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A/Prof Wong Chong Thim

Department of Physiology p phswct@nus.edu.sg @ g MD9, #03-08 6516 3232

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A/Prof CT Wong

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BLOOD Composition of blood: plasma and cellular elements of blood ll l l t f bl d Functions of Blood Blood Cells and their Production Hemostasis: prevention of blood loss e ostas s: p eve t o o b ood oss

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Blood Cells & Plasma Components

Plasma
the liquid component of the blood

Cells

Some blood parameters p

Hematocrit Hemoglobin concentration Red blood cell (rbc, erythrocyte) count White blood cell (wbc, leukocyte) count Diff Differential wbc count i l b
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Fig. 11-1 p Fig 11-1, p. 392

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Composition of blood p

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Components of plasma
Protein concentrations: i i
Total Albumin Globulins Gl b li Fibrinogen Transferrin 6.0-8.0 g/dL 3.1-4.3 g/dL 2.6-4.1 g/dL 2 6 4 1 /dL 200-450 mg/dL 3.0-6.5 mg/dL

All the plasma proteins are synthesized in liver except immunoglobulins, from Blymphocytes.

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Components of formed elements

thrombocytes

reticulocytes

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Main blood cell parameters of normal healthy adults

Cells/L (average) Approx. range Percentage of Total White Cells

Erythrocytes Males Females Total WBC Granulocytes Neutrophils Eosinophils Basophils Lymphocytes Monocytes Platelets

5.4 x 106 4.8 x 106 9000

4.4 5.8 x 106 4.1 5.2 x 106 4000 11 000 11,000

5400 275 35 2750 540 300,000

3000 6000 150 300 10 - 100 1500 4000 300 600 200,000 500,000

50 70 14 0.4 20 - 40 2-8

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Main blood parameters Test

Hematocrit (Hct) Reticulocyte count (retic) Hemoglobin concentration (Hb) RBC count

Measures
Percentage of formed elements in whole blood. Circulating percentage of reticulocytes Concentration of hemoglobin in blood Number of RBCs per l of whole blood Male

Elevated values l
Polycythemia Reticulocytosis

Depressed values l
Anemia

Anemia Erythrocytosis/ Anemia polycythemia Female 37 to 47 12 to 16

Hematocrit (%) Hemoglobin conc (g ) (g/dL) Reticulocyte count

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40 to 54 14 to 17

~0.8%
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Functions
Transport/Distribution
Oxygen and carbon dioxide Metabolic wastes Various substances (eg. minerals, vitamins, etc)

Homeostasis/Regulation
Body temperature pH H Fluid volume

Protection
Against blood loss (Hemostasis) Against infection (Immunology) *

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Immune system Leukocytes are the mobile units of the bodys immune system. They function mainly as defense agents outside the blood. They defend against the invasion of pathogens. They identify cancer cells. They remove the bodys litter by phagocytosis. They can leave the circulation and go to the sites of invasion and tissue damage damage.

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#09

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Fig. 13-26, p. 487

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Exchange of oxygen and carbon dioxide at tissue level Diffusion

Hb = hemoglobin
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Transport of oxygen

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Hemoglobin function
1 Hb molecule = 4 Hb chains 1 Hb chain = 1 heme + 1 globin

abm3s6a.mov
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Oxygen carrying capacity of blood

In hemoglobin ~ 15 g Hb/dL blood

~ 1.34 ml oxygen/g Hb (fully saturated) ~ 20 ml oxygen/dl blood

In plasma ~ 0.003 ml oxygen/dL blood/mm Hg oxygen (partial

pressure of oxygen) Partial pressure of oxygen Systemic arteries = 100 mmHg y g Systemic veins = 40 mm Hg
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Transport of carbon dioxide

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Transport of carbon dioxide

[carbaminoHb]

(~7%) (~70%)

(~23%)

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Blood Cell Production

General pattern of blood cell production p p Hormonal control of hematopoiesis:
Erythropoietin red blood cell production (erythropoiesis) Thrombopoietin platelet production p p p Colony-stimulating factors, cytokines, growth factors, and interleukins leukopoiesis (production of white blood cells)

