Lecture Notes on Blood Physio 2009

Blood Physiology 2009

OVERVIEW

Functions of Blood

Blood performs many functions in the body. The main function of the circulating blood is to carry O 2 and nutrients to the tissues and to remove CO2 and waste products from the tissues (nutritive, respiratory, excretory). In addition, blood transports other substances (e.g. hormones) from their sites of production to their sites of action and white blood cells and platelets where they are needed. (transport) Blood also aids in the distribution of water, solutes, and heat and thus contributes to homeostasis, the maintenance of a constant body environment.(homeostatic) and participates in body defenses (immunity) because it carries immune cells to sites where they are needed.

Blood Volume

This is defined as the total amount of blood in the circulation plus amount in the reservoirs. In an average adult (70 kg man), this comprises 7 -8 % of the body weight or approximately 5- 6 Liters (whole blood = 2.8 L/M2 ; plasma =1.8 L/M2)

Physiological variations in Blood Volume

Blood volume varies with age, increasing as one ages; body weight and surface area, and is larger in males than females. Acute exposure to cold reduces blood volume due to plasma water loss to the tissues. It also changes with posture, e.g. from lying to standing up. Blood volume increases in pregnancy by 20-30 % because of the increase in fetal mass. In certain circumstances like hypoxic states, e.g. high altitude, an increase in red cells (erythrocytes) causes an increase in blood volume. Exercise and extreme emotion also induces an increase in blood volume.

Composition of Blood

Blood is composed of cells and a liquid called plasma in which they are suspended. In an average healthy person, approximately 45 % of the blood volume is cells, among them erythrocytes (RBCs=99%), leukocytes (WBCs), and thrombocytes (platelets).

The plasma-fluid portion is about 5 % of the body weight. Plasma, the liquid portion of the blood, consists of a large number of organic and inorganic substances dissolved in water. Serum is plasma from which fibrinogen and other proteins involved in clotting have been removed as a result of clotting.

Most of the plasma solutes are proteins and are classified into 3 broad groups : albumins (most abundant), globulins and fibrinogen (7-9 % of plasma weight). Mineral electrolytes like sodium, calcium and potassium comprise less than 1 % of plasma weight. In addition there are other nutrients, metabolic waste products, and hormones plus gases dissolved in plasma, i.e. oxygen, carbon dioxide, nitrogen.

Albumin comprises 60-80% of the total proteins in plasma. The presence of albumin provides the osmotic pressure needed to draw water from tissue fluid into capillaries to maintain blood volume and pressure. Globulin has three subtypes : alpha and beta globulins which aid in transport of lipids and fat-soluble vitamins, and gamma globulin which acts as antibodies from lymphocytes. As mentioned, fibrinogen is a protein present in plasma which participates in blood clotting (a clotting factor) by its conversion into fibrin.

Hematopoiesis

1 DVDiaz,MDUPCM Dept of Physiology


All blood cells and certain other cells located throughout the body - particularly in the reticuloendothelial system (RES) - are continuously regenerated throughout life by the process called hematopoiesis. This refers to the development and production of all blood cells = genesis of blood cells. and begins as early as the 20 th week in fetal life and continues in the red bone marrow until death.

Most hematopoietic cells are short lived, some surviving for only a day or two. Thus hematopoiesis serves to maintain a steady renewal of these cells on physiological demand.

Hematopoiesis is believed to be the function of a single precursor cell called the pluripotent stem cell (SC) (stem cell theory). In the bone marrow, these pluripotential hematopoietic stem cells (PHSC) are present from which all the cells

in the circulating blood are derived. These cells give rise to blood cells of (1) myeloid series – mainly from bone

marrow; (2) lymphoid series – mainly from lymphoid tissues.

The early offspring cells (progenitor cells) although not yet recognized as different from the PHSC are already committed to a particular cell line and are called committed stem cells. These committed stem cells when grown in culture will produce colonies of specific types of blood cells. The committed stem cell that produces erythrocytes is called a colony-forming unit-erythrocyte (CFU-E), and so forth. They lose their capacity for self-renewal. The growth and reproduction of progenitor cells or committed stem cells are controlled by multiple proteins called growth inducers, e.g. IL-3. Other sets of proteins called differentiation inducers promote differentiation of the cells to final types of adult blood cells.

