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Tissue Chemistry & Biological Fluids Biochemistry has passed from a state of descriptive to quantifiable science. As a biochemist, you should always be.

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Presentation on theme: "Tissue Chemistry & Biological Fluids Biochemistry has passed from a state of descriptive to quantifiable science. As a biochemist, you should always be."— Presentation transcript:


2 Tissue Chemistry & Biological Fluids Biochemistry has passed from a state of descriptive to quantifiable science. As a biochemist, you should always be interested in things about metabolic sequences: The description of the enzymes & chemical changes that comprise the metabolic sequence The rate at which material can be transformed by the sequence The amount of material utilized by the sequence among living things The nature of the control mechanisms which adjust the amounts of material utilized by the sequence 2

3 Wet weight (kg)Protein content (kg) Skeletal muscle306.6 Adipose tissue Stomach & intestine Liver Brain Kidneys Heart Adrenals0.014? Blood Skin4.9? Bone Roughly, the contributions of the different tissues to the body's metabolism are proportional to the weights of the tissue and the biological fluids 3

4 From the following table… It is not the sheer mass of tissue which determines its quantitative contribution to metabolic activity Activity of tissue is determined by its enzyme content 4

5 The body can be crudely divided into two components: Circulating TissuesBiological Tissues BloodCartilage WaterBone LymphSkin Interstitial fluidMuscles Cerebrospinal fluidLiver 5

6 Vertebrates have evolved 2 principal mechanisms for supplying their cells with a continuous & adequate flow of oxygen: A circulating system that actively delivers oxygen to the cells Acquisition of oxygen The oxygen carriers in vertebrates are the proteins hemoglobin & myoglobin 6

7 Blood In a normal weight, there is about 5-6 liters of blood (12%) or 85ml/kg It circulates as a homogenous suspension of erythrocytes, leukocytes & platelets in a solution of proteins, inorganic ions, & low-molecular weight organic compounds 7

8 Functions of the Blood Transport of nutrients Exchange of respiratory gases Transport of waste products Distribution of hormones & other regulatory substances Protection against microorganisms Acid-base, electrolyte & water homeostasis Heat regulation Prevention of excessive hemorrhage by coagulation 8

9 General Composition By volume, 40-45% of the blood consists of erythrocytes, leukocytes & platelets 1 mm 3 of blood contains: 5 x 10 6 RBCs; WBCs; platelets Male AdultsFemale Adults RBC x cells/L x cells/L WBC x 10 9 cells/L x 10 9 cells/L Platelets x 10 9 cells/L 9

10 The Packed Cell Volume (PCV Hematocrit) PCV Hematocrit = Volume of Red Cells/ Volume of whole blood x 100 Expressed as volume of erythrocyte per liter of whole blood Normal adult males = 41-53; adult females = Hematocrit used to determine PCV Color of supernatant plasma gives rough idea of bilirubin content & is often a useful clue about the nature of anemia: White plasma iron deficiency anemia Lemon yellow plasma Hemolytic or Megaloblastic anemia 10

11 Blood Volume & the Hematocrit Rarely necessary to have an accurate blood volume Hematocrit (HCT) is the volume percentage of erythrocytes in whole blood HCT is obtained by centrifugation The specific gravity of WBC's intermediate between plasma & RBC, thus forming "Buffy Coat" 11

12 Errors in the Estimation of HCT Usually up to 5% of the apparent RBC mass is plasma HCT differs according to blood source: - Some particles when centrifuged tend to accumulate in the center of the tube - HCT value is affected by movements of fluid (hydrostatic pressure) 12

13 Clinical value of HCT HCT is important in the diagnosis of anemia Rough estimation of blood loss after hemorrhage 13

14 Continuation… Whole Blood Whole blood – formed elements = plasma Plasma – Clotting factors = Serum 14

15 Physical Characteristics Arterial blood is crimson Venous blood is darker red Specific gravity = & the viscosity is 5-6 times that of water Specific gravity of plasma = Ph =

