PHYSIOLOGY OF BLOOD FUNCTIONS OF BLOOD HOMEOSTATIC FUNCTION buffering thermoregulation (transport of heat) TRANSPORT OF SUBSTANCES (blood gases, nutrients, metabolites, vitamins, electrolytes…) HUMORAL CONTROL OF ORGANISM (hormones) DEFENCE OF ORGANISM (immune functions) BLOOD CLOTTING BASIC CHARACTERISTICS •Suspension character •6 - 8% total body mass –55% - fluid phase (plasma) –45% - formed phase (blood cells and platelets) •Serum: from plasma during blood clotting – after consumption of fibrinogen formed-elements-of-blood BONE MARROW Size (1600-3000 grams), activity. Red bone marrow, yellow bone marrow. Pluripotent stem cells. Unipotent (determined) stem cells – differentiated cells. Medullar haematopoiesis – ADULTS. Extra-medullar haematopoiesis ERYTROPOESIS Ontogenesis 3rd week – yolc sac 6th week – liver (formation in the yolk sac expires) 12th week – lien 20th week – bone marrow 32nd week – rearrangement from embryonic hemoglobin to HbF newborn – only in bone marrow, rearrangement HbF to HbA adult – sternum, vertebrae, ribs, clavicula, proximal epiphyses of some long bones Hematopoéza.jpg Source: Wikimedia Commons vývojové řady krvinek Regulatory function of megakaryocytes (MKs)! -control of bone marrow homeostasis -mesenchyal stem cells (MSCs) = an important regulator of MKs function via production of cytokines and soluble factors - role of MKs in modulating the replication and differentiation of osteoclasts and osteoblasts = regulation of bone formation and matrix reorganization -MKs represent an important reservoir of bioactive hemopoietic and angiogenic factors -MKs can directly regulate hemopoietic stem cells (HSCs) and next hemopoietic cells (mainly via IL-6) -Mks participate in angiogenesis Malara A, Abbonante V, Di Buduo CA, Tozzi L, Currao M, Balduini A: The secret life of a megakaryocyte: emerging roles in bone marrow homeostasis control. Cellular and Molecular Life Sciences 2015, 72(8):1517-1536. Cells Cells /ml (average) Normal range Percent of total number of leukocytes Leukocytes (total) 9000 3600 - 9600 Granulocytes Neutrophiles 5400 3000 - 6000 42 – 75 Eozinophiles 275 150 - 300 1 - 4 Basophiles 35 0 - 100 0,4 Agranulocytes Lymphocytes 2750 1200 - 3400 20 - 50 Monocytes 540 110 - 590 1,7 – 9,3 Erythrocytes woman 4,2 – 5,4 . 106 men 4,5 - 6,3 . 106 Platelets 300 000 140000 – 440000 BLOOD CELLS White blood cell count MCHC mean corpuscular haemoglobin concentration 320-360 g/l NORMOCHROMIA ¯/ HYPO/HYPERCHROMIA RBC (ERY) 3.5-5.5*1012/l POLYCYTAEMIA ¯ ERYTHROCYTOPENIA HCT haematocrite 0.38-0.49 l/l HGB concentration of hemoglobin 140-180 g/l POLYGLOBULIA ¯ ANAEMIA MCV mean corpuscular volume 80-95 fl MACKROCYTE ¯ MICROCYTE MCH mean corpuscular haemoglobin 27-32 pg – NORMOCHROMIA ¯/ HYPO/HYPERCHROMIA RED BLOOD CELLS (ERYTHROCYTES) Men Women Hematocrit (Hct) (%) 47 42 Erythrocytes (RBC) (106/ml) 4,5 - 6,3 x106 4,2–5,4x106 Haemoglobin (Hb) (g/l) 140 - 180 120 - 160 Mean volume of ery (MCV) (fl) = Hct x 10 / RBC (106/ml) 82 - 97 82 - 97 Mean content of Hb in ery (MCH) (pg) = Hb x 10 / RBC (106/ml) 27 - 33 27 - 33 Mean concentration of Hb in ery (g/100ml) = Hb x 100 / Hct 32 - 36 32 - 36 Mean diameter of ery (MCD) (mm) 7,5 7,5 Function of erythrocytes: blood gases transport RED BLOOD CELL EXAMINATION 1.Red blood cell count •normocytemia •erytrocytopenia (oligocytemia) •polyglobulia (polycytemia) 2. Concentration of haemoglobin •anaemia 3. Hematocrit SHAPE AND SIZE OF ERYTHROCYTES Shape: biconcave disc OPTIMAL RATIO OF SURFACE TO VOLUME!!! By 30% larger surface in comparison with the cell of the same size but of round shape!!! Anizocytosis – physiological, pathological. Price-Jones curve. Size: 7,5 mm in diameter, 2 mm thickness – normocytes. Microcytes (-osis): diameter below 6 mm, volume below 80 fl Macrocytes (-osis), megalocytes: diameter above 8.2 mm, volume above 95 fl Amount of haemoglobin in one red blood cell: hypochromia (below 27 pg Hb/ery), normochromia, hyperchromia Deformation of red blood cells. Fahraeus-Lindqvist effect. Gallagher PG: Abnormalities of the Erythrocyte Membrane. Pediatric Clinics of North America 2013, 60(6):1349-+. -Glycophorins A and B -major sialoglycoproteins of the human erythrocyte membrane which bear the antigenic determinants for the MN and Ss blood groups (MNS blood group) - 1.Transport proteins 2.Cell adhesion proteins 3.Structural proteins -Spectrin -the most prominent component (two isoforms α,β; a tetramer; a meshwork) -fixed to the membrane - ankyrin binding sites for several other proteins (glycophorin C, actin, band 4.1, adducin) -This organization keeps the erythrocyte shape. Transport proteins -Band 3 (Diego Blood group) -mediating the exchange of chloride (Cl−) for bicarbonate (HCO3−) across a plasma membrane -Aquaporin 1 = water channel (Colton Blood Group) -GLUT1 -Jk antigen -on a protein responsible for urea transport in the red blood cells and the kidney (aka human urea transporter 11- HUT11 or UT-B1) -Rh-associated glycoprotein (RHAG) (Rh Blood Group) -an ammonia transporter protein -Na+/K+-ATPase -Ca2+-ATPase -Na-K-Cl cotransporter -Sodium-chloride symporter -Chloride potassium symporter -Potassium intermediate/small conductance calcium-activated channel (Gardos channel) Cell adhesion proteins -ICAM-4 (Landsteiner and Wiener Blood System) -BCAM = Basal cell adhesion molecule (Lutheran blood group) Structural proteins •Establish linkages with skeletal proteins •Regulating cohesion •Ankyrin-based macromolecular complex •Protein 4.1R-based macromolecular complex –Protein 4.1 (Beatty's Protein) –Glycophorins C and D (Gerbich Blood Group) –XK (Kell blood group precursor) (Kell Blood Group) –RhD/RhCE (Rh Blood Group) –Duffy antigen/chemokine receptor (DARC) –Alpha-adducin –Dematin • Erythrocyte exceptions They lack organelles •no ATP production in oxidative phosphorylation •no ability to replace damaged lipids and proteins (low metabolic activities, with no ability to synthesize new proteins or lipids) Free radicals exposure •haemoglobin autoxidation (O2•- release) •a cell membrane rich in polyunsaturated fatty acids (susceptible to lipid peroxidation) •deformation in tiny capillaries; catalytic ions leakage (cause of lipid peroxidation) Erythrocyte metabolism 1. Glucose as a source of energy (GLUT1 transporter, insulin-independent) 2. Glycolysis generates ATP and 2,3-bisphosphoglycerate (the specific binding of 2,3-BPG to deoxyhemoglobin decreases the oxygen affinity of hemoglobin and facilites oxygen release in tissues) 3. The pentose phosphate pathway produces NADPH 4. Glutathione synthesis - the antioxidant defence system 3angl Sprague RS, Stephenson AH, Ellsworth ML: Red not dead: signaling in and from erythrocytes. TRENDS in Endocrinology and Metabolism 2007, 18(9):350-355. Poikilocytes – drop-like erythrocytes Schizocytes – fragmented erythrocytes Spherocytes – volume normal, diameter smaller, thickness bigger Eliptocytes – ecliptic shape Leptocytes – thin, centrally concentrated haemoglobin (thalasemia, after splenectomy) Akantocytes – prickly prominences MORPHOLOGICAL VARIATIONS OF ERYTHROCYTES Sicklecells FRAGILITY OF ERYTHROCYTES Haemolysis – destruction of red blood cell membrane. Types of haemolysis: a) physical b) chemical c) osmotic d) biological (toxic) e) immunological Spherocytosis - disorders of protein net responsible for shape and elasticity of erythrocyte membrane – actin, ankyrin, spectrin. Disorders of glucose-6-phosphate-dehydrogenase . Erythrocytes life span: 120 days, role of lien (double circulation), splenectomy. Reticulocytes. Gallagher PG: Abnormalities of the Erythrocyte Membrane. Pediatric Clinics of North America 2013, 60(6):1349-+. Sedimentation rate indirectly corresponds to suspension stability of blood. Method of Fahreus-Westergren (FW). Physiological values: men – women Units: mm to1 hr/2 hrs Physiological causes of increased sedimentation. Pathological causes of increased sedimentation. ERYTHROCYTE SEDIMENTATION Esrrack_350px Silverthorn, D. U. Human Physiology – an Integrated Approach. 6th. edition. Pearson Education, Inc. 2012. HAEMOGLOBIN Red pigment transporting oxygen. Protein, 64 450, 4 subunits. Hem – derivative of porphyrine containing iron, conjugated with polypeptides (globin). Embryonic haemoglobin: Gower I a Gower II (t2e2, a2e2), Portland Fetal haemoglobin: Hb F, b2g2, weaker binding of 2,3 DPG Adult haemoglobin: Hb A, a2b2 (141/146) Forms of haemoglobin: oxyhaemoglobin - O2 carbaminohaemoglobin – CO2 methaemoglobin – Fe3+ in hem carboxyhaemoglobin – CO Hemoglobin_t-r_state_ani Heme 300px-Hemoglobin Silverthorn, D. U. Human Physiology – an Integrated Approach. 6th. edition. Pearson Education, Inc. 2012. 5angl Abnormalities of haemoglobin production •haemoglobinopathy (abnormal structure of chains) •thalasemia (lower production of normal chains) •Sickle cell anaemia (Hb J) Synthesis and destruction of haemoglobin Hem: glycin a succinyl-CoA Globin: AMK Hem - globin: biliverdin, bilirubin (lumirubin – photo-therapy), bile. •Clinical aspects - Glycosylated haemoglobin (HbA1) • •formed by hemoglobin's exposure to high plasma levels of glucose •non-enzymatic glycolysation (glycation)- sugar bonding to a protein •normal level HbA1- 5%; a buildup of HbA1- increased glucose concentration •the HbA1 level is proportional to average blood glucose concentration over previous weeks; in individuals with poorly controlled diabetes, increases in the quantities of these glycated hemoglobins are noted (patients monitoring) • • •Sugar CHO + NH2 CH2 Protein • • • •Sugar CH N CH2 Protein • • • •Sugar CH2 NH CH2 Protein Schiff base Glycosylated protein Amadori reaction Glycoprotein, 39 000, a2-globulin. Recombinant erythropoetin. Small amount in plasma, urine, lymph, foetal blood. Inactivation: liver Origin: kidneys (85-90%) – endothelial cells of peri-tubular capillaries in kidney core, liver (10-15%) Stimulation of release: tissue hypoxia of any origin, alkalosis, cobalt salts, androgens, catecholamines (b-receptors) Effects: Erythropoetin responsive cell – differentiation into erythroid line: increase of synthesis of nucleic acids, increase of iron absorption in erythroid cells, stimulation of cells release from bone marrow into circulation Acclimation – adaptation to high altitude ERYTHROPOETIN HIF signaling in cells of the osteoblastic lineage regulate EPO expression in bone under physiologic and pathophysiologic conditions. Osteoblasts – next cellular source of erythropoietin In addition to regulating erythropoiesis, EPO has also been implicated in the regulation of bone formation and repair. Wu C, Giaccia AJ, Rankin EB: Osteoblasts: a Novel Source of Erythropoietin. Current Osteoporosis Reports 2014, 12(4):428-432. Lamon S, Russell AP: The role and regulation of erythropoietin (EPO) and its receptor in skeletal muscle: how much do we really know? Frontiers in Physiology 2013, 4. EPO and brain Rabie T, Marti HH: Brain Protection by Erythropoietin: A Manifold Task. Physiology 2008, 23(5):263-274. Lundby C, Olsen NV: Effects of recombinant human erythropoietin in normal humans. Journal of Physiology-London 2011, 589(6):1265-1271. ERYTHROPOESIS Substances affecting erythropoesis Need of