DEFINITION AND CLOTTING FACTORS

Factors Involved in Blood Clotting

• Coagulation of blood occurs through a series of reactions due to activation of a group of substances.
• Substances necessary for clotting → clotting factors.
• Thirteen clotting factors are identified.
• Clotting factors were named:
• After the scientists who discovered them, or
• According to their activity, except factor IX.
• Christmas factor (factor IX) → named after the patient in whom it was discovered.

Enzyme Cascade Theory

• Most clotting factors are proteins in the form of enzymes.
• Normally, all factors are present as inactive proenzymes.
• These must be activated into enzymes to enforce blood clotting.
• Activation occurs through a series of enzyme-enzyme conversion reactions.
• First enzyme → activated.
• It activates the second enzyme.
• Second enzyme → activates the third.
• This continues until the final active enzyme thrombin is formed.
• Enzyme cascade theory explains how various reactions involved in conversion of proenzymes to active enzymes occur in the form of a cascade.
• Cascade → process occurring through a series of steps, each step initiating the next, until the final step is reached.

Stages of Blood Clotting

Blood clotting occurs in 3 stages:

1. Formation of prothrombin activator.
2. Conversion of prothrombin into thrombin.
3. Conversion of fibrinogen into fibrin.

CLOTTING FACTORS

STAGE I: FORMATION OF PROTHROMBIN ACTIVATOR

• Blood clotting begins with formation of a substance called prothrombin activator.
• Formation of prothrombin activator is initiated by substances produced either:
• Within blood itself, or
• Outside the blood.
• Formation of prothrombin activator occurs through two pathways:
1. Intrinsic pathway
2. Extrinsic pathway

A. Intrinsic Pathway for Formation of Prothrombin Activator

• In intrinsic pathway, formation of prothrombin activator is initiated by platelets, which are within the blood itself.

Sequence of Intrinsic Pathway

1. During injury, blood vessel is ruptured.
   • Endothelium is damaged.
   • Collagen beneath the endothelium is exposed.
2. Factor XII (Hageman factor) comes in contact with collagen.
   • It is converted into activated factor XII in the presence of kallikrein and HMW kininogen.
3. Activated factor XII converts factor XI into activated factor XI in the presence of HMW kininogen.
4. 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
   • Calcium
6. When platelets have contact with collagen of the damaged blood vessel:
   • They get activated.
   • They release phospholipids.
7. Activated factor X reacts with:
   • Platelet phospholipid
   • Factor V
   • Calcium ions
   and forms prothrombin activator.
8. Factor V is also activated by the positive feedback effect of thrombin.

B. Extrinsic Pathway for Formation of Prothrombin Activator

• In this pathway, formation of prothrombin activator is initiated by tissue thromboplastin formed from injured tissues.

Sequence of Events in Extrinsic Pathway

1. Damaged tissues release factor III, i.e. tissue thromboplastin.
   • Thromboplastin contains:
   • Proteins
   • Phospholipid
   • Glycoprotein
   • These 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
   • Phospholipid component of tissue thromboplastin
   • Calcium ions
   and forms prothrombin activator.

STAGE II: CONVERSION OF PROTHROMBIN INTO THROMBIN

Sequence of Events in Stage II

1. Prothrombin activator formed in intrinsic and extrinsic pathways converts prothrombin into thrombin in the presence of calcium ions (factor IV).
2. Once formed, thrombin initiates formation of more thrombin molecules.
3. Initially formed thrombin activates factor V.
4. Factor V accelerates formation of both extrinsic and intrinsic prothrombin activator.
5. Prothrombin is converted into thrombin.
6. This effect of thrombin is called positive feedback effect.

STAGE III: CONVERSION OF FIBRINOGEN INTO FIBRIN

Sequence of Events in Stage III

1. Thrombin converts fibrinogen into activated fibrinogen, which is called fibrin monomer.
2. Fibrin monomer polymerizes with other monomer molecules and forms loosely arranged strands of fibrin.
3. Loose fibrin strands are modified into dense and tight fibrin threads by fibrin-stabilizing factor (factor XIII) in the presence of calcium ions.
4. All tight fibrin threads are aggregated to form a meshwork of stable clot.

