Coagulation Of Blood Notes

Coagulation Of Blood Definition

• Coagulation or clotting is defined as the process in which blood looses its fluidity and becomes a jelly-like mass few minutes after it is shed out or collected in a container.
• The clot is a mesh of thin fibrils entangling the blood cells. These fibrils consist of fibrin threads. Fibrin is formed from fibrinogen.

Factors Involved In Blood Clotting

• Coagulation of blood occurs through a series of reactions due to the activation of a group of substances.
• The substances necessary for clotting are called clotting factors. Thirteen clotting factors are identified:
• The clotting factors were named after the scientists who discovered them or as per the activity except factor 9. Factor 9 or Christmas factor was named after the patient in whom it was discovered.

Read And Learn More: Medical Physiology Notes

Sequence Of Clotting Mechanism

Enzyme Cascade Theory

• 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.
• It is carried out by a series of proenzyme-enzyme conversion reactions. The 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.
• Enzyme cascade theory explains how various reactions involved in the conversion of proenzymes to active enzymes take place in the form of a cascade. 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 Blood Clotting: 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.

Stage 1: Formation Of Prothrombin Activator: Blood clotting commences with the formation of a substance called prothrombin activator which converts prothrombin into thrombin. It is formed either within the blood itself or outside the blood. Thus, the formation of prothrombin activators occurs through two pathways:

1. Extrinsic pathway: In this, the formation of prothrombin activator is initiated by the tissue thromboplastin which is formed from the injured tissues
2. Intrinsic pathway: In this, the formation of prothrombin activator is initiated by platelets, which are within the blood itself.

• Intrinsic Pathway for the Formation of Prothrombin Activator: In this pathway, the prothrombin activator is formed within the blood, i.,e. by platelets.
• The sequence of Events in Intrinsic Pathway
  1. During the injury, the blood vessel is ruptured. The endothelium is damaged and collagen beneath the endothelium is exposed
  2. When factor 12 (Flegman factor) comes in contact with collagen, it is converted into activated factor 12 in the presence of kallikrein and high molecular weight (HMW) kininogen
  3. The activated factor 12 converts factor 11 into activated factor 11 in the presence of FIMW kininogen
  4. The activated factor 12 activates factor 12 in the presence of factor 4 (calcium)
  5. Activated factor 9 activates factor 10 in the presence of factor 8 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 10 reacts with platelet phospholipid and factor 5 to form prothrombin activator. This needs the presence of calcium ions
  8. Factor 5 is also activated by positive feedback effect of thrombin.

• Extrinsic Pathway for the Formation of Prothrombin Activator: In this pathway, the prothrombin activator is formed in the tissues (outside blood). Extrinsic pathway is initiated by tissue thromboplastin.
• Sequence of Events in Extrinsic Pathway
  • The tissues that are damaged during injury release factor 3, i.e. tissue thromboplastin. The thromboplastin contains proteins, phospholipid, and glycoprotein, which act as proteolytic enzymes
  • The glycoprotein and phospholipid components of thromboplastin convert factor 10 into activated factor 10, in the presence of factor 7
  • The activated factor 10 reacts with factor 5 and the phospholipid component of tissue thromboplastin to form a prothrombin activator. This reaction requires Blood clotting is all about thrombin formation. Once the presence of calcium ions.

Stage 2: Conversion Of Prothrombin Into Thrombin: Blood clotting is all about thrombin formation. Once thrombin is formed, it definitely leads to clot formation.

• The sequence of Events in Stage 2
  • Prothrombin activator that is formed in intrinsic and extrinsic pathways converts prothrombin into thrombin in the presence of calcium
  • Once formed thrombin initiates the formation of more thrombin molecules. The initially formed thrombin activates Factor 5. Factor 5 in turn accelerates the formation of both extrinsic and intrinsic prothrombin activator which converts prothrombin into thrombin. This effect of thrombin is called the positive feedback effect.

Stage 3: Conversion Of Fibrinogen Into Fibrin: The final stage of blood clotting involves the conversion of fibrinogen into fibrin by thrombin.