16 Erythrocyte Sedimentation Rate Rate of settling of RBCs after blood is drawn In healthy men : 1-3mm/hr; 4-7 mm/hr in young women Low ESR in patients with anemia Follows Stoke's Law (settling velocity) with an equation Where: V s is the particles' settling velocity (m/s) (vertically downwards if ρ p > ρ f, upwards if ρ p < ρ f ), r is the Stokes radius of the particle (m),Stokes radius g is the standard gravity (m/s 2 ),standard gravity ρ p is the density of the particles (kg/m 3 ),density ρ f is the density of the fluid (kg/m 3 ), anddensity η is the fluid viscosity (Pa s). 16

17 Continuation… ESR greatly increased during menstruation & normal pregnancy Increased rate also found in septicemia & pulmonary tuberculosis (increased globulin & fibrinogen content of plasma; also in elevated cholesterol & phospholipid levels Inflammation of various types that cause cell necrosis will cause rate of RBC to fall, but the viscosity remains unchanged. 17

18 Continuation… In alcoholic cirrhosis there is a rise in plasma bile acids & membrane cholesterol levels may rise by 55%. This has 2 effects: Cholesterol to phospholipid ratio is increased, reducing membrane flexibility Increased cholesterol content also raises total lipid present, expanding its surface area Increase by 8% in the total lipid is enough to cause formation of spherocytes (removed from the circulation as a result of alteration in size, shape and flexibility) Elevated levels of plasma bile acids (mainly cholic & deoxycholic acids) are observed in obstructive jaundice with similar consequences for the RBC membrane 18

19 Plasma Straw colored fluid with specific gravity from Specific gravity of plasma is related to its protein content Contains 90-92% water 19

20 Continuation… Blood owes much of its physiological importance to high water content: Maintaining blood pressure Important for heart regulation& in osmotic exchange between body fluid & compartments 20

21 Plasma Composition The solutes of the blood plasma constitute ≈ 10% of the volume Protein ≈ 7% Inorganic salts ≈ 0.9% Other organic compounds ≈ the rest other than proteins. 21

22 Separation of plasma proteins Based on the different mobility in an electric field Electrophoresis – widely used Isoelectric focusing Immunoelectrophoresis – separates proteins on the basis of electrophoretic as well as immunologic properties 22 Albumin Alpha 1 Alpha 2 Beta Gamma

23 Albumin & Globulins Comprise most of the proteins in the blood plasma Colloidal osmotic pressure (from the proteins of the plasma) is the force that opposes the hydrostatic pressure in the capillaries Better terminology should be "potential osmotic pressure" or "osmotic tendency" Albumin & Globulins 23

24 Proteins move in electric field by the charge they carry… Major fractions include: Albumin (54-58%) α 1 globulins (6-7%) α 2 globulins (8-9%) β 1 - globulins (13-14%) Gamma globulins (11-12%) 24

25 Enzymes of Plasma Most plasma enzymes do not have metabolic roles in plasma with the exception of those involved in coagulation Activity of certain plasma enzymes is useful as index of certain abnormal conditions: - Serum amylase – elevated in acute pancreatitis - Acid phosphatase – in cases of prostatic cancer - Alkaline phosphatase – in hepatic obstruction and bone diseases 25

26 Assay of tissue enzymes in plasma When organs are damaged part of their enzyme complement in released into the plasma. In a healthy persons, levels of intracellular enzymes are very low & a result of cellular turnover Tissues contain times higher content of soluble enzymes within their cells Intracellular enzymes released into the plasma are inactivated & removed within days Amount of enzyme released depends on the concentration of that enzyme & extent of tissue damage Knowledge of cellular location of enzyme provides good clinical information In practice, enzyme assays are most useful in detecting damage to the liver, muscles and blood cells 26

27 Enzyme Organ DistributionComments GOT Widespread but little in red cells Analysis of these enzyme started clinical enzymology CPK Widespread but skeletal muscle is richest source Monitoring skeletal & heart muscle disorder Γ-GT Mainly liver Marker for hepatocellular diseases LDH Widespread but has distinctive isoenzyme distribution Monitor heart & liver disease Acid phosphatase Specific activity in prostate glandMonitor prostatic cancer Alkaline phosphatase Widespread in tissuesDiagnosis of bone disease Some enzymes commonly assayed as part of clinical diagnosis 27