Nutritional requirements
General (proteins, carbohydrates, lipids) Vitamins (Vitamin B12, folate, others) Minerals (iron, others)

H Hemoglobin synthesis l bi th i
Heme synthesis Iron metabolism H Heme catabolism t b li
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Blood Cell Production

An erythrocyte in the circulation cannot reproduce, as it lacks a nucleus. Erythropoiesis (erythrocyte production) is the production of new red cells, replacing the functionally worn-out cells in the circulation. This occurs in the bone marrow in the adult. Pluripotent stem cells in the red marrow differentiate into all the different types of blood cells that we find in the circulation. Regulatory factors act on hemopoietic (blood-producing) red marrow to govern the type and number of cells produced and discharged into the circulation. The average life span of an erythrocyte is 120 days. The final demise of old erythrocytes is mainly in the spleen. The number of erythrocytes normally remains constant. y y y Cell production = cell death. However, a low level of oxygen delivery to the tissues stimulates an increased rate of erythrocyte production. This increase is controlled by erythropoietin, a hormone produced by the kidneys.
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Blood cell production in bone marrow

Two important properties of stem cells: 1. Replication 1 R li ti 2. Differentiation

Fig. 11-9, p. 404

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Erythropoiesis is controlled by erythropoietin.

If oxygen delivery to the tissues, yg y and kidneys, is decreased, the kidneys detect this and increase the output of erythropoietin. The rate of erythropoiesis increases. An increase in the rate of erythropoiesis increases the number of erythrocytes and the oxygencarrying capacity to the tissues. Once the oxygen delivery to the tissues, and kidneys, is sufficient, the kidneys detect this. They decrease their output of erythropoietin. [Negative feedback system] i f db k
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Hemoglobin is a molecule consisting of two parts.

The heme part is nonprotein. Each of its four iron atoms is bound to one of the polypeptides and can combine with one molecule of oxygen gas. Thi molecule f This l l is bright red when combined with oxygen. The l bi is f Th globin i four, folded f ld d polypeptide chains. Hemoglobin can also combine with carbon dioxide, bi ith b di id hydrogen ions, carbon monoxide, and nitric oxide. Hemoglobin can buffer pH b H l bi b ff H by binding with hydrogen ions.
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Globin synthesis

Type of globin chain determines type of hemoglobin hemoglobin. Each consist of a tetramer of globin polypeptide chains., each with an attached heme. -like chains 141 aa long -like chains 146 aa long HbF (), fetal Hb HbA (), adult Hb HbA2 ( ) adult Hb, normal (), d l Hb l variant, very small %. HbS () abn aa6 glu-val glu val HbE () abn aa26 glu-lys

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Iron metabolism
Iron absorption occurs predominantly in the duodenum and upper jejunum. Iron must traverse both the apical and basolateral membranes of absorptive epithelial cells to reach the plasma. This transport requires enzymes that change plasma the oxidation state of iron. Iron that is present in food (particularly in red meat, pulses (legumes) and dairy p products) primarily occurs as Fe(III) or as heme. There is evidence that heme )p y ( ) may be transported into the intestine by a specific carrier (heme carrier protein 1; HCP1) although the nature of the carrier is unknown. Iron from heme is better absorbed compared to non-heme iron. Iron that is exported from cells into plasma by ferroportin becomes bound to transferrin, 80-kDa transferrin an 80 kDa glycoprotein with homologous N terminal and C terminal N-terminal C-terminal iron-binding domains that is synthesized in the liver, retina, testis and brain. At the neutral pH of blood, transferrin can bind two atoms of Fe(III) with a ( ) ( ) y dissociation constant (Kd) of 1023 M. Fe(III) binds transferrin only in the presence of an anion, usually carbonate, that bridges iron and transferrin.
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Iron transport across the enterocyte (mucosal cells lining the internal wall of the intestines).