copper Ceruloplasmin – binding protein (a2-globulin) with ferroxidase activity. Oxidation of Fe2+ to Fe3+ is necessary for binding of iron to transferrin. Need of cobalt Part of vitamin B12 molecule. Vitamin B12 (cyancobalamin) Produced by bacteria in GIT. Source: liver, kidneys, meet, milk products… Resorption: necessity of s.c. intrinsic factor secreted by parietal cells of gastric fundus and body. Bound to transcobalamins in blood. Stored in liver, pancreas, kidneys, brain, myocardium. Function: synthesis of nucleic acids, co-factor in conversion of ribonucleotids to deoxyribonucleotids, production of metabolic active forms of folic acid NECESSARY FOR NORMAL DIVISION AND MATURATION OF RED BLOOD CELL LINE ELEMENTS. Symptoms of anaemia after years only!!! Pernicious anaemia. Folic acid (pteroylglutamic) Produced by higher plants and micro-organisms. Source: green vegetables, yeast, liver, kidneys… Function: part of co-enzymes during synthesis of DNA, participation in cell division and differentiation Deficiency: deficient nutrition, treatment with cytostatics (methotrexate) Symptoms of anaemia already after couple of months!!! Macrocyte hyperchromic anaemia. Other vitamins Vitamin B6 (pyridoxine) – metabolism of amino acids, synthesis of hem Vitamin B2 (riboflavin) – part of flavoprotein enzymes – reductases of erythrocytes (normal function and survival of erythrocytes). Normocyte anaemia with lower reticulocytes count. Vitamin C (ascorbic acid) – non-specific function in erythropoesis. Hormonal influences Androgens, estrogens, hormones of thyroid gland, glucocorticoids, growth hormone. ANAEMIA Disorder, in which basic and characteristic feature is lower amount of haemoglobin. Usually also haematocrit and red blood cell count in 1 litre of blood are below physiological value. CLASSIFICATION OF ANEMIAS MORPHOLOGICAL CLASSIFICATION Evaluation of erythrocyte volume and concentration of haemoglobin in erythrocytes 1. Normocyte anaemia 2. Microcyte a. 3. Macrocyte 1. Normochromic anaemia 2. Hypochromic a. PATHOPHYSIOLOGICAL CLASSIFICATION Anaemias caused by inefficient blood production Sideropenic anaemias – lack of iron Megaloblastic a. – lack of vitamin B12 or folic acid Anaemias caused by suppression of blood production Anaemias in chronic diseases and symptomatic anaemias Thalasemia Anaemias caused by increased losses Haemolytic a.– caused by increased destruction of erythrocytes Chronic posthaemorhagic anemia Acute posthaemorhagic anaemia ANTIGENS AND ANTIBODIES OF RED BLOOD CELLS 1) History of blood transfusions. 2) Posttransfusion reactions: aglutination, haemolysis (immediate or delayed), life-threatening complications (jaundice, damage of kidneys, anuria, death – in case of full blood or RBCs administration, in case of plasma – dilution of aglutinins!!! Autoimmune diseases. Paternity tests, event. transplantology. 3) Antigens of blood cells: a) 30 antigen systems (ABO, Rh, MNSs, Lutheran, Kell, Kidd, Lewis, Diego, P, Duffy…) b) hundreds of other – „weak“ – antigens (important for paternity testing, organ transplantations) 4) Aglutinogen: antigen of plasmatic membrane of cells - complex oligosaccharide - erytrocytes, salivary glands, pancreas, liver, kidney, lungs, testes - saliva, sperm, amnionic fluid, milk, urine 5) Aglutinin: antibody against aglutinogen, g-globulin (IgM –AB0 system, IgG – Rh system), produced in the same way as other antibodies - after births almost zero concentration in blood - production of aglutinins begins 2-8 months after birth: stimulation by antigens similar to aglutinogens – in food, in GIT bacteria - maximal concentration of antibodies is reached in 8-10 years, decreases gradually with age Blood group systems A-B-O SYSTEM Genotype Blood group Aglutinogen Aglutinin 00 0 (H) anti-A a anti-B 0A or AA A A anti-B 0B or BB B B anti-A AB AB A and B - Described by Landsteiner in 1901, 1930 – awarded by Nobel Price. Janský -1906. Frequency of blood groups in ABO system: O 47% (38%) A 41% (42%) B 9% (14%) AB 3% (6,5%) Subgroups in A a B blood groups. A1 (1 million copies of antigen on 1 ery), A2 (250 thousands copies). Heredity: both A and B is inherited dominantly, according to Mendel´s law. Rh SYSTEM Monkey Maccacus rhesus. 