28 Continuation… CPK (Creatinine phosphokinase) & LDH 1 (Isoenzyme of LDH) indicate amounts of myocardial infarct. If no further damage occurs, levels return to normal. Congested liver can be due to inefficient pumping of the right side of the heart. Most patients with metastatic prostatic carcinoma have elevated plasma phosphatase levels. RIA is used for the detection of this enzyme. Enzyme assays may also reveal other organ involvement… 28

29 Erythrocytes Circulating erythrocytes are derived from erythropoietic cells (or erythron), the precursors of erythrocytes. RBCs arise from mesenchymal cells present in bone marrow Major functions Transport of oxygen from the lungs to the tissues Controls blood pH (CO 2 ) is converted to bicarbonate by carbonic anhydrase = major buffering system) RBCs lack nucleus & other organelles; utilizes anaerobic metabolism 29

30 30

31 Structure & Composition RBC s have a biconcave disc shape (6-9 µm in diameter; 1 µm thick; µm at the periphery) Most of the solid matter is hemoglobin ( the conjugated protein responsible for the red color of the blood) Behaves like an osmometer 31

32 The Erythrocyte Membrane Composed largely of protein (49%) & lipid (43%) with a small amount of carbohydrate (8%) Has a cytoskeleton which controls the shape of the membrane & limits the lateral mobility of some intrinsic proteins Some of the protein is glycoprotein covalently linked to CHO (Sialic acid) 32

33 Membrane Changes in Diseases Mature RBCs synthesize very little lipids but: Sphingomyelin Phosphatidylcholine in the outer half of the bilayer those in plasma lipoproteins Cholesterol exhanges freely with serum cholesterol The important factor that affects this exchange is the activity of the plasma enzyme Lecithin- Choelsterol Acyl Transferase (LCAT) – responsible for the formation of majority of esterified cholesterol and is inhibited by bile acids 33

34 Erythropoiesis During gestation, erythrocytes are formed in various tissues occurring successively in: Yolk sac – main site for the 1 st weeks of gestation Liver & Spleen - from 6 weeks to 6-7 months & can continue to produce until about 2 weeks after birth Lymph Nodes 34

35 Continuation… From 6-7 months of fetal life onwards… the bone marrow is the only source of new blood cells 35

36 Continuation… Erythroid cells in the bone marrow are called normoblast (a large cell with dark blue cytoplasm, a central nucleus with nucleoli & slightly clumped chromatin Reticulocytes A reticulocyte stage results when the nucleus is finally extruded from the late normoblast. In this stage it still contains some ribosomal RNA and can still synthesize Hb Reticulocytes spends 1-2 days each in the circulation & bone marrow before it matures mainly in the spleen when RNA is completely lost A single pronormoblast usually gives rise to 16 mature red cells 36

37 Substances needed for erythropoiesis The bone marrow requires many precursors to synthesize new cells: Metals: Iron, manganese, cobalt Vitamins: B 12, folate, ascorbic acid Amino acids Hormones 37

38 Hemolysis Maybe produced by substances that dissolve or change the state of membrane lipids (ether, chloroform, bile salts & soaps). Certain biological toxins (venomous snakes & hemolytic bacteria) Physical forces (UV rays, freezing, thawing) Aging – this is why whole citrated blood cannot be used after 5-7 days 38

39 Red Cell Metabolism The components required for these include: 1. ATP – maintenance of membrane function 2. 2,3 –diphosphoglycerate (2,3 – DPG) to modulate O 2 affinity 3. NADPH – to prevent hemoglobin denaturation 4. NADH – to maintain the heme in the Fe(II) state 39

40 Continuation… The predominant metabolic fuel is glucose where they serve as gluconeogenic precursors The 2 ATP molecules are utilized in the ion pump in the cell membrane Failure to produce enough ATP results in an ability to maintain ionic balance leading to accumulation of Ca 2+ and shape change 40

