Lecture 6

Secondary Hemostasis: Formation of a Blood Clot

Secondary Hemostasis

• Blood clotting is the process of transforming blood into a solid gel called a clot or thrombus.

• The clot consists of a protein called fibrin.

• The essential part of clot formation is the formation of fibrin.

• Secondary hemostasis:

  • Occurs following platelet plug formation.

  • Involves a cascade of enzyme (clotting factors) activation.

  • Activation of enzymes occurs by proteolytic cleavage.

  • Results in the formation of a gel-like fibrin clot, in which red blood cells become trapped (red thrombus).

Factors Involved in Blood Clotting

• There are 13 factors involved in blood clotting.

• All are present as inactive plasma proteins in the blood.

• Factors are expressed as Roman numerals.

• Factor IV is ionized calcium (not a protein).

• Many activation reactions occur on the surface of platelets.

• Factors are synthesized in the liver.

• The first 4 factors are most important:

  • Factor I: Fibrinogen

  • Factor II: Prothrombin

  • Factor III: Tissue thromboplastin (tissue factor)

  • Factor IV: Calcium

Blood Clotting: Key Step

• The key step is the generation of the active thrombin enzyme.

  • Inactive prothrombin is converted to thrombin by the prothrombinase complex.

  • Thrombin converts inactive fibrinogen to active fibrin.

  • Fibrin is an insoluble protein that forms strong strands and a mesh-work (fibrin clot) upon stabilization, trapping cells.

Activation of Thrombin

• Two pathways activate clotting factors to form a common activated factor (factor Xa).

  • Factor Xa is directly involved in the formation of active thrombin.

  • Intrinsic pathway: All factors come from within the blood and blood vessel.

  • Extrinsic pathway: Initiated by factors outside the blood vessel.

• Both pathways meet and activate the common factor in the common pathway, leading to blood clot formation.

Traditional/Classical Mechanism of Blood Clotting

• Pathways are activated sequentially and interact.

• Intrinsic and extrinsic pathways merge at the formation of active factor X and lead to the formation of active thrombin in the common pathway.

  • Intrinsic pathway: Everything necessary is found in the blood.

  • Extrinsic pathway: A cellular element outside the blood is needed.

Intrinsic Pathway:

• Begins with the activation of factor XII (contact activation factor) when it contacts the surface of a damaged blood vessel wall, forming XIIa.

• Factor XII triggers the intrinsic pathway.

• In blood collection, the rough inner surface of a test tube acts like a damaged blood vessel wall, activating factor XII.

• Silicone-coated tubes prevent clotting in vitro because of the smooth inner surface.

Extrinsic Pathway:

• Activated when factor VII contacts tissue products and tissue factors outside a blood vessel.

• Tissue factors are on the outer plasma membrane of tissue cells or cells in the blood vessel walls outside the endothelium.

• Blood is exposed to these subendothelial cells when the vessel is damaged and the endothelial lining is disrupted.

• In the presence of phospholipids exposed on activated platelets and calcium, factor VII is activated by binding to tissue factor.

• Factor VIIa then activates factor X to produce activated factor X (factor Xa).

• Activation of factor X leads to the common pathway of fibrin formation.

Physiological Pathway of Blood Clotting

• Clotting is initiated in the extrinsic pathway when Factor VII is activated in the presence of tissue factors exposed by a cut.

• Factor VIIa produces a small amount of active factor Xa, which activates a small amount of thrombin, resulting in a small amount of fibrin.

• The amount of thrombin produced by the extrinsic pathway is too small for sustained coagulation.

• This initial thrombin provides a positive feedback effect to factors V, VIII, XI, and XIII of the intrinsic pathway, initiating further thrombin formation.

• The activated intrinsic pathway produces a large amount of thrombin and a large fibrin clot.

• Pathways are activated sequentially, with thrombin linking them.

Effect of Various Clotting Factor Deficiencies on Clotting

• Deficiency in factor VII causes serious bleeding.

• Lack of factor VIII causes severe bleeding (hemophilia).

• Lack of factor XI causes moderate bleeding.

