Hemostasis & BloodClotting Introduction .
Hemostasis Introduction .
Hemostasis is defined as arrest or stoppage of bleeding.
Stage of Hemostasis .
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| States of hemostasis. |
When a blood vessel is injured, the injury initiates a series of reactions, resulting in hemostasis. It occurs in three stages :
1. Vasoconstriction
2. Platelet plug formation
3. Coagulation of blood.
Vasoconstriction .
[1]. Immediately after injury, the blood vessel constricts and decreases the loss of blood from damaged portion. Usually, arterioles and small arteries constrict.
[2]. Vasoconstriction is purely a local phenomenon. When the blood vessels are cut, the endothelium is damaged and the collagen is exposed.
[3]. Platelets adhere to this collagen and get activated. The activated platelets secrete serotonin and other vasoconstrictor substances which cause constriction of the blood vessels.
[4]. Adherence of platelets to the collagen is accelerated by von Willebrand factor. This factor acts as a bridge between a specific glycoprotein present on the surface of platelet and collagen fibrils.
Platelet plug Formation .
[1]. Platelets get adhered to the collagen of ruptured blood vessel and secrete adenosine diphosphate (ADP) and thromboxane A2 .These two substances attract more and more platelets and activate them.
[2]. All these platelets aggregate together and form a loose temporary platelet plug or temporary hemostatic plug, which closes the ruptured vessel and prevents further blood loss. Platelet aggregation is accelerated by platelet-activating factor (PAF).
Coagulation of Blood .
[1]. During this process, the fibrinogen is converted into fibrin.
[2]. Fibrin threads get attached to the loose platelet plug, which blocks the ruptured part of blood vessels and prevents further blood loss completely.
BloodClotting Introduction .
Coagulation or clotting is defined as the process in which blood loses its fluidity and becomes a jelly-like mass few minutes after it is shed out or collected in a container.
Factor Involved in BloodClotting .
Coagulation of blood occurs through a series of reactions due to the activation of a group of substances. Substances necessary for clotting are called clotting factors. Thirteen clotting factors are :-
[1]. Factor I Fibrinogen .
[2]. Factor II Prothrombin .
[3]. Factor III Thromboplastin (Tissue factor) .
[4]. Factor IV Calcium Factor .
[5]. V Labile factor (Proaccelerin or accelerator globulin) .
[6]. Factor VI Presence has not been proved .
[7]. Factor VII Stable factor .
[8]. Factor VIII Antihemophilic factor (Antihemophilic globulin) ,.
[9]. Factor IX Christmas factor
[10]. Factor X Stuart-Prower factor .
[11]. Factor XI Plasma thromboplastin antecedent .
[12]. Factor XII Hageman factor (Contact factor) .
[13]. Factor XIII Fibrin-stabilizing factor (Fibrinase).
Clotting factors were named after the scientists who discovered them or as per the activity, except factor IX. Factor IX or Christmas factor was named after the patient in whom it was discovered.
Enzyme Cascade Theory .
[1]. Most of the clotting factors are proteins in the form of enzymes. Normally, all the factors are present in the form of inactive proenzyme. These proenzymes must be activated into enzymes to enforce clot formation.
[2]. It is carried out by a series of proenzyme-enzyme conversion reactions. First one of the series is converted into an active enzyme that activates the second one, which activates the third one; this continues till the final active enzyme thrombin is formed.
[3]. Enzyme cascade theory explains how various reactions, involved in the conversion of proenzymes to active enzymes take place in the form of a cascade.
[4]. Cascade refers to a process that occurs through a series of steps, each step initiating the next, until the final step is reached.
Stages of BloodClotting
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| Stages of blood coagulation . |
In general, blood clotting occurs in three stages:
1. Formation of prothrombin activator .
2. Conversion of prothrombin into thrombin .
3. Conversion of fibrinogen into fibrin.
1. Formation of prothrombin activator .
[1]. Blood clotting commences with the formation of a substance called prothrombin activator, which converts prothrombin into thrombin.