41 41

42 Continuation…  2,3 DPG is a metabolite unique to the RBC. At a concentration of 4-5mM, it is almost equimolar to Hb 20-25% of 1, 3 DPG pass to 2, 3 DPG by mutase, therefore ATP yield decreases from glucose 2,3 DPG depends on the relative rates of the mutase& phosphatase reactions 42

43 Glutathione Can be used for the removal of H This reaction protects the membrane from oxidative damage. A deficiency of any enzyme of the glycolytic, phosphogluconate or GSH-GSSG pathway may seriously compromise the energy dependent maintenance of membrane integrity. 43

44 Hereditary Hemolytic Anemias Have been associated with deficiencies of the following enzymes: Enolase enzyme – deficiency leads to decreased ATP required to maintain the biconcave shape of RBC Glucose-6-P-Dehydrogenase – deficiency may result in increased hemolysis & severe hemolytic anemia Pyruvate Kinase - deficiency may lead to bizarre model which is extremely fragile and readily hemolyzed Other enzymes such as hexokinase,glucose – phosphoisomerase, phosphofructokinase, triose phosphate isomerase, 2-3 diphosphoglycerate dismutase 44

45 Erythrocyte Destruction Senescent erythrocytes are engulfed primarily in the reticuloendothelial cells of the spleen Free hemoglobin is released and binds to plasma proteins (e.g. haptoglobin) Complex is transported to liver where Hb portion is split Heme portion is transported to plasma & converted to bilirubin; excreted in the bile Iron is released & stored in the liver for reuse 45

46 Hemoglobin 1 liter of blood usually contains 150g of hemoglobin; each gram can combine with 1.34ml of oxygen 46

47 Continuation… 1 liter of blood can carry 200ml of oxygen, 87 times higher than plasma alone. Each RBC contains ≈ 640 million Hb molecules 47

48 Hemoglobin Structure The 4 chains are held together by non-covalent bonds There are 4 binding sites for oxygen The Hb molecule is nearly spherical; packed together in a tetrahedrical way 48

49 Continuation… The amino acid sequence of hemoglobin is known for 20 species. However there are 9 positions in the sequence that contain the same amino acid in nearly or all species studied. These conserved positions are especially important for the function of hemoglobin: 1. Some of them are involved in oxygen binding sites 2. Stabilizing the molecule via forming H-bond between the helix 3. Some (e.g. GLY) for easy contact between the chains 4. Some (e.g. PRO) to terminate the elix 5. The non-polar residues (Alanine, Isoleucine) are important because reversible oxygenation of heme group depends on its location where it is protected from water. 49

50 Continuation… Normal hemoglobin is of several types containing 4 sub-units made up of various combinations of 4-5 different related peptide chains HemoglobinStructureStage of Life% in Adult % in Newborn Gower Iζ2ε2ζ2ε2 0-5 weeks embryoNoneUp to 40 Gower IIα2ε2α2ε weeks embryo NoneUp to 35 Portlandζ2γ2ζ2γ weeks embryo NoneUp to 35 Fetal (F)α2γ2α2γ2 Newborn & adult< A1α2β2α2β2 Newborn & adult9720 A2α2δ2α2δ2 Newborn & adult2.5<

51 Biosynthesis of Hemoglobin It has been estimated that there are 30 trillion erythrocytes in the circulating blood & ≈ 3 million/sec are destroyed Globin moiety is formed from amino acids from the body pool in amounts of about 8g/day in the normal adult 14% of the amino acids from an average daily protein intake are used for globin formation 51

52 52

53 Availability of Fe ++ Total body content of iron is about 2-6g & is not excreted in this form Found in porphyrin ring of the heme complex The first type of compounds (Hb, myoglobin, cytochromes,catalase) are associated with the physiology The second type is concerned with absorption, transport & storage of iron 53