• Lack of factor XII causes no bleeding problem in vivo, but blood does not clot in a glass test tube in vitro.

  • Clotting is initiated via activation of factor VII, not factor XII.

• If a person lacks factor VII, there is a serious bleeding problem.

• Blood from a healthy individual will clot when placed in a glass test tube without anticoagulant.

  • Factor XII is activated upon contact with glass, initiating clot formation.

  • Silicon-coated glass does not trigger factor XII activation due to its smooth surface.

What Does Thrombin Do In the Clotting Pathway?

• Thrombin:

  • Activation of platelets.

  • Converts soluble fibrinogen into insoluble fibrin.

  • Activates several other clotting factors, including factors V, VIII, XI, and XIII.

  • Plays a role in anti-clotting pathways (anti-coagulant activity).

Regulation of Blood Clotting

• Anticoagulants: Prevent clot formation where and when it is not required.

• Fibrinolysis: Enzymatic breakdown of fibrin in blood clots.

Prevention of Clot Formation: Natural Anticoagulants

• Natural anticoagulants in our body:

  • TFPI or tissue factor pathway inhibitor: Inhibits tissue factor (factor III), which is involved in the activation of factor VII. Tissue factor initiates the extrinsic pathway.

  • Antithrombin III: Inhibits the actions of thrombin.

  • Thrombomodulin: A protein expressed on the surface of undamaged, healthy endothelial cells; it binds to thrombin to prevent clot formation.

    • Bound thrombin activates inactive protein C to form active protein C.

    • Protein C interacts with protein S, and they inhibit factors Va and factor VIIIa. Factor Va is involved in the formation of thrombin, while factor VIIIa is closely involved in the formation of factor Xa.

Actions of Thrombin As an Anticoagulant

• Healthy endothelial cells express thrombomodulin.

• Thrombomodulin binds to thrombin and activates protein C.

• Activated protein C inhibits Factors VIIIa and Va.

Prevention of Clot Formation: Clinical Anticoagulants

• Clinical anticoagulants:

  • Calcium chelators (e.g., sodium citrate) bind ionized calcium, preventing the activation of clotting factors.

  • Heparin increases the activity of antithrombin III.

  • Vitamin K antagonists inhibit the synthesis of vitamin K-dependent clotting factors (factors II, VII, IX, and X).

Breakdown of Fibrin Clot: Fibrinolysis

• Fibrinolysis: Breakdown of fibrin in a clot.

• Plasminogen activators break down a clot.

  • tPA (tissue plasminogen activator) is a plasminogen activator.

  • tPA converts inactive plasminogen to the active enzyme plasmin.

  • Plasmin breaks down insoluble fibrin strands into soluble fibrin degradation products to dissolve the clot.

Fibrinolysis: Tissue Plasminogen Activator (tPA)

• Released from healthy endothelial cells.

• Clinical clot busters or thrombolytic drugs are used to treat patients with heart attacks to dissolve clots.

• Thrombolytics dissolve major clots quickly to restart blood flow to the heart and help prevent damage to the heart muscle.

Abnormal Hemostasis: Imbalance of PRO- and ANTI-hemostatic Factors

• When pro-hemostatic factors fail:

  • Severe bleeding disorders, leading to hemorrhage, may occur.

  • Defects in pro-hemostatic pathways may include:

    • Problems with platelets: Thrombocytopenia (deficiency in the number of platelets).

    • Abnormal platelet function due to a deficiency of von Willebrand’s factor (a plasma protein secreted by endothelial cells and platelets that promotes adhesion).

    • Problems with clotting factors: Hereditary deficiencies of clotting factors.

    • A lack of vitamin K may cause deficiencies of factors requiring vitamin K for their synthesis.

• When anti-hemostatic factors fail:

  • Blood clots remain in the circulation for a long time, leading to thrombosis (formation of a blood clot in the vessel, blocking blood flow).

  • Deficiencies of natural anticoagulants and fibrinolytic factors due to hereditary disorders may cause thrombosis.

  • Acquired disorders, such as decreased blood flow, may also lead to the formation of blood clots.