• Sequence of Events in Stage 3
  • Thrombin converts fibrinogen into activated fibrinogen due to the loss’ of 2 pairs of polypeptides from each fibrinogen molecule. The activated fibrinogen is called a fibrin monomer.
  • Fibrin monomer polymerizes with other monomer molecules and forms loosely arranged strands of fibrin
  • Later these loose strands are modified into dense and tight fibrin threads by fibrin stabilizing factor (factor 13) in the presence of calcium ions. All the tight fibrin threads are aggregated to form a meshwork.

Blood Clot

Definition And Composition Of Clot

• Blood clot is defined as a mass of coagulated blood which contains RBCs, WBCs, and platelets entrapped in fibrin meshwork.
  RBCs and WBCs are not necessary for clotting process.
• However, when the clot is formed, these cells are trapped in it along with platelets. The trapped RBCs are responsible for the red color of the clot.
• The external blood clot is also called a scab. It adheres to the opening of damaged blood vessel and prevents blood loss.

Clot Retraction

• After the formation, the blood clot starts contracting. And after about 30-45 minutes, a straw-colored fluid called serum oozes out of the clot.
• The process involving the contraction of blood clot and oozing of serum is called clot retraction.
• The contractile proteins namely, actin, myosin, and thrombasthenia in the cytoplasm of platelets are responsible for clot retraction.

Fibrinolysis: The lysis of blood clots inside the blood vessel is called fibrinolysis. It helps to remove the clot from the lumen of the blood vessel. This process requires a substance called plasmin or fibrinolysin.

• Formation of Plasmin
  • Plasmin is formed from inactivated glycoprotein called plasminogen. Plasminogen is synthesized in liver and it is incorporated with other proteins in the blood clot.
  • Plasminogen is converted into plasmin by tissue plasminogen activator (t-PA), lysosomal enzymes, and thrombin. The t-PA and lysosomal enzymes are released from damaged tissues and damaged endothelium.
  • Thrombin is derived from blood. The t-PA is always inhibited a substance called a t-PA inhibitor. It is also inhibited by factors 5 and 8.
  • Besides t-PA, there is another plasminogen activator called urokinase plasminogen activator (u-PA). It is derived from blood.
• The sequence of Events Involved in the Activation of Plasminogen
  1. During intravascular clotting, the endothelium of the
     blood vessel secretes a thrombin-binding protein, thrombomodulin, it is secreted by the endothelium of ail the blood vessels except minute vessels of the run.
  2. Thrombomodulin combines with thrombin and forms a thrombomodulin-thrombin complex
  3. Thrombomodulin — thrombin complex activates protein C
  4. Activated protein C inactivates factor 5 and 8 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.
• Action of Plasmin: Plasmin causes lysis of clot by dissolving and digesting the fibrin threads.
• Significance of Lysis of Clot
  • In vital organs, particularly the heart, the blood dot obstructs the minute blood vessel leading to myocardial infarction. The lysis of blood clot allows reopening of affected blood vessels and prevents the development of infarction.
  • Fibrinolytic enzymes like streptokinase are used for the lysis of blood clot during the treatment in early stages of myocardial infarction.

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. The smooth endothelial lining of the blood vessels.
2. Chemical Factors
   1. Presence of natural anticoagulant heparin that is produced by the liver
   2. Production of thrombomodulin by the endothelium of the blood vessels (except in brain capillaries). Thrombomodulin is a thrombin-binding protein.
   3. It binds with thrombin and forms a thrombomodulin-thrombin complex. This complex activates protein C.
   4. Activated protein-C along with its cofactor protein-S inactivates Factor 5 and Factor 8. Inactivation of these two clotting factors prevents clot formation
   5. All the clotting factors are in an inactive state.

Anticoagulants

The substances, which prevent or postpone coagulation of blood, are called anticoagulants.

1. Anticoagulants are of three types:
2. Anticoagulants are used to prevent blood clotting inside the body, i.e. in vivo
3. Anticoagulants used to prevent clotting of blood that is collected from the body, i.e. in vitro
4. Anticoagulants used to prevent blood clotting both in vivo and in vitro.