[2]. Its formation is initiated by substances produced either within the blood or outside the blood. Thus, formation of prothrombin activator occurs through two pathways:
i. Intrinsic pathway
ii. Extrinsic pathway.
i. Intrinsic Pathway for the Formation of Prothrombin Activator
The formation of prothrombin activator is initiated by platelets, which are within the blood itself .
Sequence of Events in Intrinsic pathway .
[1]. During the injury, the blood vessel is ruptured. Endothelium is damaged and collagen beneath the endothelium is exposed.
[2]. When factor XII (Hageman factor) comes in contact with collagen, it is converted into activated factor XII in the presence of kallikrein and high molecular weight (HMW) kinogen.
[3]. The activated factor XII converts factor XI into activated factor XI in the presence of HMW kinogen.
[4]. The activated factor XI activates factor IX in the presence of factor IV (calcium).
[5]. Activated factor IX activates factor X in the presence of factor VIII and calcium.
[6]. When platelet comes in contact with collagen of damaged blood vessel, it gets activated and releases phospholipids.
[7]. Now the activated factor X reacts with platelet phospholipid and factor V to form prothrombin activator. This needs the presence of calcium ions.
[8]. Factor V is also activated by positive feedback effect of thrombin .
ii. Extrinsic Pathway for the Formation of Prothrombin Activator .
The formation of prothrombin activator is initiated by the tissue thromboplastin, which is formed from the injured tissues.
Sequence of Events in Extrinsic Pathway .
[1]. Tissues that are damaged during injury release tissue thromboplastin (factor III). Thromboplastin contains proteins, phospholipid and glycoprotein, which act as proteolytic enzymes.
[2]. Glycoprotein and phospholipid components of thromboplastin convert factor X into activated factor X, in the presence of factor VII.
[3]. Activated factor X reacts with factor V and phospholipid component of tissue thromboplastin to form prothrombin activator. This reaction requires the presence of calcium ions.
2. Conversion of prothrombin into thrombin .
Blood clotting is all about thrombin formation. Once thrombin is formed, it definitely leads to clot formation.
Sequence of Events .
[1]. Prothrombin activator that is formed in intrinsic and extrinsic pathways converts prothrombin into thrombin in the presence of calcium (factor IV).
[2]. Once formed thrombin initiates the formation of more thrombin molecules. The initially formed thrombin activates Factor V. Factor V in turn accelerates formation of both extrinsic and intrinsic prothrombin activator, which converts prothrombin into thrombin. This effect of thrombin is called positive feedback effect .
3. Conversion of fibrinogen into fibrin.
The final stage of blood clotting involves the conversion of fibrinogen into fibrin by thrombin.
Sequence of Events .
[1]. 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.
[2]. Fibrin monomer polymerizes with other monomer molecules and form loosely arranged strands of fibrin.
[3]. 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.
Blood clot .
[1]. Blood clot is defined as the mass of coagulated blood which contains RBCs, WBCs and platelets entrapped in fibrin meshwork.
[2]. RBCs and WBCs are not necessary for clotting process. However, when clot is formed, these cells are trapped in it along with platelets.
[3]. The trapped RBCs are responsible for the red color of the clot.
[4]. The external blood clot is also called scab. It adheres to the opening of damaged blood vessel and prevents blood loss.
Clot Retraction .
[1]. After the formation, the blood clot starts contracting. And after about 30 to 45 minutes, the straw-colored serum oozes out of the clot.
[2]. The process involving the contraction of blood clot and oozing of serum is called clot retraction. Contractile proteins, namely actin, myosin and thrombosthenin in the cytoplasm of platelets are responsible for clot retraction.
Fibrinolysis .
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| Fibrinolysis . |
[1]. Lysis of blood clot inside the blood vessel is called fibrinolysis.
[2]. It helps to remove the clot from lumen of the blood vessel. This process requires a substance called plasmin or fibrinolysin.
Formation of Plasmin .
[1]. Plasmin is formed from inactivated glycoprotein called plasminogen. Plasminogen is synthesized in liver and it is incorporated with other proteins in the blood clot.
[2]. Plasminogen is converted into plasmin by tissue plasminogen activator (t-PA), lysosomal enzymes and thrombin.
