1). Spherocytosis is a hereditary deficiency in the amount of spectrin hen or abnormalities of its structure. H°Cfl^t The spherocytes are more susceptible to osmotic lysis than are normal Ere. dimer of spectrin 24 Metabolism of the red blood cell Anaerobic glycolysis, producing lactate, is the energy source. The synthesis of 2,3-bisphosphoglycerate, closely associated to glycolysis, affects the affinity of haemoglobin for dioxygen. The pentose phosphate pathway is efficient, it metabolizes up to 10 % of the total flux of glucose. NADPH produced is required for the reduction of oxidized glutathione and methaemoglobin In the adult RBC, glycogenesis, synthesis of fatty acids, cholesterol, proteins, and nucleic acids cannot occur, as well as catabolism of fatty acids and ketone bodies. Some lipids (e.g. phospholipids, cholesterol) from the red cell membrane can exchange with corresponding lipids of plasma lipoproteins. 25 Erythrocytes and oxidative stress High partial pressure of 02 and the presence of Fe" in haemoglobin represent a menace to processes and structures within erythrocytes. Efficient antioxidants protect RBC from damage caused by oxidative stress. - Superoxide dismutase and catalase decompose superoxide anion and hydrogen peroxide. - Glutathione peroxidase catalyzes reduction of hydrogen peroxides by GSH (reduced glutathione). GSH is regenerated by NADPH in the reaction catalyzed by glutathione reductase: ROOH (or H202) x 2 G-SH x NADP+ H20 + ROH (or 2 H20) G-S-S-G NADPH + H+ glutathione peroxidase glutathione reductase 26 NADPH is required for regeneration of glutathione to its reduced form GSH. NADPH is generated in two reaction of the pentose phosphate pathway catalyzed by glucose-6-P dehydrogenase and 6-phosphogluconate dehydrogenase. Deficiency of glucose-6-phosphate dehydrogenase is the most common of all inherited enzymopathies, caused by point mutations within the gene located in chromosome X. It is extremely frequent in some regions of the world: in tropical Africa, the Mediterranean, in certain parts of Asia, and, for example, among Afroamericans (11 % incidence). The deficiency is quite benign in the absence of oxidative stress. However, an exposure to oxidants (e.g. drugs - antimalarial pamaquine, sulfonamides, chemicals - naphthalene, consumption of fava beans, some infections) may result in a severe attack of haemolytic anaemia, because namely RBC are sensitive to increase in production of oxygen radicals and peroxides. On the other hand, this enzyme deficiency protect against falciparum malaria. The parasites causing this disease require reduced glutathione and the products of the pentose phosphate cycle for optimal growth. 27 - Methaemoglobin reductase (cytochrome b5 reductase) is a component of the NADH-cytochrome b5 methaemoglobin reductase system, which reduces methaemoglobin-Fe111 back to haemoglobin-Fe that is able to transport dioxygen. In the blood of healthy individuals, less than 1 % of total haemoglobin is present in the form of methaemoglobin. Inherited methaemoglobinaemia - inherited deficiency of MetHb reductase. Acquired methaemoglobinaemia occurs after ingestion of certain drugs (e.g. sulfonamides) or chemicals (e.g. aniline, nitrites, in sucklings also nitrates). Evident cyanosis appears usually when more than 10 % of total haemoglobin is oxidized to methaemoglobin. Hb(Fe") NADH + H+ methaemoglobin methaemoglobin reductase (cyt b5 reductase) 28 Polymorphonuclear leukocytes (PMN) Neutrophils are the most numerous circulating leukocytes (50 - 70 %). They have an important role in non-specific defence mechanisms - they can move along a chemical gradient of leucotactic substances to the site of a tissue injury or bacterial infection. Neutrophils are microphages. Metabolism Considerable activities of glycolysis, glycogenesis, and the pentose phosphate pathway. Due to low number of mitochondria, only slight activity of the citric acid cycle and oxidative phosphorylation. The proteosynthetic apparatus is developed less perfectly than in other cells. Some special enzyme activities e.g. NADPH oxidase and myeloperoxidase. The biological half-life of neutrophils is about 6 -7 hours in the blood, a few days in the connective tissue. Neutrophils can survive even under anaerobic conditions. 29 Phagocytosis - the role of neutrophils in antibacterial defence After bacterial invasion into a tissue, neutrophils begin migration from the capillaries to the site of infection. Their movements are initiated and directed by chemotaxis. Leucotactic substances (attractants) are, for example, various complement components, small bacterial peptide fragments, and eicosanoids, namely leukotriene LTB4 Neutrophils adhere to endothelial cells of the capillary wall, the process supported by membrane proteins integrins and selectins is called margination of neutrophils, and penetrate through the capillary wall -diapedesis - to the site of infection. Then they actively engulf microorganisms or other small particles by phagocytosis. Bacterium or a foreign particle is encompassed by pseudopodia and phagosome originates after complete closure. Phagosome fuses with lysosomes and specific granules into phagolysosome, vacuolar H+-ATPase maintain the content at pH about 4, and hydrolases catalyze digestion of organic components. 30 Examples of important proteins in neutrophils: Primary granules (lysosomes) Hydrolases cathepsin B - an acid proteinase elastase - a neutral proteinase able to split elastin p-glucuronidase - an acid specific glycosidase, absent in other cell types lysozyme - splits muramic acid, a peptidoglycan of bacterial walls Myeloperoxidase - catalyzes formation of HCIO from peroxide and chloride Defensins - small basic peptides that easily invade into lipid dilayers Secondary (specific) granules Hydrolases collagenase - a metalloproteinase hydrolyzing collagen lysozyme - muramidase Lactoferrin - a protein that binds firmly ions of iron 31 The respiratory burst of phagocytic cells is the sole profitable utilization of reactive oxygen species production - it helps kill bacteria engulfed by phagocytic cells. Interaction of neutrophils with bacteria, binding of chemotactic factors or immunocomplexes onto specific receptors in plasma membrane activate motility of neutrophils, secretion of granules, and the activity of an membrane enzyme NADPH oxidase (a flavoprotein) and cytochrome b558 that initiate the respiratory burst: 2 02 + NADPH NADPH oxidase 2 .02- + NADP+ + H+ Cyt fc558 The consumption of 02 by the cell rises steeply due to superoxide production, which results in formation of hydrogen peroxide (a spontaneous dismutation of superoxide anion): 2 -CV -► H202 + 02 Myeloperoxidase catalyzes the production of hypochlorous acid - an effective microbicidal agent H202 + CI- + H+ -► HCIO + H20 In a similar way, peroxynitrous acid HO-O-NO is formed from nitroxide NO. 32