1. Heparin:

• Heparin is a naturally produced anticoagulant in the body, it is produced by mast cells which are situated immediately outside the capillaries in large number of tissues or organs that contain more connective tissue.
• These wandering cells are abundant in liver and lungs. Basophils also secrete heparin.
• Heparin is a conjugated polysaccharide. Commercial heparin is prepared from the liver and other organs of animals.
• The 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 3 (a protease inhibitor present in circulation) and removes thrombin from circulation
  3. Activates antithrombin 3
  4. Inactivates the active form of other clotting factors like 9, 10, 11, and 12.
• Uses of Heparin: Heparin is used as an anticoagulant both in vivo and in vitro.
  • Clinical use
    • Intravenous injection of heparin (0.5-1 mg/kg body weight) postpones clotting for 3-4 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:
      1. Heparin is used generally to prevent intravascular blood clotting during surgery
      2. It is used during dialysis when blood is passed through artificial kidney
      3. It is used during cardiac surgery, which involves passing the blood through a heart-lung machine
      4. It is used as an anticoagulant to preserve the blood before transfusion.
• Use in the laboratory: Heparin is also used as an anticoagulant in vitro while collecting blood for various investigations. About 0.10.2 mg is sufficient for 1 ml of blood. It is effective for 8-12 hours. After that blood will clot because heparin only delays clotting and does not prevent it. Heparin is the most expensive anticoagulant.

2. Coumarin Derivatives: Dicoumoral and warfarin are the derivatives of coumarin.

• Coumarin Derivatives Mechanism of Action: The derivatives of coumarin prevent blood clotting by inhibiting the action of vitamin K. Vitamin K is essential for the formation of various clotting factors namely, 2, 7, 9, and 10.
• Coumarin Derivatives Uses: Dicoumoral and warfarin are the commonly used oral anticoagulants in clinical practice (in vivo).

3. EDTA: Ethylenediaminetetra acetic 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
  • Commonly administered intravenously in cases of lead poisoning.
  • Used as an anticoagulant in the laboratory (in vitro). 0.5-2.0 mg of EDTA per ml of blood is sufficient to preserve the blood for at least 6 hours. On refrigeration, it can preserve the blood up to 24 hours.

4. Oxalate Compounds

• Oxalate compounds prevent coagulation by forming calcium oxalate, which is precipitated later. Thus, these compounds reduce the blood calcium level.
• Earlier sodium and potassium oxalates were used. Nowadays, mixture of ammonium oxalate and potassium oxalate in the ratio of 3:2 is used. Each salt is an anticoagulant by itself. But potassium oxalate alone causes shrinkage of RBCs.
• Ammonium oxalate alone causes swelling of RBCs. But together, these substances do not alter the cellular activity.
• Oxalate Compounds Mechanism of Action: Oxalate combines with calcium and forms insoluble calcium oxalate. Thus, oxalate removes calcium from blood and lack of calcium prevents coagulation.
• Oxalate Compounds Uses: Oxalate compounds are used as in vitro anticoagulants. 2 mg of the mixture is necessary for 1 ml of blood. Oxalate is poisonous so it can not be used in vivo.

5. Citrates: Sodium, ammonium, and potassium citrates are used as anticoagulants.

• Citrates Mechanism of Action: Citrate combines with calcium in the blood to form insoluble calcium citrate. Like oxalate, citrate also removes calcium from blood and lack of calcium prevents coagulation.
• Citrates Uses
  • Used to store blood in the blood bank. It is available in two forms:
    • Acid citrate dextrose (ACD) – 1 part of ACD with 4 parts of blood
    • Citrate phosphate dextrose (CPD) – 1 part of CPD with 4 parts of blood
  • Used in laboratory in vitro. Formol-citrate solution (Dacie’s solution) is used for RBC and platelet counts.

6. Other Substances Which Prevent Blood Clotting: Peptone, proteins from the venom of copperhead snake, and hirudin (from leach) are the known anticoagulants.

Physical Methods To Prevent Blood Clotting

The coagulation of blood is postponed or prevented by the following physical methods:

1. Cold: Reducing the temperature to about 5°C postpones coagulation of blood.
2. Collecting Blood In A Container With Smooth Surface: Collecting the blood in a container with smooth surface like a silicon-coated container prevents clotting. The smooth surface inhibits the activation of factor 12 and platelets. So, the formation of prothrombin activator is prevented.

Procoagulants: Procoagulants or hemostatic agents are the substances, which accelerate the process of blood coagulation. Procoagulants are:

1. Thrombin: The thrombin is sprayed upon bleeding surface to arrest bleeding by hastening blood clotting.
2. Snake Venom: The venom of some snakes (vipers, cobras and rattle snakes) contains proteolytic enzymes which enhance blood clotting by activating the clotting factors.
3. Extracts Of Lungs And Thymus: The extract obtained from the lungs and thymus has thromboplastin, which causes rapid blood coagulation.
4. Sodium Or Calcium Alginate: These substances enhance blood clotting process by activating the Hegman factor.
5. Oxidized Cellulose: It causes clotting of blood by activating the Hegman factor.