In adults, blood stem cells are normally found in the red bone marrow inside the bones. Blood cells are made in the bone marrow in the skull, ribs, sternum (breast bone), spine and pelvis. The stem cells divide and multiply to make the blood cells. These cells differentiate (develop and mature) as they grow into white cells, red cells or platelets

THE BLOOD CELLS

Red Blood Cells (RBCs) or Erythrocytes

The most abundant of blood cells are the red blood cells or erythrocytes, comprising 5,000 million/mm 3 of blood. They appear as flattened biconcave discs approx 7 m in diameter and 2.2 m thick.

The main function of RBC is to transport oxygen via hemoglobin. Their unique shape serves the purpose of increasing the surface area through which gas can diffuse. They lack nuclei and mitochrondria and obtain energy through anaerobic metabolism.

Hematopoeisis of RBCs is called erythropoeisis and has the following phases/sites :- Intrauterine life

o Intravascular phase : up to 3rd month : endothelial cells RBCso Hepatic phase : 3rd to 5th month : liver and spleen ; nRBCs from mesenchymal cellso Myeloid phase : 5th month onwards

- Postnatal life: o Children : red bone marrow of axial and appendicular skeletono Adults : red bone marrow of axial skeleton

Until 5 yrs old, the bone marrow of all bones produces RBCs. After the age of 20 years the marrow becomes quite fatty and stops producing RBCs except for proximal portions of the humeri and tibia. Beyond 20, most are produced in the marrow of the membranous bones such as vertebrae, sternum, ribs and ilia. Even in these bones, the marrow becomes less



productive as age increases. Normally, 75 % of the cells in the marrow belong to the WBC-producing myeloid series and only 25 % are maturing red cells.

RBC Stages of differentiation

The stages of differentiation of RBCs are as follows: the first belonging to the RBC series is the proerythroblast which are formed from the CFU-E (colony-forming unit-erythrocyte). This proerythroblast divides multiple times eventually forming many mature RBCs. The first generation is called basophil erythroblast because they stain basic dyes and has very little hemoglobin. In the succeeding generations, the cells become filled with hemoglobin, the nucleus condenses to a small size and its final remnant is extruded from the cell and the endoplasmic reticulum is reabsorbed.These cells are called a reticulocyte and they containing basophilic material. These reticulocytes have a short life and the concentration among all RBCs of the blood is normally slightly less than 1 %. These pass from the bone marrow into the blood capillaries by diapedesis (the process of squeezing through the pores of the capillary membrane). The remaining basophilic material in the reticulocyte normally disappears within 1 to 2 days and the cell that emerges is the mature erythrocyte.

RBCs Functions

The major function of the erythrocytes is to transport hemoglobin which in turn carries oxygen from the lungs to the tissues. Other functions are : because they carry hemoglobin in the cells, they are responsible for the acid base buffering power of the whole blood. They also contain a large quantity of carbonic anhydrase which catalyzes the reversible reaction between CO2 and water increasing the rate of this reaction several thousand times. The rapidity of this reaction makes it possible for the water of the blood to transport enormous quantities of CO 2 from the tissues to the lungs in the form of bicarbonate.

The Hemoglobin molecule and oxygen saturation

Hemoglobin (Hgb) is the red,O2-carrying pigment in the RBCs of vertebrates. It is a protein with a MW of 64,450; a globular molecule made up of 4 subunits. Each subunit contains a heme moiety conjugated to a polypeptide. Heme is an iron-containing porphyrin derivative; a complex made up of a porphyrin and one atom of ferrous iron. Each of the four iron atoms can bind reversibly to one O2 molecule. The polypeptides are collectively referred to as the globin portion of the hemoglobin molecule. There are two pairs of polypeptides in each molecule.

In the normal adult human Hgb, the two types of polypeptide are called alpha chains each of which contains 141 amino acid residues; and the beta chains, each of which contains 146 amino acid residues. Thus hemoglobin A is designated as a2b2 .

Hematocrit is defined as the percentage of blood volume that is occupied by erythrocytes. It is measured by centrifuging a sample of blood. Normally it is approximately 45 % in men and 42 % in women.

Hemoglobin binds to oxygen to form oxyhemoglobin with oxygen attaching to the iron in the heme. When fully saturated, each gram of normal Hgb contains 1.39 ml of O2, however, blood normally contains small amounts of inactive Hgb derivatives and the measure values is lower at 1.34 ml of O2. The Hgb concentration in normal blood is about 15 gm/dL. Therefore, 1 dL of blood contains 20.1 ml (1.34ml x 15) of O2 bound to Hgb when the Hgb is 100 % saturated.