40th of the 20th century, Wiener a Landsteiner. Frequency: 85% - Rh+, 15% - Rh-. Antigens D, C, E, d, c, e. Present only on erythrocytes. D – the „strongest“ antigen: Rh – positive, Rh – negative (produces anti-D aglutinin after contact with D-erythrocytes). Aglutinins production: only after the contact with D-erythrocytes (transfusion, foetal erythroblastosis). High concentration of anti-D antibodies lasts for many years!!! HAEMOLYTIC JAUNDICE OF NEWBORNS Rh-negative mother x Rh-positive foetus. First pregnancy – immunisation of mother during delivery (or interruption or miscarriage!!!). Next pregnancy – anti-D aglutinins (IgG) cross foetoplacental barrier. Foetus damage: approx. in 17% of next pregnancies Haemolysis of foetal erythrocytes – haemolyti disease of newborn (erythroblastosis fetalis): •anaemia •jaundice •oedemas – event. hydrops fetalis •CNS damage (icterus) –bile acids enter CNS (no haematoencephalic barrier!) •deaths of foetus in utero Prevention of foetal damage: 1) administration of small doses of anti-D antibodies to mother during pregnancy 2) administration of one dose of anti-D antibodies during postpartum period Success of therapy: up 90%. http://tmedweb.tulane.edu/pharmwiki/lib/exe/fetch.php/rhod.png Section of yellow bone marrow. This bone marrow is yellow in its fresh state because of the abundance of yellowish adipocytes present. The hemopoietic (*) tissue is comparatively less abundant than in red bone marrow. The adipocytes, or fat cells, (Ad) appear as large circular clear spaces in this field. A megakaryocyte (M) and venous sinuses (S) are also labelled. Source: http://audilab.bmed.mcgill.ca/HA/html/blood_7_E.html http://audilab.bmed.mcgill.ca/HA/HAimage/blood_fig_6.jpg This bone marrow is referred to as red marrow because it contains few adipocytes, or fat cells, among an abundance of hemopoietic cells. It is difficult to identify the individual precursors of red and white blood cells because they are closely packed and condensed during the fixation of the tissue (*). The following elements are identified: a megakaryocyte (M), which is a very large polyploid cell responsible for the production of blood platelets one adipocyte (Ad) two blood sinuses (S). The walls of these vessels are the sites where newly formed erythrocytes and leukocytes pass from the connective tissue into the blood circulation. Silverthorn, D. U. Human Physiology – an Integrated Approach. 6th. edition. Pearson Education, Inc. 2012. Morrison SJ, Scadden DT: The bone marrow niche for haematopoietic stem cells. Nature 2014, 505(7483):327-334. Ho MSH, Medcalf RL, Livesey SA, Traianedes K: The dynamics of adult haematopoiesis in the bone and bone marrow environment. Br J Haematol 2015, 170(4):472-486. Kopp HG, Avecilla ST, Hooper AT, Rafii S: The bone marrow vascular niche: Home of HSC differentiation and mobilization. Physiology 2005, 20:349-356. Morrison SJ, Scadden DT: The bone marrow niche for haematopoietic stem cells. Nature 2014, 505(7483):327-334. bone marrow contains endothelial cell precursors Schatteman GC, Dunnwald M, Jiao C: Biology of bone marrow-derived endothelial cell precursors. American Journal of Physiology-Heart and Circulatory Physiology 2007, 292(1):H1-H18.