Tests For Clotting

Tests available to evaluate the process of blood coagulation are:

1. Bleeding time
2. Clotting time
3. Prothrombin time
4. Partial prothrombin time
5. Thrombin time.

1. Bleeding Time: Breeding time is the time interval from oozing of blood after a cut or injury till arrest of bleeding. Usually, it is determined by the Duke method using blotting paper or filter paper method. Its normal duration is 3-6 minutes. It is prolonged in purpura.

2. Clotting Time: Clotting time is the time interval from oozing of blood after a cut or injury till the formation of clot. It is usually determined by capillary tube method. Its normal duration is 3-8 minutes. And it is prolonged in hemophilia.

3. Prothrombin Time

• It is the time taken by blood to clot after adding tissue thromboplastin to it. 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. Calcium nullifies the effect of oxalate.
• The tissue thromboplastin activates prothrombin and blood clotting occurs.
• During this procedure, the time taken by blood to clot after adding tissue thromboplastin is determined. Prothrombin time indicates the total quantity of prothrombin present in the blood.
• The normal duration of prothrombin time is about 12 seconds. It is prolonged in deficiency of prothrombin and other factors like factors 1, 5, 7, and 10. However, it is normal in hemophilia.

4. Partial Prothrombin Time

• Partial prothrombin time is the time taken for blood to clot after adding phospholipids and calcium to it. It is also called activated prothrombin time.
• This test is done to investigate the bleeding disorders and to detect the presence of heparin in patients treated with heparin (heparin therapy).
• It is carried out by observing clotting time after adding phospholipid, a surface activator, and calcium to patient’s plasma. Phospholipid serves as a platelet substitute.
• Commonly used surface activator is kaolin. Normal duration of partial prothrombin time is 30-50 seconds, it is prolonged in heparin therapy (since heparin inhibits clotting) and deficiency or inhibition of factors 2, 5, 8, 9, 10, 11, and 12.

5. Thrombin Time

• Thrombin time is the time taken for the blood to clot after adding thrombin to it.
• It is done to investigate the presence of heparin in plasma or to detect fibrinogen abnormalities.
• This test involves observation of clotting time after adding thrombin to the patient’s plasma. Normal duration of thrombin time is 12-20 seconds.
• It is prolonged in heparin therapy and during dysfibrinogenemia (abnormal function of fibrinogen with normal fibrinogen level).

Applied Physiology

Bleeding Disorders: Bleeding disorders are diseases characterized by prolonged bleeding time or clotting time. Bleeding disorders are of three types

1. Hemophilia
2. Purpura
3. von Willebrand disease.

1. Hemophilia

• Hemophilia is a group of sex-linked inherited blood disorders featured by prolonged clotting time. Usually, it affects the males, 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 a prothrombin activator. That is why the coagulation time is prolonged. Bleeding time and prothrombin time are normal. The formation of prothrombin activator is affected due to the deficiency of factor 8, 9, or 11.
• Types of hemophilia: Depending upon the deficiency of the factor involved, hemophilia is classified into three types:
  1. Hemophilia A or classic hemophilia: It is due to the deficiency of factor 8. 85% of people with hemophilia are affected by hemophilia A.
  2. Hemophilia B or Christmas disease: It is due to the deficiency of factor 9. 15% of people with hemophilia are affected by hemophilia B.
  3. Hemophilia C or factor 11 deficiency: It is due to the deficiency of factor 11. It is a very rare blood disorder.
• Symptoms of hemophilia
  1. Spontaneous bleeding
  2. Prolonged bleeding due to cuts, tooth extraction, and surgery
  3. Hemorrhage in gastrointestinal and urinary tracts
  4. Bleeding in joints followed by swelling and pain
  5. The appearance of blood in urine
• Treatment for hemophilia: The effective therapy for classical hemophilia involves replacement of missing clotting factors.