A distinct feature of oxygen and hemoglobin is that of cooperative binding, that is, binding of one molecule to another progressively facilitates the binding of progressive molecules. The combination of oxygen and hemoglobin in the lung is called oxygen loading while the release of oxygen from hemoglobin to the tissue is called unloading. The formation of oxyhemoglobin from the combination of oxygen and hemoglobin is a reversible reaction: Deoxy + O2 « OxyHgb



The oxyHgb dissociation curve is the curve relating percentage saturation of the O2-carrying power of Hgb to pO2. It has a characteristic sigmoid shape. The affinity of the Hgb for O2 is affected by pH, temperature and the concentration in the red cells of 2,3-diphosphoglycerate(2,3-DPG). A rise in temperature or fall in pH shifts the curve to the right. When the curve is shifted in this direction, a higher pO2 is required for Hgb to bind a given amount of O2; there is decreased affinity of Hgb for O2. 2,3-DPG is found in RBCs, formed from 3-phosphoglyceraldehyde which is a product of glycolysis via the Embden-Meyerhof pathway. It is a highly charged anion that binds to the beta chains of the deoxyHgb. An increase in the concentration of 2,3-DPG shifts the reaction to the right, causing more O2 to be liberated. Concentration is increased in anemia and a variety of diseases in which there is chronic hypoxia. This facilitates the delivery of oxygen to the tissues by raising pO2 at which O2 is released in peripheral capillaries. The opposite occurs in hypothermia, alkalemia such that hemoglobin has increased affinity for oxygen.

Factors regulating Erythropoiesis

An estimated 2.5 million erythrocytes are produced every second in order to replace those that are continuously destroyed by the spleen and liver. The average life span of RBCs is ~ 120 days. The direct control of erythrocyte production (erythropoiesis) is exerted primarily by a hormone called erythropoietin which is secreted into the blood mainly by a particular group of hormone-secreting connective-tissue cells primarily in the kidney. 90 % of all erythropoietin is formed in the kidneys, the remainder formed mainly in the liver. It is a glycoprotein with a MW of about 34,000.

Erythropoietin acts by binding to membrane receptors on cells that will become erythroblasts which later become reticulocytes and develop into mature erythrocytes. Erythropoeitin thus increases the number of nucleated precursors in bone marrow and reticulocytes and mature erythrocytes in the blood.

Regulation of RBC production

In the normal person, the total volume of circulating erythrocytes remains remarkably constant because of reflexes that regulate the bone marrow’s production of these cells. Erythropoietin is normally secreted in small amounts at a rate adequate to replace the usual loss. Erythropoeitin production is influenced by the level of tissue oxygenation. Any condition that causes the quantity of oxygen transported to the tissues to decrease increases the rate of RBC production. The secretion rate is increased markedly above basal values when there is a decreased oxygen delivery to the kidneys. As a result, there is an increase in plasma erythropoietin concentration, erythrocyte production, and the oxygen-carrying capacity of the blood. Therefore, the oxygen delivery to the tissues returns toward normal.

Hemorrhage or anemia or any other condition immediately causes the bone marrow to produce large quantities of RBCs. Destruction of major portions of the bone marrow by any means esp x-ray therapy causes hyperplasia of the bone marrow thereby attempting to supply the demand for RBCs in the body. At very high altitudes, where the quantity of oxygen in the air is greatly decreased, insufficient oxygen is transported to the tissues, and red cell production is greatly increased. Various diseases of the circulation that cause decreased blood flow through the peripheral vessels and particularly those that cause failure of oxygen absorption by the blood as it passes through lungs can also increase the red cell production e.g. prolonged cardiac failure, hypoxia from lung disease. The tissue hypoxia increases the rate of RBC production with a resultant increase in the hematocrit and usually the total blood volume as well.

Other factors that affect RBCs include vitamins and iron. Final maturation of the RBCs as well as the rate of production are affected by the person’s nutritional status.Especially important are vitamin B12 and folic acid which are essential for the synthesis of DNA (each is required for the formation of thymidine triphosphate, one of the essential building blocks of DNA). Lack of either causes failure of nuclear maturation and division. In addition, the erythroblastic cells of the bone marrow produce mainly larger than



normal red cells called macrocytes with flimsy membranes and irregular, large, oval shape with greater fragility. The fragility causes them to have a short life, one-half to one-third of normal.