54 Iron Absorption No iron absorption takes place in the stomach Stomach acid is essential for iron reduction Duodenum contains "apoferritin" (converts Fe ++ to Fe +++ ) Ferritin may then act as an iron store or transported to the serosal side where it is released in the ferrous form In the blood, iron is bound by specific α-1 Globulin (transferrin) 54 Iron is stored in the liver & bone marrow in 2 forms: Ferritin & Hemosiderin (agglomeration of ferritin molecules)

55 Transport of Oxygen If arterial blood is analyzed for its oxygen content, it is found to contain 18-20% volume Hb is an allosteric protein: the binding of additional O 2 Hb enters the binding of additional O 2 to the same Hb molecule 55

56 Continuation… The sigmoidal property of the curve is believed to be due to heme-heme interactions Heme-heme interaction means binding at one heme facilitates the binding of oxygen at the other hemes on the same tetramer & vice versa 56

57 Cooperative Property When environmental oxygen levels are high, partially saturated hemoglobin molecules exhibit enhanced affinity for binding additional oxygen molecules, a specialized behavior referred to as cooperativity. Hb has the capacity to bind between 1 and 4 O 2 molecules, ranging from fully "desaturated" Hb (deoxyHb) to fully "saturated" Hb (oxyHb). As part of this process, Hb also serves to replenish the "oxygen stores" maintained by myoglobin (Mb), the O 2 -binding protein in muscle which releases its oxygen in response to high levels of muscle activity. 57

58 2, 3 Bispospoglycerate (2,3 –DPG) Is very important for long-term regulation of Hb affinity to O 2 2,3 BPG shunt is a pathway derived from glycolysis. Competition with oxygen for binding site on ß-subunits Hypoxia stimulates 2,3 BPG synthesis, i.e. improve O 2 release 2,3 DPG binds electrostatically to β- subunits through Lys 82, His 143 & N-term. The juxtaposition of these groups is favorable for 2,3-DPG binding only in the T-state. DPG stabilizes the deoxyHb by cross-linking the β chains. In other words, DPG shifts the equilibrium to tense form. 58

59 Clinical Significance of DPG It has been approved that DPG levels decrease from 4.5mM to 5.0mM Ill patients may take longer time to regain DPG when blood is transfused Inosine can be converted to DPG inside RBC. Inosine can now be used to preserve integrity of stored blood In hypoxia (e.g. emphysema), airflow in the bronchioles is blocked so the pressure increases as DPG increases. DPG levels lead to 27% increase in the amount of oxygen due to pressure changes. (Also in high altitude adaptations) Fetal Hb has high affinity to transfer oxygen from maternal to fetal circulation. HbF binds DPG less strongly than does HbA & consequently has higher oxygen affinity. 59

60 Life span of RBCs RBCs have a limited life span of 120 days The " Red Cell Theory of Aging " is based on observed Ca 2+ that occurs in old erythrocytes Ca 2+ rises to 0.5mM, enough to activate transglutaminase present in cell membrane Transglutaminase form cross-links by creating iso-peptide bonds Red cells with cross-linked membrane proteins are less flexible & are removed in the circulation by the spleen 60

61 RBC Destruction Hemoglobin from senescent erythrocytes (phagocytosed in the reticuloendothelial cells of the spleen) are transported to the liver bound to the plasma protein haptoglobin The globin portion is reused as amino acids & the heme moiety is converted to several steps to the bile pigments Bilirubin, Urobilin & Stercobilin are colored (BILE PIGMENTS) 61

62 Continuation… The pigments (biliverdin & bilirubin) are extracted in bile The iron of heme is removed & the process, bound to plasma transferrin & either recycled as new hemoglobin or stored in the liver as ferritin In the liver, bilirubin, either from Hb or from other hemoporteins is transported bound or loosely associated with plasma albumin In the small intestine… Conjugates with glucoronic acid to form bilirubin diglucoronide which is water soluble & is readily excreted by means of the bile into the intestine Hydrolysis in the intestine by a β-glucoronidase into bilirubin & glucoronic acid Reduction of bilirubin by bacterial floral action to colorless D or L- Urobilinogen 62

