HEMOSTASIS_043930.pdf

LECTURE NOTES ON HEMOSTASIS BY ADAOBI P. OKEKE HEMOSTASIS Hemostasis is the process of forming clots in the walls of damaged blood vessels and preventing blood loss while maintaining blood in a fluid state within the vascular system. When a small blood vessel is transected or damaged...

LECTURE NOTES ON HEMOSTASIS BY ADAOBI P. OKEKE HEMOSTASIS Hemostasis is the process of forming clots in the walls of damaged blood vessels and preventing blood loss while maintaining blood in a fluid state within the vascular system. When a small blood vessel is transected or damaged, the injury initiates a series of reactions that lead to hemostasis. This seals off the damaged region and prevents further blood loss. Hemostasis occurs in three stages: 1. Vascular Constriction 2. Platelet Plug Formation 3. Blood Coagulation 1. Vascular Constriction Once a blood vessel is injured, the trauma to the vessel wall causes the smooth muscle in the vessel wall to contract leading to an immediate stoppage of blood flow from the injured/ruptured vessel. This contraction is usually due to : i. local myogenic spasm- which initiates most of the vasoconstriction ii. local autacoid factors from the traumatized tissues and blood platelets- platelets releases vasoconstrictor substances like thromboxane A2 and serotonin which is responsible for most of the vasoconstriction that occurs in smaller blood vessels. iii. nervous reflexes- these are initiated by pain nerve impulses or other sensory impulses that originate from the traumatized vessel or nearby tissues. The more severely a vessel is traumatized, the greater the degree of vascular spasm. The spasm can last for many minutes or even hours, during which time the processes of platelet plugging and blood coagulation can take place. 2. Platelet Plug Formation Normally, many very small vascular holes develop throughout the body each day. So, if this small cut does occur, it is often sealed by a platelet plug. When platelets come in contact with a damaged vascular surface especially with collagen fibers in the vascular wall, they become activated which is evident in the immediate change in their characteristics viz: 1. they begin to swell 2. they assume irregular forms with numerous irradiating filopods protruding from their surfaces; 3. their contractile proteins contract forcefully and cause the release of granules that contain multiple active factors; 4. they become sticky enabling them to adhere to collagen in the tissues and to a protein called von Willebrand factor that leaks into the traumatized tissue from the plasma; 5. they secrete large quantities of ADP; and their enzymes form thromboxane A2. Nearby platelets are also activated by the ADP and Thromboxane (secreted by the initially activated platelets) so that they’re able to stick to and adhere to the original activated platelets. Therefore, at the site of any opening in a blood vessel wall, the damaged vascular wall activates successively increasing numbers of platelets that themselves attract more and more additional platelets, thus forming a platelet plug. This is at first a loose plug, but it is usually successful in blocking blood loss if the vascular opening is small. Then, during the subsequent process of blood coagulation, fibrin threads form. These attach tightly to the platelets, thus constructing an unyielding plug.. 3. Coagulation of Blood The third and last stage of hemostasis is formation of blood clot. During this process, the fibrinogen is converted into fibrin. Fibrin threads get attached to the loose platelet plug, which blocks the ruptured part of blood vessels and prevents further blood loss completely. The clot begins to develop in 15 to 20 seconds if the trauma to the vascular wall has been severe, and in 1 to 2 minutes if the trauma has been minor. Activator substances from the traumatized vascular wall, from platelets, and from blood proteins adhering to the traumatized vascular wall initiate the clotting process. Within 3 to 6 minutes after rupture of a vessel, if the vessel opening is not too large, the entire opening or broken end of the vessel is filled with clot. After 20 minutes to an hour, the clot retracts; this closes the vessel still further. FACTORS INVOLVED IN BLOOD CLOTTING The clotting process is initiated by activator substances called clotting factors. The clotting factors include:. Factor I Fibrinogen Factor II Prothrombin Factor III Thromboplastin (Tissue factor) Factor IV Calcium Factor V Labile factor (Proaccelerin or accelerator globulin) Factor VI Presence has not been proved Factor VII Stable factor Factor VIII Antihemophilic factor A (Antihemophilic globulin) Factor IX Christmas factor (plasma thromboplastic component (PTC) or antihaemophilic factor B)) Factor X Stuart-Prower factor Factor XI Plasma thromboplastin antecedent (antihaemophilic factor C), Factor XII Hageman factor (glass factor or contact factor) Factor XIII Fibrin-stabilizing factor Prekallikrein Fletcher factor High-molecular-weight kininogen Fitzgerald factor; (HMWK) Platelets Mechanism of Blood Coagulation Normally, circulating blood does not clot spontaneously. Clot formation is initiated under the following situations: Trauma to the vascular wall and adjacent tissues, Trauma to blood Contact of blood with damaged endothelial cells or collagen or other tissue elements outside the vessel. The process of coagulation involves a cascade of reactions in which activation of one factor leads to activation of the next clotting factor. It is important to note that each factor activates (compared to its own amount) a huge quantity of the other factor, i.e. at each