A common cause of maturation failure is failure to absorb vitamin B12 from the gastrointestinal tract which may occur in pernicious anemia. In this condition, the basic abnormality is an atrophic gastric mucosa that fails to secrete normal gastric secretions : mainly intrinsic factor which combines with vitamin B 12 in food and makes it available for absorption. Lack of intrinsic factor, therefore, causes loss of much of the vitamin because of both digestive enzyme action in the gut and failure of its absorption. Folic acid, although a normal constituent of green vegetables, some fruits, liver and other meats is easily destroyed during cooking. People with gastrointestinal absorption abnormalities such as the small intestinal disease called sprue often have serious difficulty in absorbing both folic acid and vitamin B12.

Iron is essential for synthesis of hemoglobin. Its deficiency causes the most common form of anemia : microcytic, hypochromic anemia.

RBC destruction

There are about 3 x 1013 RBCs and about 900 gm of Hgb in the circulating blood of an adult man. RBCs normally circulate an average of 120 days before being destroyed. When they burst and release their hemoglobin, the Hgb is phagocytized almost immediately by macrophages in many parts of the body, but esp. by the Kupffer cells of the liver and macrophages of the spleen and bone marrow. Iron released from Hgb is passed back into the blood to be carried by transferrin either to the bone marrow for production of new RBCs or to the liver and other tissues for storage in the form of ferritin. The porphyrin portion of the Hgb molecule is converted by the macrophages through a series of stages into the bile pigment bilirubin. This is released into the blood and later secreted by the liver into the bile.

Iron transport and metabolism

Iron is important for the formation of hemoglobin,myoglobin, cytochromes, cytochrome oxidase, peroxidase, catalase. When iron is formed from the small intestine, it immediately combines in the blood plasma with a beta globulin, apotransferrin, to form transferrin which is then transported in the plasma. The iron is loosely bound in the transferrin and then can be released to any of the tissue cells in the body. In the receiving cell cytoplasm, the iron combines mainly with a protein, apoferritin to form ferritin. The iron stored as ferritin is called storage iron. Smaller quantities in the storage pool are stored in an extremely insoluble form called hemosiderin which can be stained and observed microscopically as large particles in tissue slices. When RBCs have lived their life span and are destroyed, the Hgb released from the cells is ingested by the cells of the monocyte-macrophage system. The free iron is liberated and it then is mainly stored in the ferritin pool or reused for formation of new hemoglobin. About 0.6 mg of iron are excreted each day, mainly into the feces. Additional quantities are lost whenever bleeding occurs; a menstrual loss of blood brings the iron loss to an average of about 1.3 mg/day.

Anemia

Anemia refers to any condition in which there is an abnormally low hemoglobin concentration and/or red blood cell count. Major causes of anemias are the ff:

- Dietary deficiencies of iron, vitamin B12 or folic acid : iron deficiency anemia, pernicious anemia- Bone marrow failure due to toxic drugs or cancer : aplastic anemia- Blood loss from the body : hemorrhage- Inadequate secretion of erythropoietin in chronic kidney disease- Excessive destruction of erythrocytes :hemolytic anemiaIron deficiency anemia



Because of the recycling of iron, dietary requirements for iron are usually quite small (1 mg/day); women with average menstrual blood loss (up to 2 mg/day); pregnant women (4 mg/day). Iron deficiency anemia in adults is usually not due to a dietary deficiency but rather to blood loss which reduces the amount of iron that can be recycled.

Leukocytes

White blood cells (WBCs) or leukocytes or polymorphonuclear granulocytes (PMNs)are the mobile units of the body’s protective system formed partially in the bone marrow and partially in the lymph tissue. The total leucocytes = 5,000 - 10,000 /mm3. The real value is that they are specifically transported to areas of serious infection and inflammation thereby providing a rapid and potent defense against any infectious agent that might be present.

Regulation of leucopoeisis

A variety of cytokines stimulate different stages of leukocyte development.Cytokines with general effects are :

- Multipotent growth factor- 1- Interleukin- 1 (IL-1)- Interleukin- 3 (IL-3)

Cytokines with specific effects are :- Granulocyte colony-stimulating factor (G-CSF) stimulates neutrophils- Granulocyte-monocyte colony-stimulating factor (GM-CSF) stimulates development of monocytes and eosinophils

Types of WBCs

The first three types of cells, the PMNs all have a granular appearance for which reason they are called granulocytes and in clinical terminology, “polys” because of the multiple nuclei. If appropriate dyes are added to a drop of blood, which is then examined under a microscopy, the various classes of leukocytes are clearly visible and are classified according to their structure and affinity for the various dyes. Granules of one group take up the red dye eosin and are called eosinophils; the second class take up the “basic dye” and are called basophils and the third have little affinity for either dye and are therefore called neutrophils.