BLOOD CLOT

Definition and Composition of Blood Clot

• RBCs and WBCs are necessary for the clotting process.
• However, when clot is formed:
• These cells get trapped in it along with platelets.
• Entrapped RBCs → responsible for red color of clot.
• External blood clot called scab adheres to the opening of damaged blood vessel and prevents blood loss.

Clot Retraction

• Clot retraction → contraction of blood clot 30–45 minutes after formation.
• It causes oozing of serum out of the clot.
• Contractile proteins:
• Actin
• Myosin
• Thrombosthenin
• These are present in cytoplasm of platelets and are responsible for clot retraction.

FIBRINOLYSIS

• It helps to remove the clot from lumen of blood vessel.
• Fibrinolysis requires a substance called plasmin or fibrinolysin.

Formation of Plasmin

• Plasmin is formed from an inactivated glycoprotein called plasminogen.
• Plasminogen is synthesized in the liver.
• It is incorporated with other proteins in the blood clot.
• Plasminogen is converted into plasmin by:
• Tissue plasminogen activator (t-PA)
• Lysosomal enzymes
• Thrombin
• t-PA and lysosomal enzymes are released from:
• Damaged tissues
• Damaged endothelium
• Thrombin is derived from blood.
• t-PA is always inhibited by a substance called t-PA inhibitor.
• It is also inactivated by factors V and VIII.
• Besides t-PA, another plasminogen activator derived from blood is called urokinase plasminogen activator (u-PA).

Sequence of Events in Activation of Plasminogen

1. During intravascular clotting, endothelium of blood vessels secretes a thrombin-binding protein called thrombomodulin.
   • It is secreted by endothelium of blood vessels, except 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 factors V and VIII in the presence of a cofactor called protein S.
5. Protein S also inactivates the t-PA inhibitor.
6. Now t-PA becomes activated.
7. Activated t-PA and lysosomal enzymes activate plasminogen to form plasmin.
8. Plasminogen is also activated by thrombin and u-PA.

Significance of Lysis of Clot

• In vital organs, particularly the heart, blood clot obstructs minute blood vessels leading to myocardial infarction.
• Lysis of blood clot allows reopening of affected blood vessels and prevents development of infarction.

Fibrinolytic Enzymes

• Fibrinolytic enzymes such as streptokinase are used for lysis of blood clot during treatment in the early stages of myocardial infarction.

ANTICLOTTING MECHANISM IN BODY

Factors Preventing Blood Clotting During Circulation

1. Smooth surface of endothelium
   • Prevents activation of clotting factors.
2. Glycocalyx layer
   • Present on inner surface of endothelium.
   • Repels platelets and clotting factors.
   • Therefore, initiation of blood clotting is prevented.
3. Continuous flow of blood
   • Does not allow aggregation of platelets.
   • Prevents blood clotting.
4. Presence of natural anticoagulants in blood
   • Heparin
   • Protein C:
     • Activated by thrombomodulin-thrombin complex.
     • Activated protein C along with factor protein S inactivates factor V and factor VIII.
     • Inactivation of these clotting factors prevents clot formation.

ANTICOAGULANTS

Three Types

1. Anticoagulants used to prevent blood clotting inside the body → in vivo anticoagulants.
2. Anticoagulants used to prevent clotting of blood collected from the body → in vitro anticoagulants.
3. Anticoagulants used to prevent blood clotting both in vivo and in vitro.

I. HEPARIN

• Heparin is a naturally produced anticoagulant in the body.
• It is produced by mast cells.
• Mast cells are wandering cells situated immediately outside capillaries in tissues or organs containing more connective tissue.
• Mast cells are abundant in:
• Liver
• Lungs
• Basophils also secrete heparin.
• Heparin is a conjugated polysaccharide.
• Commercial heparin is prepared from liver and other organs of animals.
• Commercial preparation is available in liquid or dry form as:
• Sodium salts
• Calcium salts
• Ammonium salts
• Lithium salts

Mechanism of Action of Heparin

1. Prevents blood clotting by its anti-thrombin activity.
   • Directly suppresses activity of thrombin.
2. Combines with antithrombin III present in circulation.
   • Removes thrombin from circulation.
3. Activates the active form of other clotting factors like:
   • IX
   • X
   • XI
   • XII

Uses of Heparin

• Used as anticoagulant:
• In vivo
• 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.
• Therefore, it is used as an anticoagulant in clinical practice.