[3]. The t-PA and lysosomal enzymes are released from damaged tissues and damaged endothelium. Thrombin is derived from blood.
[4]. The t-PA is always inhibited by a substance called t-PA inhibitor. It is also inhibited by factors V and VIII.
[5]. Besides t-PA, there is another plasminogen activator called urokinase plasminogen activator (u-PA). It is derived from blood.
Sequence of Events Involved in the Activation of Plasminogen .
[1]. During intravascular clotting, the endothelium of the blood vessel secretes a thrombin-binding protein, the thrombomodulin. It is secreted by the endothelium of all the blood vessels, except the minute vessels of brain. .
[2]. Thrombomodulin combines with thrombin and forms a thrombomodulin-thrombin complex .
[3]. Thrombomodulin-thrombin complex activates protein C .
[4]. Activated protein C inactivates factor V and VIII in the presence of a cofactor called protein S .
[5]. Protein C also inactivates the t-PA inhibitor .
[6]. Now, the t-PA becomes active .
[7]. Activated t-PA and lysosomal enzymes activate plasminogen to form plasmin.
Plasminogen is also activated by thrombin and u-PA .
Anticlotting Mechanism in the Body .
Under physiological conditions, intravascular clotting does not occur. It is because of the presence of some physicochemical factors in the body.
1. Physical Factors .
[1]. Continuous circulation of blood.
[2]. Smooth endothelial lining of the blood vessels.
2. Chemical Factors – Natural Anticoagulants .
[1] . Presence of natural anticoagulant called heparin that is produced by the liver
[2] . Production of thrombomodulin by endothelium of the blood vessels (except in brain capillaries). Thrombomodulin is a thrombin-binding protein. It binds with thrombin and forms a thrombomodulin-thrombin complex. This complex activates protein C. Activated protein C along with its cofactor protein S inactivates Factor V and Factor VIII. Inactivation of these two clotting factors prevents clot formation
[3] . All the clotting factors are in inactive state.
Anticoagulants .
Substances which prevent or postpone coagulation of blood are called anticoagulants.
Anticoagulants are of three types:
[1] . Anticoagulants used to prevent blood clotting inside the body, i.e. in vivo.
[2] . Anticoagulants used to prevent clotting of blood that is collected from the body, i.e. in vitro.
[3] . Anticoagulants used to prevent blood clotting both in vivo and in vitro.
1. Heparin .
[1]. Heparin is a naturally produced anticoagulant in the body. It is produced by mast cells which are the wandering cells present immediately outside the capillaries in many tissues or organs that contain more connective tissue.
[2]. These cells are abundant in liver and lungs. Basophils also secrete heparin. Heparin is a conjugated polysaccharide.
[3]. Commercial heparin is prepared from the liver and other organs of animals.
[4]. Commercial preparation is available in liquid form or dry form as sodium, calcium, ammonium or lithium salts.
Mechanism of Action of Heparin .
[1]. Prevents blood clotting by its antithrombin activity. It directly suppresses the activity of thrombin .
[2]. Combines with antithrombin III (a protease inhibitor present in circulation) and removes thrombin from circulation .
[3]. Activates antithrombin III
[4]. Inactivates the active form of other clotting factors like IX, X, XI and XII .
Uses of Heparin .
Heparin is used as an anticoagulant both in vivo and in vitro.
Clinical use
Intravenous injection of heparin (0.5 to 1 mg/kg body weight) postpones clotting for 3 to 4 hours (until it is destroyed by the enzyme heparinase). So, it is widely used as an anticoagulant in clinical practice. In clinics, heparin is used for many purposes such as:
[1]. To prevent intravascular blood clotting during surgery.
[2]. While passing the blood through artificial kidney for dialysis.
[3]. During cardiac surgery, which involves heart lung machine.
[4]. To preserve the blood before transfusion.
Use in the laboratory
[1]. Heparin is also used as anticoagulant in vitro while collecting blood for various investigations. About 0.1 to 0.2 mg is sufficient for 1 mL of blood. It is effective for 8 to 12 hours.