63 Urobilinogen First part is reabsorbed & excreted in the urine as oxidized orange-yellow pigment L-Urobilin Second part is reduced in the intestine to L-Stercobilinogen & excreted as an oxidized pigment L-Stercobilin in the feces (Faecal urobilinogen) Urinary urobilin is increased if… Hemolysis is excessive when large amounts of bilirubin enter the bowel & are converted to stercobilinogen There is liver damage (impairs re-excretion of normal amounts of urobilinogen into the bile) 63

64 Erythrocyte Abnormalities A large number of human diseases are associated with abnormal function of the erythrocytes including Altered rates of erythrocyte production & destruction Defects in iron or heme metabolism Combination of these conditions These diseases are called ANEMIAs… 64

65 Nutritional Deficiency Anemia Hematopoietic precursor cells are particularly sensitive to any insult that impairs DNA synthesis. This leads to appearance of characteristic megaloblasts - corresponds to normoblasts (characterized by increased ratio of RNA to DNA). The cause can be deficiencies in metal traces, folic acid & vitamin B12 Larger amount of hemoglobin than other proteins, & constant daily losses of Hb must be replaced by resynthesis 65

66 Continuation… Iron-deficiency – lack of dietary iron or excess blood loss (e.g menstruation) Folic acid deficiency – its deficiency leads to megaloblastic anemia as it is a co-factor for a variety of reactions to 1-carbon metabolism (synthesis of purines & thymines) Vitamin B 12 deficiency – deficiency leads to pernicious anemia. Based on malabsorption of Vitamin B 12 due to failure of the gastric mucosa to secrete adequate intrinsic factors 66

67 Hemolytic anemias  Anemias associated with increased destruction of erythrocytes, characterized by shortened life span of cells Isoimmune hemolytic disease – in newborns; caused by transplacental transfer of maternal blood-group Abs capable of reacting with fetal erythrocytes Hereditary spherocytosis - associated with the presence of spherical erythrocytes that are more fragile to hypotonic solutions; nature of defect unknown 67

68 Continuation… Paroxysmal nocturnal hemoglobinuria – erythrocytes are abnormally sensitive to lysis by complement Sickle Cell anemia – abnormal Hb, HbS aggregates on deoxygenation & the aggregates deform the shape of the cell, rendering susceptible to lysis Thalassemias – caused by defective synthesis of α & β globin chains 68

69 Sickle Cell Anemia 1. Characterized by the sickle-cell or crescent shape of the erythrocytes when the oxy HBs is converted to deoxy HbS at low PO At intracellular concentrations, molecules of deoxy HbS aggregate to form filaments on tubules of indeterminately high molecular weight 3. The sickle-cell causes severe anemia since they have increased mechanical fragility 4. Sickle cells also impede blood flow through capillaries 5. It is genetically transmitted 6. Vigorous physical activity at high altitude, air travel in unpressurized plane, & anesthesia can be potentially hazardous to a person with this disease 69

70 Characterization of HbS  HbS has between 2 & 4 more net + charges per molecule than net HbA  Non-polar residue on the outside of HbS (due to Val) causing low solubility  Sticky patch on the outside of its β chains & are present on both deoxy HbS & oxy HbS but not on HbA 70 AS pI of Oxy Hb = 0.22 pI of deoxy Hb = 0.23

71 Thalassemias  Normally the rates of synthesis of the α & β chains of Hb must be virtually identical. α -Thalassemia In α-Thalassemia, there is deficiency in α chains & hence β chains precipitate; β- thalassemia is the reverse Results from deletion of the α-globulin gene (Homozygotes with α-Thalassemia exhibit a syndrome known as hydrops fetalis) 71

72 Continuation… β- thalassemia  β- thalassemia are heterogenous  β- globin gene is deleted  β- globin gene remains intact & β- globin mRNA is synthesized but not translated  In many β- thalassemia, the β- globin gene is present but very little β- globin mRNA is produced 72

73 Blood Groups Isoagglutinins (blood group substances) are found on the erythrocyte surfaces & are responsible for the major immunological reactions of erythrocytes (Blood Types) Surface of RBCs carry antigens (agglutinogens), the plasma carry agglutinins 73