Heparin is Used For

1. To prevent cardiovascular blood clotting during surgery.
2. During dialysis when blood is passed through an artificial kidney.
3. During cardiac surgery involving passing blood through heart-lung machine.
4. To preserve blood before transfusion.

Use in Laboratory

• Heparin is also used as an anticoagulant in vitro while collecting blood for various investigations.
• Heparin is the most expensive anticoagulant.

II. COUMARIN DERIVATIVES

• Dicoumarol and warfarin are derivatives of coumarin.

Mechanism of Action

• Coumarin derivatives prevent blood clotting by inhibiting the action of vitamin K.
• Vitamin K is essential for formation of clotting factors:
• II
• VII
• IX
• X

Uses

• Dicoumarol and warfarin are commonly used oral anticoagulants (in vivo).

III. EDTA

• Ethylenediaminetetraacetic acid (EDTA) is a strong anticoagulant.
• Available in two forms:
1. Disodium salt (Na₂ EDTA)
2. Tripotassium salt (K₃ EDTA)

Mechanism of Action

• EDTA prevents blood clotting by removing calcium from blood.

Uses of EDTA

• Used as an anticoagulant:
• In vivo
• In vitro
• It is administered intravenously in cases of lead poisoning (in vivo).

IV. OXALATE COMPOUNDS

• Oxalate compounds prevent coagulation by forming calcium oxalate, which is precipitated later.
• Oxalate compounds reduce the blood calcium level.
• A mixture of ammonium oxalate and potassium oxalate in the ratio 3:2 is used.
• Each salt is anticoagulant by itself.
• Potassium oxalate alone causes shrinkage of RBCs.
• Ammonium oxalate alone causes swelling of RBCs.
• Together, both substances do not alter cellular activity.

Mechanism of Action

• Oxalate combines with calcium and forms insoluble calcium oxalate.
• Thus:
• Oxalate removes calcium from blood.
• Lack of calcium prevents blood coagulation.

Uses

• Used as in vitro anticoagulants.
• Cannot be used in vivo.

V. CITRATES

• Sodium, ammonium and potassium citrates are used as anticoagulants.

Mechanism of Action

• Citrate combines with calcium in blood to form insoluble calcium citrate.
• Like oxalate, citrate also removes calcium and prevents coagulation.

Uses

• Used as anticoagulant:
• In vivo
• In vitro
• Used to store blood in blood bank.
  • Acid citrate dextrose (ACD)
  • Citrate phosphate dextrose (CPD)
• Used in laboratory in vitro for:
  • RBC counts
  • Platelet counts.

VI. OTHER SUBSTANCES WHICH PREVENT BLOOD CLOTTING

• Peptone
• Proteins from venom of copper-head snake
• Hirudin from leech
• These are known as anticoagulants.

Physical Methods to Prevent Blood Clotting

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

1. Cold
   • Reducing temperature to about 5°C postpones coagulation of blood.
2. Collecting Blood in a Container with Smooth Surface
   • Collecting blood in a container with smooth surface like a silicon-coated container prevents clotting.
   • Smooth surface inhibits activation of factor XII and platelets.
   • Therefore, formation of prothrombin activator is prevented.

PROCOAGULANTS

Procoagulants

1. Thrombin
   • Thrombin is sprayed upon the bleeding surface to arrest bleeding by hastening blood clotting.
2. Snake venom
   • Venom of some snakes:
   • Cobras
   • Rattle snakes
   • Contains proteolytic enzymes which enhance blood clotting by activating clotting factors.
3. Extracts of lungs and thymus
   • Extract obtained from lungs and thymus has thromboplastin.
   • Causes rapid blood coagulation.
4. Sodium or calcium alginate
   • Enhances blood clotting process by activating Hageman factor.
5. Oxidized cellulose
   • Causes clotting of blood by activating Hageman factor.

TESTS FOR BLOOD CLOTTING

• Blood clotting tests are used to diagnose blood disorders.
• Some tests are also used to monitor patients treated with anticoagulant drugs such as:
• Heparin
• Warfarin
• Six tests are available for blood clotting:

1. Bleeding time
2. Clotting time
3. Prothrombin time
4. Partial prothrombin time
5. International normalized ratio
6. Thrombin time

1. BLEEDING TIME

• Bleeding time → time interval from oozing of blood after a cut or injury until arrest of bleeding.
• Usually determined by Duke method using:
• Blotting paper
• Filter paper
• Normal bleeding time → 3–6 minutes.
• Prolonged in purpura.