Neutrophils are by far the most abundant kind of leukocytes (55-70% of all leukocytes are neutrophils). They have segmented nuclei, typically with 2 to 5 lobes connected together by thin strands of chromatin which can be difficult to see; the cell may thus appear to have multiple nuclei. The nuclear chromatin is condensed into coarse clumps. Small numbers of immature neutrophils or band form neutrophils may be seen in a blood smear. These are incompletely segmented and often have a 'C-shaped' nucleus. The cytoplasm of a neutrophil contains two types of granules. Type A granules are non-specific and contain lysosomal enzymes, defensins, and some lysosyme. The granules are azurophilic (violet color) with Wright's stain. Type B granules are specific to neutrophils and stain light pink ('neutral stain'). They contain collagenase, to help the cell move through connective tissue, and lactoferrin, which is toxic to bacteria and fungi.

Neutrophils are attracted to sites of injury and infection, where they adhere to vessel walls in a process known as margination. They then migrate into surrounding tissue and engulf bacteria by phagocytosis. Specific granules fuse with the phagosomes containing the bacteria and release their contents. Then azurophilic granules release their contents, lowering the pH and killing both bacteria and neutrophil. The life span in circulating blood is only about 4-8 hours but survive in tissues for 4-5 days. In times of serious tissue infection, this total life span is often shortened to


http://www.mc.vanderbilt.edu/histo/blood/band-form.jpg


only a few hours because they proceed rapidly to the infected are, perform their functions, and in the process are themselves destroyed.

Eosinophils make up 2-5% of total leukocytes, and are distinguished by their prominent acidophilic (red/orange) granules containing a compound known as major basic protein (MBP), which is toxic to many parasitic larvae. The nucleus usually has only 2 to 3 lobes. The functions of this type of granulocyte are associated with allergic responses and defense against parasites.

Basophils make up less than 1% of total leukocytes, and are distinguished by prominent dark blue specific granules containing histamine, heparin and other compounds. The nucleus is usually obscured by the density of granules. Basophils are associated with the immediate immune response to external antigens, such as that which occurs in asthma, hay fever, and anaphylaxis.

The monocytes are larger with a single oval or horseshoe nucleus and few granules. They are the largest cell type seen in blood smears, and constitute 5 to 8% of total leukocytes. Their nuclei are not multilobular like granulocytes, but may be deeply indented or U-shaped, with reticular-appearing chromatin. They also have a short transit time of 10-20 hours in the blood before wandering through the capillary membrane into the tissues where they swell and become tissue macrophages and where they can live for months unless they are destroyed while performing phagocytic function. These macrophages form the basis of the tissue macrophage system. The cytoplasm of monocytes contains numerous lysosomal granules which give it a uniform grayish-blue "ground-glass" appearance. Monocytes eventually leave the bloodstream and become tissue macrophages, which are responsible for removal of debris as well as defense against certain types of invaders such as fungi and TB, which cannot be dealt with effectively by neutrophils. Unlike neutrophils, macrophages are able to regenerate their lysosomal granules and may thus have a longer lifespan than neutrophils. Monocytes also serve as precursors for several other cells in the body, many of which also serve a phagocytic function (Kupffer cells of liver, osteoclasts of bone, etc.).

Lymphocytes have large nuclei with scanty cytoplasm. Lymphocytes are distinguished by having a deeply staining nucleus which may be eccentric in location, and a relatively small amount of cytoplasm. They are continually in circulation between the lymphatic system and the blood; can survive for weeks to months. Their life span depends on the body’s need for these cells because they are essential in specific immune defense. Lymphocytes are specialized white blood cells whose function is to identify and destroy invading organisms such as bacteria and viruses. Some T lymphocytes directly destroy invading organisms, whereas other T lymphocytes regulate the immune system by directing immune responses.

The two major types of lymphocytes in blood are B cells, which produce specific antibody when activated, and T cells, which have various roles in cell-mediated immunity and in immunoregulation. T and B cells cannot be distinguished from one another in routine smears; immunostains for specific membrane antigens must be used for this purpose.

The combination of monocytes, mobile macrophages, fixed tissue macrophages and a few specialized endothelial cells in the bone marrow, spleen and lymph nodes is called the reticuloendothelial system (RES). All or almost all of these cells originate from the monocytic stem cells and thus RES is synonymous with monocytic-macrophage system. This system is essentially a generalized phagocytic system located in all tissues esp where large quantities of particles, toxins and other unwanted substances must be destroyed.