stage amplification of result is obtained. Thus, the process can be viewed as a bioamplifier. The process of coagulation can be divided into three main steps: A. Formation of prothrombin activator, B. Conversion of prothrombin to thrombin, and C. Conversion of fibrinogen into fibrin. A. Formation of Prothrombin Activator Blood coagulation commences with the formation of a complex of activated substances called prothrombin activator, which converts prothrombin into thrombin. The formation of prothrombin activator involves two mechanisms viz: 1. Extrinsic pathway, and 2. Intrinsic pathway. Extrinsic pathway The extrinsic pathway of formation of prothrombin activator begins with trauma to the vascular wall or the tissues outside the blood vessel. It includes the following three basic steps 1. Release of tissue thromboplastins Several substances are released from the traumatized tissues all of which constitute tissue thromboplastin (factor III). This includes phospholipids of cell membranes of the tissues and lipoprotein complex containing glycoprotein which acts as an enzyme. 2. Activation of factor X to form activated factor X Tissue thromboplastin combines with factor VII (stable factor) to form the tissue thromboplastin -factor VII complex, which in the presence of Ca2+ activates factor X to form activated factor X (Xa). 3. Effect of activated factor X to form prothrombin activator The activated factor X, along with tissue phospholipids or phospholipids released from platelets, factor V (labile factor) and Ca2+ forms a complex which is called prothrombin activator. Intrinsic pathway The second mechanism for initiating formation of prothrombin activator, and therefore for initiating clotting, begins in the blood itself following trauma to the blood or exposure of blood to collagen in a traumatized vascular wall. The steps in this pathway are as follows: 1. Activation of factor XII Trauma to blood or exposure to collagen fibres underlying damaged vascular endothelium (or electronegatively charged wettable surface such as glass, in vitro) activates plasma factor XII to form XIIa (a proteolytic enzyme) and initiates the intrinsic pathway. The blood trauma also damages the platelets because of adherence to either collagen or a wettable surface (or by damage in other ways), and this releases platelet phospholipids that contain the lipoprotein called platelet factor 3, which also plays a role in subsequent clotting reactions. 2. Activation of factor XI The activated Factor XII acts enzymatically on Factor XI to activate this factor as well, which is the second step in the intrinsic pathway. This reaction also requires HMW (high-molecular- weight) kininogen and is accelerated by prekallikrein 3. Activation of factor IX The activated Factor XI then acts enzymatically on Factor IX to activate this factor also. 4. Activation of factor X Factor IXa in the presence of activated factor VIII, and Ca2+ as well as phospholipids and factor 3 (released from traumatized platelets) activate factor X to form Xa. When either Factor VIII or platelets are in short supply, this step is deficient. Factor VIII is the factor that is missing in a person who has classic hemophilia, for which reason it is called antihemophilic factor. Platelets are the clotting factor that is lacking in the bleeding disease called thrombocytopenia. 5. Formation of prothrombin activator This step in the intrinsic pathway is the same as the last step in the extrinsic pathway. That is, activated Factor X combines with Factor V and platelet or tissue phospholipids to form the complex called prothrombin activator. Interaction between the Extrinsic and Intrinsic Pathways Although clotting occurs by both pathways simultaneously after blood vessels rupture, there are a few differences between the two pathways:’ Extrinsic pathway begins with trauma to the vascular wall or the tissue outside the vessel wall, while intrinsic pathway begins in the blood itself. Extrinsic pathway is explosive in nature, with severe tissue trauma clotting occurring in as little as 15 s, while intrinsic pathway is much slower to proceed, usually requiring 2–6 min to cause clotting. The extrinsic and intrinsic pathways are initiated by different factors, though, both converge at factor X and then follow the same common pathway. B. Conversion of Prothrombin to Thrombin Prothrombin Prothrombin (factor II) is a plasma protein (an α2 globulin) with following features:  It is the inactive precursor of enzyme thrombin (which is not present normally in the circulating blood). Its molecular weight is about 68,700 It is synthesized in liver in the presence of vitamin K (Note: Hepatic synthesis of factor VII, IX and X is also dependent on the presence of vitamin K). Its concentration in plasma of an adult is 15 mg/dL which falls in liver diseases: In a newborn baby, plasma concentration of prothrombin is lower. Conversion of prothrombin to thrombin This is caused by the prothrombin activator in the presence of Ca2+. This occurs on the surface of platelets which form the platelet plug at the site of injury. Thus, clot formation starts at the site of injury. The rate of formation of thrombin is directly proportional to the quantity of prothrombin activator available, which in turn is proportional to degree of trauma to the vessel wall or to the blood. Thrombin Thrombin so formed acts as proteolytic enzyme. Its molecular weight is 33,700, almost exactly one half that of prothrombin. The amount of thrombin produced is in excess of the need. It has been estimated that the amount of thrombin produced during clotting of only 1 mL of blood is sufficient to coagulate 