[2]. After that, blood will clot because heparin only delays clotting and does not prevent it. Heparin is the most expensive anticoagulant.
2. Coumarin Derivatives .
Warfarin and dicoumoral are the derivatives of coumarin.
Mechanism of Action .
[1]. Coumarin derivatives prevent blood clotting by inhibiting the action of vitamin K.
[2]. Vitamin K is essential for the formation of various clotting factors, namely II, VII, IX and X.
Uses .
[1]. Dicoumoral and warfarin are the commonly used oral anticoagulants (in vivo).
[2]. Warfarin is used to prevent myocardial infarction (heart attack), strokes and thrombosis.
3. EDTA .
Ethylenediaminetetraacetic acid (EDTA) is a strong anticoagulant.
It is available in two forms:
[1]. Disodium salt (Na2 EDTA).
[2]. Tripotassium salt (K3 EDTA).
Mechanism of Action .
These substances prevent blood clotting by removing calcium from blood.
Uses .
EDTA is used as an anticoagulant both in vivo and in vitro.
[1]. It is Commonly administered intravenously, in cases of lead poisoning.
[2]. Used as an anticoagulant in the laboratory (in vitro).
[3]. 0.5 to 2.0 mg of EDTA per mL of blood is sufficient to preserve the blood for at least 6 hours.
[4]. On refrigeration, it can preserve the blood up to 24 hours.
4. Oxalate Compounds .
[1]. Oxalate compounds prevent coagulation by forming calcium oxalate, which is precipitated later. Thus, these compounds reduce the blood calcium level.
[2]. Earlier sodium and potassium oxalates were used. Nowadays, mixture of ammonium oxalate and potassium oxalate in the ratio of 3 : 2 is used.
[3]. Each salt is an anticoagulant by itself. But potassium oxalate alone causes shrinkage of RBCs.
[4]. Ammonium oxalate alone causes swelling of RBCs. But together, these substances do not alter the cellular activity.
Mechanism of Action .
[1]. Oxalate combines with calcium and forms insoluble calcium oxalate.
[2]. Thus, oxalate removes calcium from blood and lack of calcium prevents coagulation.
Uses .
[1]. Oxalate compounds are used only as in vitro anticoagulants.
[2]. 2 mg of mixture is necessary for 1 ml of blood. Since oxalate is poisonous, it cannot be used in vivo.
5. Citrates .
Sodium, ammonium and potassium citrates are used as anticoagulants.
Mechanism of Action .
[1]. Citrate combines with calcium in blood to form insoluble calcium citrate.
[2]. Like oxalate, citrate also removes calcium from blood and lack of calcium prevents coagulation.
Uses .
Citrate is used as in vitro anticoagulant.
[1]. It is used to store blood in the blood bank as:
a. Acid citrate dextrose (ACD): 1 part of ACD with 4 parts of blood
b. Citrate phosphate dextrose (CPD): 1 part of CPD with 4 parts of blood
[2]. Citrate is also used in laboratory in the form of formol-citrate solution (Dacie’s solution) for RBC and platelet counts.
Physical methods to prevent Blood Clotting .
Coagulation of blood is postponed or prevented by the following physical methods:
Cold .
Reducing the temperature to about 5°C postpones the coagulation of blood.
Collecting blood in a Container with Smooth Surface .
[1]. Collecting the blood in a container with smooth surface like a silicon-coated container prevents clotting.
[2]. The smooth surface inhibits the activation of factor XII and platelets. So, the formation of prothrombin activator is prevented.
Procoagulant .
Procoagulants or hemostatic agents are the substances which accelerate the process of blood coagulation. Procoagulants are:
Thrombin .
Thrombin is sprayed upon the bleeding surface to arrest bleeding by hastening blood clotting.
Snake Venom.
Venom of some snakes (vipers, cobras and rattle snakes) contains proteolytic enzymes which enhance blood clotting by activating the clotting factors.
Extracts of Lungs & Thymus .
Extract obtained from the lungs and thymus has thromboplastin, which causes rapid blood coagulation.
Sodium or calcium alginate .