Macrophage and Neutrophil Responses during Inflammation

These are the 4 lines of defense of the macrophage and neutrophil response during inflammation:



• Within minutes after inflammation begins, the macrophages already present in the tissues immediately begin their phagocytic actions. Many become sessile macrophages and break loose and become mobile during the first hour or so.

• Within the first hour or so, large numbers of neutrophils begin to invade the inflamed area from the blood. There is margination, diapedesis and chemotaxis.

• Also within a few hours, the number of neutrophils in the blood increases 4-5 fold.• Along with neutrophils, monocytes from the blood enter the inflamed tissue and enlarge to become macrophages• The fourth line of defense is greatly increased production of both granulocytes and monocytes by the bone

marrow which take 3-4 days to reach the stage of leaving the bone marrow

Platelets or thrombocytes

Thrombocytes, or platelets, are the smallest cellular component of blood. The circulating platelets are round or oval discs, colorless cell fragments, 1-4 um in diameter which contain numerous granules. They are produced when the cytoplasmic portions of large bone marrow cells called megakaryocytes become pinched off and enter the circulation.The normal concentration in the blood is between 150,000-300,000/L. The platelets are active structures with a half-life in the blood of 8-12 days. It is eliminated from the circulation mainly by the tissue macrophage system (macrophages in the spleen).

The major function is in blood clotting. They circulate inactivated, about 250,000 per cubic mm of blood, until they come into contact with a damaged blood vessel. At this point, the platelets form a clump, adhering to each other and to the blood vessel wall. They secrete chemicals that alter a blood-borne protein, fibrinogen, so that it forms a mesh of fibers at the damage site. A clot forms when platelets and red and white blood cells become trapped in the fibers. Blood clotting begins within seconds of injury. The same process can produce unwelcome clots in undamaged blood vessels.

Regulation of Thrombopoesis

Thrombopoeitin is the regulatory molecule that stimulates proliferation of megakaryocytes and their maturation into platelets. The gene that codes for thrombopoeitin has been cloned so that recombinant thrombopoeitin is now available for medical research and applications.

HEMOSTASIS

Hemostasis

The ability of the body to control the flow of blood following vascular injury is paramount to continued survival. The process of blood clotting and then the subsequent dissolution of the clot, following repair of the injured tissue, is termed hemostasis. Hemostasis is the arrest or stoppage of bleeding; some define it as prevention of blood loss. When blood vessels are damaged, bleeding occurs. Three processes act to stem the flow of blood : vasoconstriction, platelet aggregation and blood coagulation. The fourth process, clot dissolution is important to dissolve the clot.

Hemostasis is composed of 4 major events that occur in a set order following the loss of vascular integrity: 1. The initial phase of the process is vascular constriction. This limits the flow of blood to the area of injury. 2. Next, platelets become activated by thrombin and aggregate at the site of injury, forming a temporary, loose platelet plug. The protein fibrinogen is primarily responsible for stimulating platelet clumping. Platelets clump by binding to collagen that becomes exposed following rupture of the endothelial lining of vessels. Upon activation, platelets release adenosine-5'-diphosphate (ADP), and TXA2 (which activate additional platelets and cause them to adhere), serotonin, phospholipids, lipoproteins, and other proteins important for the coagulation cascade. In addition to induced secretion, activated platelets change their shape to accommodate the formation of the plug. The aggregation of platelets may continue in this manner until some of the small blood vessels become blocked by the mass of aggregated platelets. Platelets are prevented from aggregating along the length of a normal vessel by the anit-aggregation action of prostacylin. This substance is



released from the normal endothelial cells in the adjacent, uninjured part of the vessel. Platelets also release serotonin (5-hydroxytryptamine) which enhances vasoconstriction, and thromboplastin, which hastens blood coagulation.3. To insure stability of the initially loose platelet plug, a fibrin mesh (also called the clot) forms and entraps the plug. If the plug contains only platelets it is termed a white thrombus; if red blood cells are present it is called a red thrombus. 4. Finally, the clot must be dissolved in order for normal blood flow to resume following tissue repair. The dissolution of the clot occurs through the action of plasmin

Process 1 : VasoconstrictionPhysical injury to a blood vessel elicits a contractile response of the vascular smooth muscle and thus a narrowing of the vessel. This is called vasoconstriction. This process can completely close the lumen of the vessel and stop the flow of blood. The contraction results from local myogenic spasm initiated by direct damage to the vascular wall. Nervous reflexes are initiated by pain nerve impulses or other impulses that originate from the traumatized vessel or from nearby tissues. For smaller vessels, the platelets are responsible for much of the vasoconstriction by releasing the vasoconstrictor substance thromboxane A2. Permanent closure of the vessel by constriction occurs only in the very smallest of vessels of the microcirculation and hemostasis is ultimately dependent upon two other processes of formation of the platelet plug and blood clotting.In both processes, the platelets are involved. So to understand these, it is important to first discuss the nature of platelets themselves. Platelets have many functional characteristics of whole cells and contain in their cytoplasm active factors such as…