74 Continuation… ABO system is more complicated than the outline given. The ABO groups actually have 6 groups: A1A1 A2A2 BABA2BA2BO The following rules must be observed in blood transfusion: If the recipient's ABO group is known, give blood of the same group if possible Give Blood group O if the ABO group is unknown If the recipient's blood group is AB, neither antibodies are found are found in plasma so any red cells can be given 74

75 Rhesus Groups The term Rhesus (Rh) blood group system refers to the 5 main Rhesus antigens (C, c, D, E and e) as well as the many other less frequent Rhesus antigens. The terms Rhesus factor and Rh factor are equivalent and refer to the Rh D antigen only. Rh + gene is the dominant gene; Rh – is the recessive gene antigens GenotypeSymbolRh(D) status cde/cderr- CDe/cdeR1rR1r+ CDe/CDeR1R1R1R1 + cDE/cdeR2rR2r+ CDe/cDER1R2R1R2 + cDE/cDER2R2R2R2 + 75

76 Blood Coagulation  Injury to a blood vessel initiates a series of reaction involving 3 separate processes: 1. The damage end contracts 2. Platelets begin to adhere to the injured endothelium & form a plug 3. Blood clot formation 76

77 Clotting When the vessel is punctured or cut, the endothelium brings blood into contact with sub-endothelial collagen Platelets at the site of injury are influenced to stick As the platelets aggregate they release vasoactive amines (serotonin & epinephrine) & prostaglandin metabolites (thromboxane A 2 ) which stimulate vasoconstriction The plug is called thrombus & it is a major chemical defense against blood loss The actual blood clotting processes that lead to a proper clot are set into motion by 2 mechanisms: intrinsic & extrinsic pathways: 77 Must be initiated rapidly when the vascular system is damaged but must occur when the circulatory system is intact

78 The Coagulation Cascade 78

79 The Intrinsic (Intravascular system)  So termed because all factors involved are present in the vascular system  The 3 factors involved lead to the activation of factor X & in turn to the conversion of prothrombin to thrombin A. The Hageman factor binds to collagen or to vasoactive peptide such as Kallikrein, resulting to a proteolytically active form XIIa B. XIIa activates XI by hydrolyzing an internal peptide bond C. In the presence of Ca 2+, IX is activated to IXa. This activation is vitamin K dependent. D. In the final step factor X is converted to Xa by IXa in the presence of VIII (hemophilia A factor), platelet phospholipids and Ca 2+ ions 79

80 The Extrinsic (Extravascular system) The factors involved are supplements of the intrinsic to ensure more rapid coagulation Factor VII is converted to active form VIIa by factor III in the presence of Ca 2+ 80

81 Conversion of factor II (Prothrombin) to factor IIa (Thrombin) The rest of the reactions are common in both patrhways Factor Va, platelets, phospholipids & Ca 2+ to promote the reaction 81

82 Conversion of Factor I (Fibrinogen) to factor Ia (Fibrin) By thrombin, ARG-GLY in α-A & β-B chains of fibrinogen is released in a form of fibrinopeptide A & B from NH2 terminal ends of the chains. The gamma chain is not affected Factor VIIIa (the fibrin-stabilizing factors FSF is present in human platelets & in plasma Factor VIIIa (fibrinoligase) is a trans glutaminase that catalyzes the formation of cross-linked peptide bonds 82

83 Continuation… Once the clotting cascade is initiated, mechanisms must operate to prevent clotting from spreading throughout the intravascular system. Plasmin & fibrinolysin prevent such spread & dissolve any clots that do form. Plasmin is derived from inactive plasminogen in a reaction that requires blood & tissue factors including factor XIIa & Kallikrein 83

84 ANTICOAGULANTS In vitro… A number of substances prevent coagulation [oxalate, fluoride, citrate, EDTA – they precipitate Ca 2+ and bind to it] Bile salts are inhibitors of thromboplastin Dicumarol – is an antagonist of vitamin K by impairing its biosynthesis Heparin – is a complex polysaccharide β- diglucoronic acid & α-D-glucosamine 84

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