3 litres of blood. Thus, this presents a dangerous possibility of extensive intravascular clotting whenever the haemorrhage mechanism is in operation (even in a trivial cut). However, practically it does not occur so, since there exist adequate mechanisms in the body to prevent it. C. CONVERSION OF FIBRINOGEN INTO FIBRIN Fibrinogen Fibrinogen is a high-molecular-weight protein (MW = 340,000) that occurs in the plasma in quantities of 100 to 700 mg/dl. Fibrinogen is formed in the liver, and liver disease can decrease the concentration of circulating fibrinogen, as it does the concentration of prothrombin, pointed out above. The final stage of blood clotting involves the conversion of fibrinogen into fibrin by thrombin and it takes place as follows: i. Thrombin converts inactive fibrinogen into activated fibrinogen due to loss of 2 pairs of polypeptides from each fibrinogen molecule. The activated fibrinogen is called fibrin monomer. ii. Fibrin monomer polymerizes with other monomer molecules and form loosely arranged strands of fibrin. iii. Later these loose strands are modified into dense and tight fibrin threads by fibrin-stabilizing factor (factor XIII) in the presence of calcium ions. All the tight fibrin threads are aggregated to form a meshwork of stable clot. MECHANISM OF BLOOD COAGULATION BLOOD CLOT RETRACTION The blood clot formed at the end of the coagulation process is composed of a meshwork of fibrin threads running in all directions along with the entrapped blood cells, platelets and plasma. The fibrin threads adhere to the damaged surface of blood vessels. At this juncture, it is important to note that coagulation is the property of plasma alone. The RBCs and WBCs do not take part in it. They only become caught up in the meshwork of the clot. Within a few minutes after a clot is formed, it begins to contract and usually squeezes out most of the fluid called serum (plasma without fibrinogen and other clotting factors) within 30–60 min. Platelets are essential for clot retraction. Platelets are attached to fibrin fibres of the clot in such a way that they actually bind different fibres together. The contractile proteins (platelet thrombosthenin, actin and myosin) present in the cytoplasm of platelets cause strong contraction of platelet spicules attached to fibrin fibres. This helps to compress the fibrin meshwork, i.e. cause clot contraction. This contraction is activated by thrombin and calcium ions. The process of contraction of blood clot and oozing of serum is called clot retraction. If a blood clot is kept for several hours, the clot retracts to about 40% of its original volume. Clot retraction is impaired if blood platelets have been removed. Role of calcium in blood coagulation From the study of mechanism of blood coagulation, it is quite clear that except for the first two steps in the intrinsic pathway, calcium ions are required for promotion of all the reactions. Therefore, in the absence of calcium ions, blood clotting will not occur. Thus, coagulation of blood can be prevented in vitro (e.g. for storage in the blood bank or for separation of plasma) by reducing the calcium ion concentration below the threshold level for clotting. The use of oxalates and citrates as in vitro anticoagulants is based on this principle. In essence, calcium can be deionized by causing it to react with substances such as citrate ion or it can be precipitated by causing it to react with substances such as oxalate ion. However, in vivo the degree of hypocalcaemia (e.g. due to deficiency of vitamin D or hypoparathyroidism) is compatible with life and does not cause bleeding disorder. APPLIED PHYSIOLOGY BLEEDING DISORDERS Bleeding disorders are the conditions characterized by prolonged bleeding time or clotting time. Bleeding time (BT) is the time interval from oozing of blood after a cut or injury till arrest of bleeding. Usually, Its normal duration is 3 to 6 minutes. It is prolonged in purpura. Clotting time (CT) is the time interval from oozing of blood after a cut or injury till the formation of clot. Its normal duration is 3 to 8 minutes. It is prolonged in hemophilia. Bleeding disorders are of three types: 1. Hemophilia. 2. Purpura. 3. von Willebrand disease. Hemophilia Hemophilia is a group of sex-linked inherited blood disorders, characterized by prolonged clotting time. However, the bleeding time is normal. Usually, it affects the males, with the females being the carriers. Because of prolonged clotting time, even a mild trauma causes excess bleeding which can lead to death. Damage of skin while falling or extraction of a tooth may cause excess bleeding for few weeks. Easy bruising and hemorrhage in muscles and joints are also common in this disease. Causes of hemophilia Hemophilia occurs due to lack of formation of prothrombin activator. That is why the coagulation time is prolonged. The formation of prothrombin activator is affected due to the deficiency of factor VIII, IX or XI. Types of hemophilia Depending upon the deficiency of the factor involved, hemophilia is classified into three types: i. Hemophilia A or classic hemophilia: This type of hemophilia occurs as a result of deficiency of factor VIII. 83% of people with hemophilia are affected by hemophilia A. The majority of patients with haemophilia A have blood levels of factor VIII below 5%, and so usually bleed severely on minor trauma. Relationships of levels of factor VIII with status of haemostasis are as: Factor VIII activity Status of haemostasis >50% Normal hemostasis 25-50% Excessive bleeding after severe trauma 5-25% Excessive bleeding after minor trauma 1-5% Severe bleeding on minor trauma