- Actin and myosin and thrombasthenin which cause platelets to contract- Residuals of ER and GA which synthesize various enzymes and store calcium- Other systems which are capable of forming ATP, ADP, prostaglandins (PGs) involved in the clotting process- Fibrin-stabilizing factor and growth factors

In their cell membrane, glycoproteins causes adherence to injured areas of the vessel wall and phospholipids play several activating roles at multiple points in the blood clotting process.

Process 2: Formation of Platelet PlugWhen platelets come in contact with a damaged vascular surface, they begin to swell, assume irregular forms with pseudopods, their contractile proteins contract to release granules that contain multiple active factors. They become sticky and adhere to collagen in the tissues and to von Willebrand factor that spreads throughout the plasma. They also secrete large quantities of ADP and enzymes which form thromboxane A2 which in turn activate nearby platelets which become sticky and adhere to other platelets. This results in the formation of a platelet plug. Then through the process of blood coagulation, fibrin threads form and attach to the platelets constructing an unyielding plug.

This is the sequence of events leading to the formation of a platelet plug and vasoconstriction following damage to a blood vessel wall: (Note two positive feedbacks in the pathways). Injury to a vessel disrupts the endothelium and exposes the underlying connective tissue collagen molecules. Platelets adhere to the collagen via an intermediary vWF, a plasma protein secreted by endothelial cells and platelets. Binding of the platelets to collagen triggers the platelets to release the contents of their secretory vesicle which contain a variety of chemical agents including ADP and serotonin. These cause platelet activation and platelet aggregation that rapidly creates a platelet plug inside the vessel. Adhesion of platelets rapidly induces them to synthesize thromboxane A2 which when released to the extracellular fluid (ECF) acts locally to further stimulate platelet aggregation and release of their secretory vesicle contents. While the platelet plug is being built up and compacted, the vascular smooth muscle in the damaged vessel is being stimulated to contract thereby decreasing the blood flow to the area and the pressure within the damaged vessel. This vascoconstriction is also mediated by TxA2 and other chemical mediators.

Process 3 : Blood coagulation or clot formationBlood clotting is the transformation of blood into a solid gel termed a clot or thrombus and consisting mainly of a protein polymer known as fibrin. Clotting occurs locally around the original platelet plug and is the dominant hemostatic defense. Its functions are to support and reinforce the plug and to solidify the blood that remains in the



wound channel. This begins to develop in 15-20 seconds if trauma to the vascular wall has been severe and in 1-2 minutes if minor. Within 3-6 minutes after rupture of a vessel, if the vessel opening is not too large, the entire opening or broken end is filled with clot. After 20 minutes to an hour, the clot retracts and this closes the vessel still further. The fluid expressed is called serum which lacks fibrinogen and most of the other clotting factors. Platelets are also necessary for clot retraction to occur.

Clotting FactorsClotting Factors SynonymsFibrinogen Factor IProthrombin Factor IITissue Factor Factor III ; Tissue thromboplastinCalcium Factor IVFactor V Proaccelerin ; Labile factorFactor VII Serum prothrombin conversion accelerator (SPCA) ;

Proconvertin ; Stable factorFactor VIII Anti-hemophilic Factor (AHF) ; Anti0hemophilic globulin (AHG) ;

Anti-hemophilic factor A Factor IX Plasma thromboplastin component (PTC) ; Christmas factor ;

Anti-hemophilic factor BFactor X Stuart factorFactor XI Plasma thromboplastin antecedent (PTA) ;

Anti-hemophilic factor C

Factor XII Hageman factorFactor XIII Fibrin –stabilizing factorPrekallikrein Fletcher factorHMW kininogen Fitzgerald factor ; HMWKPlatelets

Blood clotting is a complex process consisting of sequential activation of various factors that are present in an inactive state in the blood. The cascade of reactions involves three steps :

In response to rupture of the vessel or damage to the blood itself, there is the formation of a complex of activated substances collectively called prothrombin activator. This catalyzes the conversion of prothrombin to thrombin. The thrombin acts as an enzyme to convert fibrinogen to fibrin fibers that enmesh platelets, blood cells and plasma to form the clot.