2. CLOTTING TIME

• Clotting time → time interval from oozing of blood after a cut or injury until formation of clot.
• Usually determined by capillary tube method.
• Normal clotting time → 3–8 minutes.
• Prolonged in hemophilia.

3. PROTHROMBIN TIME

• Prothrombin time → time taken by blood to clot after adding tissue thromboplastin to it.
• Blood is collected and oxalated so that:
• Calcium is precipitated.
• Prothrombin is not converted into thrombin.
• Blood clotting is prevented.
• A large quantity of tissue thromboplastin with calcium is added to this blood.
• Calcium nullifies the effect of oxalate.
• Tissue thromboplastin activates prothrombin.
• Blood clotting occurs.
• Time taken by blood to clot after adding tissue thromboplastin is determined.
• Prothrombin time indicates the total quantity of prothrombin present in blood.
• Normal prothrombin time → 10–12 seconds.
• Prolonged in deficiency of prothrombin and other factors such as:
• I
• V
• VII
• X
• However, it is normal in hemophilia.

4. PARTIAL PROTHROMBIN TIME

• Partial prothrombin time (PPT) or activated prothrombin time → time taken for blood to clot after adding:
• An activator such as phospholipid
• Calcium
• Also called activated partial prothrombin time (APPT).
• Useful in monitoring patients taking anticoagulant drugs.
• It is carried out by observing clotting time after adding:
• Phospholipid
• Surface activator
• Calcium
• to patient’s plasma.
• Phospholipid serves as platelet substitute.
• Commonly used surface activator → kaolin.
• Normal partial prothrombin time → 30–45 seconds.
• Prolonged in:
• Heparin
• Warfarin
• Deficiency or inhibition of factors:
• VII
• VIII
• IX
• XI
• XII

5. INTERNATIONAL NORMALIZED RATIO

• International normalized ratio (INR) → rating of a patient’s PT when compared to an average.
• It measures extrinsic clotting pathway system.
• INR is useful in monitoring the impact of anticoagulant drugs such as warfarin and adjusting the dosage of anticoagulants.
• 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.
• Blood takes longer time to clot if INR is higher.
• Normal INR → about 1.
• In patients taking anticoagulant therapy for atrial fibrillation:
• Normal INR should be between 2 and 3.
• For patients with heart valve disorders:
• INR should be between 3 and 4.
• INR greater than 4 indicates blood is clotting too slowly and there is a risk of uncontrolled blood clotting.

6. THROMBIN TIME

• Thrombin time (TT) → time taken for blood to clot after adding thrombin to it.
• Done to investigate:
• Presence of heparin in plasma
• Fibrinogen abnormalities
• Test involves observing clotting time after adding thrombin to patient’s blood.
• Normal thrombin time → 12–20 seconds.
• Prolonged in:
• Heparin therapy
• Dysfibrinogenemia (abnormal function of fibrinogen with normal fibrinogen level).

APPLIED PHYSIOLOGY: THROMBOSIS

• Normally, blood does not clot in blood vessels because of some factors already explained.
• Some abnormal conditions can cause thrombosis.

Causes of Thrombosis

1. Injury to blood vessels
   • Damage of endothelial lining during:
   • Infection
   • Mechanical obstruction
   • Initiates blood clotting.
2. Roughened endothelial lining
   • During infection, damage or arteriosclerosis:
   • Endothelium becomes rough.
   • Initiates clotting.
3. Sluggishness of blood flow
   • Decreased rate of blood flow causes:
   • Aggregation of platelets
   • Formation of thrombus
   • Slowness of blood flow occurs during:
   • Reduced cardiac action
   • Hypotension
   • Low metabolic rate
   • Prolonged confinement to bed
   • Immobility of limbs
4. Agglutination of RBCs
   • Occurs by:
   • Foreign antigens
   • Toxic substances
   • Leads to thrombosis.
5. Toxic thrombi
   • Occur due to action of chemical poisons such as:
   • Arsenic compounds
   • Mercury
   • Poisonous mushrooms
   • Snake venom
6. Congenital absence of protein C
   • Causes thrombosis and death in infancy.