Amount of available iron is dependent on p (1)Amount of iron in food we ingest main source is meat (2)Form of iron present in the food
Ferric iron (Fe(III)) in the diet is converted to ferrous iron (Fe(II)) by a ferroreductase (DCYTB, duodenal cytochrome B) that is located on the apical surface of enterocytes of the duodenal mucosa.

Amount of iron absorbed is well controlled by feedback system (many hypotheses, no complete agreement), absorbing enough to balance daily loss. loss Liver stores a ready supply of iron.
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Endocytosis of the complex of iron, transferrin and the transferrin receptor. The plasma protein transferrin (Tf) binds Fe(III) with high affinity. At the neutral pH (7.2) in plasma, TfFe(III) binds to the transferrin receptor (TfR) on the cell surface from where it is internalized by receptormediated endocytosis through clathrin-coated pits. The internalized vesicle (an endosome) becomes acidified (pH 5.5) by the action of an H+ ATPase (not shown). As the p of the endosome decreases, the structure of the ( ) pH , TfTfR complex changes and Fe(III) is released from TfFe(III). Fe(III) is converted to Fe(II) by the endosomal reductase STEAP3 (six-transmembrane epithelial antigen of the prostate 3) and is then transported out of the endosome into the cytosol by divalent metal transporter-1 (DMT1) F (II) can b stored i f iti i nonerythroid cells or incorporated t t 1 (DMT1). Fe(II) be t d in ferritin in th id ll i t d into haemoglobin in erythroid cells. The TfTfR complex is exocytosed by a recycling endosome.
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In liver

In maturing rbc
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Synthesis of Porphobilinogen and Heme

1* ALA synthase; 2 PBG synthase/ALA dehydratase; 3 PBG deaminase/Uroporphyrinogen III synthase; 4 Uroporphyrinogen decarboxylase; 5 coproporphyrinogen III oxidase; 6 protoporphyrinogen IX oxidase; 7 Ferrochelatase (Addition of iron occurs at this step)
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Heme Catabolism

The largest repository of heme in the human body is in red blood cells, which have a life span of about 120 days. There is thus a f b t d Th i th turnover of about 6 g/day of hemoglobin, which presents 2 problems. Breakdown of rbc normally occurs in the reticuloendothelial system, mainly in the spleen and liver (Kupffer cells). First, the porphyrin ring is hydrophobic and must be solubilized to be excreted. Second, iron must be conserved for new heme synthesis. Normally, senescent red blood cells and heme from other sources are engulfed by cells of the reticuloendothelial system. The globin is recycled or converted into amino acids, which in turn are recycled or catabolized as required. I i d Iron i released into the circulation is l d i t th i l ti for reuse by the body.

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Heme i oxidized, with the heme ring being opened by the H is idi d i h h h i b i db h endoplasmic reticulum enzyme, heme oxygenase. The oxidation step requires heme as a substrate, and any hemin (Fe3+) is reduced to heme (Fe2+) prior to oxidation by heme oxygenase. oxygenase

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In the next reaction a second bridging methylene (between rings III and IV) is reduced by biliverdin reductase, producing ,p g bilirubin. Bilirubin is released into the circulation. It is transported in the plasma bound to albumin. This is the free or unconjugated bilirubin, and it is quite insoluble.

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Albumin bound bilirubin is transport to hepatocytes, where UDP glucuronyl transferase adds 2 equivalents of glucuronic acid to bilirubin to produce the more water soluble, bilirubin diglucuronide derivative. The increased water solubility of Th i d l bili f the tetrapyrrole facilitates its excretion with the remainder of the bile as the bile pigments pigments.

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Destruction of rbc and reutilization of breakdown products

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Jaundice
In normal individuals, intestinal bilirubin is acted on by individuals bacteria to produce the final porphyrin products, urobilinogens and urobilins, that are found in the feces. Bilirubin and its catabolic products are collectively known as the bile pigments. In individuals with abnormally high red cell lysis, or liver y g y , damage or obstruction of the bile duct, the bilirubin and its precursors accumulate in the circulation; the result is hyperbilirubinemia, hyperbilirubinemia the cause of the abnormal body pigmentation known as jaundice.