2. Purpura

• It is a disorder characterized by prolonged bleeding time. However, the clotting time is normal.
• The characteristic feature of this disease is spontaneous bleeding under the skin from ruptured capillaries. It causes small tiny hemorrhagic spots in many areas of the body.
• The hemorrhagic spots under the skin are called purpuric spots (purple-colored patch-like appearance). That is why this disease is called purpura.
• Blood also sometimes collects in large areas beneath the skin which are called ecchymoses.
• Types and causes of purpura: The purpura is classified into different types depending upon the causes.
• Thrombocytopenic purpura: Thrombocytopenic purpura is due to the deficiency of platelets (thrombocytopenia). In bone marrow disease, platelet production is affected leading to deficiency of platelets.
• Idiopathic thrombocytopenic purpura: Purpura due to some unknown cause is called idiopathic thrombocytopenic purpura. It is believed that platelet count decreases due to the development of antibodies against platelets, which occurs after blood transfusion.
• Thrombasthenic purpura: It is due to structural or functional abnormality of platelets. However, the platelet count is normal. It is characterized by normal clotting time, normal or prolonged bleeding time but defective clot retraction.

3. von Willebrand Disease

• von Willebrand disease is a bleeding disorder characterized by excess bleeding even with a mild injury. It is due to inherited deficiency of von Willebrand factor which is a protein secreted by the endothelium of damaged blood vessels and platelets.
• This protein is responsible for adherence of platelets to the endothelium of blood vessels during hemostasis after an injury. It is also responsible for the survival and maintenance of factor 8 in plasma.
• The deficiency of von Willebrand factor suppresses platelet adhesion. It also causes deficiency of factor 8. This results in excess bleeding which resembles the bleeding that occurs during platelet dysfunction or hemophilia.

Thrombosis: Thrombosis or intravascular blood clotting refers to coagulation of blood inside the blood vessels. Normally, blood does not clot in the blood vessel because of some factors which are already explained.

• Causes of Thrombosis
  1. Injury to blood vessels: During infection or mechanical obstruction, the endothelial lining of the blood vessel is damaged and it initiates thrombosis.
  2. Roughened endothelial lining: In infection, damage, or arteriosclerosis, the endothelium becomes rough and this initiates clotting.
  3. Sluggishness of blood flow: Decreased rate of blood flow causes aggregation of platelets and formation of thrombus. The slowness of blood flow occurs in reduced cardiac action, hypotension, low metabolic rate, prolonged confinement to bed, and immobility of limbs.
  4. Agglutination of RBCs: Agglutination of the RBCs leads to thrombosis. Agglutination of RBCs occurs by foreign antigens or toxic substances.
  5. Toxic thrombosis: Thrombosis is common due to action of chemical poisons like arsenic compounds, mercury, poisonous mushrooms, and snake venom.
  6. Congenital absence of protein C: Protein C is a circulating anticoagulant, which inactivates factors 5 and 8. Thrombosis occurs in the absence of this protein. Congenital absence of protein C causes thrombosis and death in infancy.
• Complications of Thrombosis

1. Thrombus: During thrombosis, the lumen of blood vessels is occluded. The solid mass of platelets, red cells, and or clot, which obstructs the blood vessel, is called thrombus. The thrombus formed due to the agglutination of RBC is called an agglutinative thrombus.
2. Embolism and embolus: Embolism is the process in which the thrombus or part of it is detached and carried in the bloodstream and occludes the small blood vessels resulting in arrests of blood flow to any organ or region of the body. Embolus is the thrombus or part of it, which arrests the blood flow. The obstruction of blood flow by embolism is common in lungs (pulmonary embolism), brain (cerebral embolism), or heart (coronary embolism).
3. Ischemia: Insufficient blood supply to an organ or area of body by the obstruction of blood vessels is called ischemia. Ischemia results in tissue damage because of hypoxia (lack of oxygen). Ischemia also causes discomfort, pain, and tissue death. Death of body tissue is called necrosis.
4. Necrosis and infarction: Necrosis is a general term that refers to tissue death caused by loss of blood supply, injury, infection, inflammation, physical agents, or chemical substances.
   • Infarction means tissue death due to loss of blood supply. Loss blood supply is usually caused by occlusion of an artery by thrombus or embolus and sometimes by atherosclerosis.
   • The area of tissue that undergoes infarction is called an infarct. Infarction commonly occurs in heart, brain, lungs, kidneys, and spleen.