Complications of Thrombosis

1. Thrombus

• During thrombosis, lumen of blood vessels is occluded.
• Solid mass of:
• Platelets
• Red cells
• White cells
• Which obstructs the blood vessel → called thrombus.
• Thrombus formed due to agglutination of RBC is called agglutinative thrombus.

2. Embolism and Embolus

• Embolism → process during which thrombus or part of it becomes detached and is carried into bloodstream and occludes small blood vessels, resulting in arrest of blood flow to any organ or region of body.
• Embolus → thrombus or part of it which becomes detached and arrests blood flow.
• Obstruction of blood flow by embolism is common in:
• Lungs → pulmonary embolism
• Brain → cerebral embolism
• Heart → coronary embolism

3. Ischemia

• Insufficient blood supply to an organ or area of body due to obstruction of blood vessels → ischemia.
• Ischemia results in tissue damage because of hypoxia (lack of oxygen).
• Ischemia also causes:
• Discomfort
• Pain
• Tissue death
• Death of body tissue → necrosis.

DISSEMINATED INTRAVASCULAR COAGULATION

• It is caused by:
• Infection
• Inflammation
• Cancer
• DIC is a serious condition.
• It often results in development of multiple organ dysfunction syndrome (MODS).

BLEEDING DISORDERS

Bleeding disorders are of 3 types:

1. Hemophilia
2. Purpura
3. von Willebrand disease

1. HEMOPHILIA

• Hemophilia → group of sex-linked inherited blood disorders characterized by prolonged clotting time.
• However, bleeding time is normal in hemophilia.
• In this disorder:
• Males are affected.
• Females are carriers.
• Because of prolonged clotting time:
• Even a small trauma causes excessive bleeding.
• This can lead to death.
• Damage of skin while falling or extraction of a tooth can cause excessive bleeding for a few weeks.
• Easy bruising and hemorrhage in muscles and joints are also common.

Cause of Hemophilia

• Lack of prothrombin activator is the cause of hemophilia.
• Formation of prothrombin activator is affected due to deficiency of:
• Factor VII
• Factor IX
• Factor XI

Types of Hemophilia

Depending upon deficiency of the factor involved, hemophilia is classified into 3 types:

1. Hemophilia A
   • Also called classic hemophilia.
   • Due to deficiency of factor VIII.
   • 85% of people with hemophilia are affected by hemophilia A.
2. Hemophilia B
   • Also called Christmas disease.
   • Due to deficiency of factor IX.
   • 15% of people with hemophilia are affected by hemophilia B.
3. Hemophilia C
   • Due to deficiency of factor XI.
   • It is a very rare blood disorder.

2. PURPURA

• Purpura → disorder characterized by prolonged bleeding time.
• However, clotting time is normal.
• Characteristic feature:
• Spontaneous bleeding under the skin from ruptured capillaries.
• It causes small hemorrhagic spots under the skin called purpuric spots.
• Purpuric spots have:
• Purple-colored
• Patch-like appearance
• Hence the disease is called purpura.

Types and Causes of Purpura

1. Thrombocytopenic Purpura
   • Due to deficiency of platelets (thrombocytopenia).
   • In bone marrow disease:
   • Platelet production is affected.
   • Leads to deficiency of platelets.
2. Idiopathic Thrombocytopenic Purpura
   • Due to some unknown cause.
   • Platelet count decreases due to development of antibodies against platelets.
   • This occurs after blood transfusion.
3. Thrombasthenic Purpura
   • Due to structural or functional abnormality of platelets.
   • Platelet count is normal.
   • Characterized by:
   • Normal clotting time
   • Normal or prolonged bleeding time
   • Defective clot retraction.

3. VON WILLEBRAND DISEASE

• von Willebrand disease → bleeding disorder characterized by excess bleeding even with a mild injury.
• Due to inherited deficiency of von Willebrand factor.
• von Willebrand factor is a protein secreted by endothelium of damaged blood vessels and platelets.
• This protein is responsible for:
• Adherence of platelets to endothelium of blood vessels during hemostasis after an injury.
• It is also responsible for survival and maintenance of factor VIII in plasma.
• Deficiency of von Willebrand factor:
• Suppresses platelet adhesion.
• Causes deficiency of factor VIII.
• This results in excess bleeding resembling bleeding during:
• Platelet dysfunction
• Hemophilia.