The key step is the formation of fibrinogen to fibrin by thrombin.



Coagulation

cascade (diagram)

The early portions of the clotting cascade has two pathways : intrinsic and extrinsic pathways.Intrinsic because everything necessary for it is in the blood and extrinsic because a cellular element outside the blood is needed. The intrinsic cascade is initiated when contact is made between blood and exposed endothelial cell surfaces.

- The intrinsic pathway starts with the contact activation of factor XII to XIIa. This catalyzes the activation of factor XI to XIa which activates factor IX to IXa.

- Factor IXa activates factor X to Xa, the enzyme that converts prothrombin to thrombin.- Note that factor VIIIa serves as a cofactor in the factor IXa-mediated activation of factor X.

The extrinsic pathway is initiated upon vascular injury which leads to exposure of tissue factor (TF) (also identified as factor III), a subendothelial cell-surface glycoprotein that binds phospholipid.

- The extrinsic pathway begins with a protein called tissue factor which binds factor VII which becomes activated to factor VIIa.

- The complex of tissue factor and factor VIIa catalyzes the activation of factor X and in addition catalyzes the activation of factor IX which can then help activate even more factor X by plugging into the intrinsic pathway.

- Thrombin also contributes to the activation of factors XI and VIII in the intrinsic pathway and factor V with factor Va then serving as a cofactor for factor Xa.

- Not shown is the fact that thrombin also activates platelets.- Under physiological conditions, the two pathways play sequentially.

Clotting pathway : Conversion of Prothrombin to Thrombin and Polymerization of Fibrinogen to form fibrin

This is the diagram depicting the conversion of fibrinogen to fibrin by thrombin.


IX IXa IX

VIII VIIIa

X Xa X

Prothrombin Thrombin

V Va

Vessel damage

Subendothelial cells exposed to bloodTissue factor

VIIa VII

Contact activation

Activatedplatelets

Activatedplatelets

Ca ++

Vessel damageExposed collagen

XII XIIa

XI XIa


The rate limiting factor is the formation of prothrombin activator. This activator in the presence of sufficient amounts of ionic calcium causes this conversion of prothrombin to thrombin which in turn causes polymerization of fibrinogen molecules into fibrin fibers within 10-15 seconds. Prothrombin is a plasma protein , an alpha 2-globulin, with a MW of 68,700 present in a concentration of about 15 mg/dl. It is unstable and can easily split into smaller compounds one of which is thrombin. Prothrombin is formed by the liver and requires vitamin K for normal formation. Thrombin has a MW of 33,700 while fibrinogen’s is 340,000. Fibrinogen occurs in plasma in 100-700 mg/dl formed from the liver.

Bleeding deficiencies

- Vitamin K deficiency : needed for liver formation of five (5) clotting factors :o Prothrombino Protein Co Factor VIIo Factor IXo Factor X

- Hemophilia : genetic absence of factor VIII- Thrombocytopenia : decreased platelets

Anti-clotting systems

The body also has mechanisms for limiting clot formation and for dissolving a clot after it has formed. There are at least 3 different mechanisms that oppose clot formation thereby helping limit this process and preventing it from spreading excessively:

- The tissue factor pathway inhibitor is secreted mainly by endothelial cells binds to tissue factor-factor VIIa complexes and inhibits their ability to generate factor Xa.

- The second anticoagulant mechanism is triggered by thrombin which can bind to an endothelial cell receptor called thrombomodulin. This binding eliminates all of thrombin’s clot-producing effects and causes bound thrombin to bind a particular plasma protein , protein C. Protein C is activated and in combination with another plasma protein inactivates factors VIIIa and Va.

- A third naturally occurring anticoagulant mechanism is a plasma protein called antithrombin III which inactivates thrombin and several other clotting factors. Antithrombin III is activated when it binds to heparin, a substance that is present on the surface of endothelial cells.

Process 4 : Fibrinolytic SystemThe previous substances limit clot formation but there is also a fibrinolytic system that dissolves the clot after it is formed and is the principal effector of clot removal.It constitutes a plasma proenzyme, plasminogen which can be activated to the active enzyme plasmin by protein plasminogen activators. Once formed, plasmin digests fibrin, thereby dissolving the clot.

References :

1. Human Physiology by Vander, Sherman, Luciano, 8th edition 2001.2. Physiology by Berne & Levy, 5th edition, 2004.3. Human Physiology by Stuart Ira Fox, 6th edition, 2001.


Documents

Lecture Notes on Blood Physio 2009