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Anemia
Anemia is a reduction below the normal capacity in the blood to carry oxygen. Th are many kinds of anemia. There ki d f i Nutritional anemia is caused by a dietary deficiency of a factor needed for erythropoiesis (eg. iron, vitamins). Aplastic anemia is due to the failure of the bone marrow to make adequate numbers of RBCs. Renal anemia is due to kidney disease; lack of Epo. Hemorrhagic anemia is due to the loss of significant amounts of blood (and production cannot keep up with loss). Hemolytic anemia is due to the rupture of many RBCs. Sickle cell y p y disease can make red cells fragile and vulnerable to hemolysis.

PRODUCTION vs LOSS/DESTRUCTION
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Polycythemia
Polycythemia is an excess in circulating erythrocytes. Normal indication is an elevated hematocrit or hemoglobin levels. levels Primary polycythemia is caused by an tumor-like condition in the bone marrow. Secondary polycythemia is an erythropoietin induced adaptive erythropoietin-induced mechanism to improve the oxygen-carrying capacity in the blood. Relative (spurious) polycythemia: Other conditions can elevate the hematocrit, hematocrit such as dehydration or a decrease in plasma volume. volume

PRODUCTION vs LOSS/DESTRUCTION
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Leukocytes are produced at varying rates.

Their rates change depending on the changing defense needs of the body. They are produced from pluripotent stem cells in the bone marrow. These cells can differentiate and proliferate into different cell lines, producing the different kinds of white blood cells. ll Granulocytes and monocytes are produced only in the bone marrow. Lymphocytes are originally produced from precursor cells in the bone marrow. Most new ones are produced from lymphocytes in lymphoid tissue (eg lymph nodes).

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Hemostasis e os s s
Primary hemostasis
V Vessel walls l ll Platelets

Coagulation
Coagulation factors Extrinsic pathway Intrinsic pathway Common pathway

C t l of hemostasis Control f h t i
Anticoagulants Fibrinolysis
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Hemostasis prevents blood loss from damaged blood vessels.

The first two steps to stop escaping blood from a vessel are:
vascular spasm - This reduces blood flow through a damaged vessel. platelet plugging - An aggregation of platelets forms a plug. Platelets aggregate on contact with exposed collagen in the da aged wall o vesse . e p ate et p ug sea s b ea damaged wa of a vessel. The platelet plug seals a break in a vessel.

Platelets releases numerous substances when activated. Some of these substances will enhance the aggregation process. A Among these are A h ADP, thromboxane A2, calcium h b A2 l i ions. Other substances from the endothelium of a blood vessel inhibit platelet aggregation, keeping the process under control control.
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Normal and damaged vessel wall

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The clotting cascade is a series of steps involving twelve clotting factors. They lead to the final conversion of fibrinogen into fibrin.
The clotting factors serve as proteolytic enzymes in a series of ser e proteol tic en mes reactions, the clotting sequence. One factor in the sequence is activated which in turn activates another factor and so on. This sequence is called the clotting cascade. cascade The last two steps in the cascade are: prothrombin is converted to thrombin fib i fibrinogen is converted to fibrin i d fib i intrinsic pathway - This uses the sequence of factors in the cascade. extrinsic pathway - This requires only four steps and requires contact with tissue factors external to the blood.

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Fig. 11-13, p. 408 LSM3212 Blood 48

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Coagulation factors
*
I II III IV V VII VIII IX X XI XII XIII HMW-K Pre-K Ka PL Fibrinogen Prothrombin Thromboplastin/tissue factor Calcium Proacclerin, labile factor, accelerator globulin Proconvertin, SPCA, stable factor Antihemophilic factor (AHF), antihemophilic factor A, antihemophilic globulin (AHG) ih hili l b li Plasma thromboplastic component (PTC), Christmas factor, antihemophilic factor B Stuart-Prower factor Plasma thromboplastin antecedent (PTA), antihemophilic factor C Hageman factor, glass factor Fibrin-stabilizing factor, Laki-Lorand factor factor High-molecular-weight kininogen, Fitzgerald factor Prekallikrein, Fletcher factor Kallikrein Platelet h h li id Pl l phospholipid

* * *

* Vitamin K dependent coagulation factors

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Vitamin K acts as a cofactor in the formation of gcarboxyglutamyl residues (gla components) from g glutamyl residues (glu y (g components) in the coagulation factors listed. These residues are important in the formation of calcium binding sites. The next slide show the events that will h h h ill lead to the conversion of p prothrombin to thrombin.