Sodium or calcium alginate substances enhance blood clotting process by activating the Hageman factor.
Oxidized cellulose.
Oxidized cellulose causes clotting of blood by activating the Hageman factor.
Tests for Blood Clotting .
Blood clotting tests are used to diagnose blood disorders. Some tests are also used to monitor the patients treated with anticoagulant drugs such as heparin and warfarin.
1. Bleeding time .
2. Clotting time .
3. Prothrombin time.
4. Partial prothrombin time .
5. International normalized ratio.
6. Thrombin time.
Bleeding time (BT) .
[1]. Bleeding time (BT) is the time interval from oozing of blood after a cut or injury till arrest of bleeding.
[2]. Usually, it is determined by Duke method using blotting paper or filter paper method.
[3]. Its normal duration is 3 to 6 minutes. It is prolonged in purpura.
Clotting time (CT).
[1]. Clotting time (CT) is the time interval from oozing of blood after a cut or injury till the formation of clot.
[2]. It is usually determined by capillary tube method. Its normal duration is 3 to 8 minutes. It is prolonged in hemophilia.
Prothrombin time (PT).
[1]. Prothrombin time (PT) is the time taken by blood to clot after adding tissue thromboplastin to it.
[2]. Blood is collected and oxalated so that, the calcium is precipitated and prothrombin is not converted into thrombin. Thus, the blood clotting is prevented. Then a large quantity of tissue thromboplastin with calcium is added to this blood.
[3]. Calcium nullifies the effect of oxalate. The tissue thromboplastin activates prothrombin and blood clotting occurs.
[4]. During this procedure, the time taken by blood to clot after adding tissue thromboplastin is determined.
[5]. Prothrombin time indicates the total quantity of prothrombin present in the blood.
[6]. Normal duration of prothrombin time is 10 to 12 seconds.
[7]. It is prolonged in deficiency of prothrombin and other factors like factors I, V, VII and X. However, it is normal in hemophilia.
Partial Prothrombin Time or activated Prothrombin Time .
[1]. Partial prothrombin time (PPT) is the time taken for the blood to clot after adding an activator such as phospholipid, along with calcium to it. It is also called activated partial prothrombin time (APTT).
[2]. This test is useful in monitoring the patients taking anticoagulant drugs. It is carried out by observing clotting time after adding phospholipid, a surface activator and calcium to a patient’s plasma.
[3]. Phospholipid serves as platelet substitute. Commonly used surface activator is kaolin .
[4]. Normal duration of partial prothrombin time is 30 to 45 seconds.
[5]. It is prolonged in heparin or warfarin therapy (since heparin and warfarin inhibit clotting) and deficiency or inhibition of factors II, V, VIII, IX, X, XI and XII.
International normalized ratio (INR).
[1]. International normalized ratio (INR) is the rating of a patient’s prothrombin time when compared to an average. It measures extrinsic clotting pathway system.
[2]. INR is useful in monitoring impact of anticoagulant drugs such as warfarin and to adjust the dosage of anticoagulants.
[3]. Patients with atrial fibrillation are usually treated with warfarin to protect against blood clot, which may cause strokes. These patients should have regular blood tests to know their INR in order to adjust warfarin dosage.
[4]. Blood takes longer time to clot if INR is higher. Normal INR is about 1 .
1. In patients taking anticoagulant therapy for atrial fibrillation, INR should be between 2 and 3.
2. For patients with heart valve disorders, INR should be between 3 and 4.
3. But, INR greater than 4 indicates that blood is clotting too slowly and there is a risk of uncontrolled blood clotting.
Thrombin time (TT) .
[1]. Thrombin time (TT) is the time taken for the blood to clot after adding thrombin to it.
[2]. It is done to investigate the presence of heparin in plasma or to detect fibrinogen abnormalities.
[3]. This test involves observation of clotting time after adding thrombin to patient’s plasma.
[4]. Normal duration of thrombin time is 12 to 20 seconds. It is prolonged in heparin therapy and during dysfibrinogenimia (abnormal function of fibrinogen with normal fibrinogen level).
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