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Coagulation cascade

Common pathway

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(Prothrombin activator)

The figure on the left is the situation at the start of the common pathway. Factor Xas gla components are attracted to the ionised Ca that are in contact with platelet factor 3 (also called platelet phospholipids). These platelet phospholipids have negative charges that attract the Ca in the plasma. This builds up the concentration of Xa in the damaged area. Prothrombin, which also possesses gla components are also attracted by the Ca present. This thus increases its concentration and also brings it in close proximity with Xa (see figure on right). Xa then can act on the prothrombin and convert it to thrombin. Same sort of reaction occurs in the activation of factor X by IXa (intrinsic pathway) or VIIa (extrinsic). Next 2 slides show how inactive coagulation factors are activated mainly by loss of part of the protein molecule, change in 3D structure, exposure of active sites in the molecule.

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( (Either VIIa complex or IXa complex) p p )

(active site)

Figure on left is the normal circulating inactive factor X. Removal of part of the molecule by either IXa (intrinsic pathway) or VIIa complexes (extrinsic pathway) allows for changes in 3D conformation, leading to exposure of active site, converting it into an active enzyme. Similar events occur during activation of IX, VII and conversion of prothrombin to thrombin.

( = gla components)
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(Xa, V, Ca, PL) ( g ( = gla components) p )

(active site)

(Thrombin removes part of the fibrinogen molecule, which allows the fibrin monomer to bind to one another)

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Anticoagulants
Heparin/heparan
Heparin in plasma; heparan on endothelial surface Functions as co-factor to anti-thrombin III Normal constituent of plasma

Anti-thrombin III
Plasma protein that binds to and inactivates thrombin, IXa, Xa One of the many plasma proteins normally present in blood

Warfarin (coumarin) drugs

Drugs affecting vitamin K dependant coagulation factors

Removal of calcium ions (in vitro)

Chelates/binds to calcium ions (eg. citrate, oxalate, ( g , , EDTA)
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Fibrinolysis

UPA = urinary PA/ urokinase PAI = PA inhibitor 2PI-Plasmin = plamin inhibitor FDPs = fibrin degradation products
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The clot is not a permanent solution for injury to a vessel. To ensure smooth i j l T h flow of blood in the vessels, clot will have to be removed. Fibroblasts form scar tissue for vessel repair. The clot is slowly dissolved by the enzyme plasmin. Plasminogen is made in the liver and is a plasma protein normally present in the circulation. At the site of damage, it is converted to g plasmin by tissue plasminogen activator (tPA), released from damage endothelial cells. Phagocytic white blood cells remove the products of clot dissolution.

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Additional notes on blood clotting: g

1. Clot retraction occurs after the clot is formed. 2. Amplification occurs in the clotting process. One molecule can activate one hundred molecules in the next step and so forth. f th 3. Whether a clot will form or not is dependent on the balance of th factors promoting clot formation and that of clot f the f t ti l tf ti d th t f l t destruction (fibrinolysis)

http://www.mhhe.com/biosci/esp/2002_general/Esp/folder_str ucture/tr/m1/s7/trm1s7_3.htm / / / / h

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Balance between clot formation and destruction

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Aims & Objectives

1. Describe the composition of blood. 2. 2 Explain the main functions of blood. blood 3. Describe the process of normal erythropoiesis. Explain the functions of nutritional and hormonal factors required for normal erythropoiesis. 4. 4 Define hemostasis Explain the mechanisms hemostasis. involved in hemostasis.

Reference: Sherwood L 2010 Human Physiology. 7th ed Chapter 11

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