HEMOSTASIS & COAGULATION.pptx
- 1. NORMAL HAEMOSTASIS & COAGULATION CASCADE DR YOGESH AGRAWAL RESIDENT MD TRANSFUSION MEDICINE SMS MEDICAL COLLEGE
- 2. HAEMOSTASIS: (blood+ stable) Haemostasis is defined as a set of well-regulated processes designed to rapidly and specifically form a clot at sites of vascular injury. It is the first stage of wound healing. • Normal haemostasis accomplish two important functions: • Maintenance of blood in a fluid, clot free state. • A rapid & localized haemostatic plug formation at a site of vascular injury.
- 3. SEQUENCE OF EVENTS IN HEMOSTASIS: 1. VASOCONSTRICTION: After initial injury, there is brief period of arteriolar vasoconstriction attributable to: 1. Reflex neurogenic mechanisms. 2. Local factors derived from endothelium like ENDOTHELIN, a potent vasoconstrictor. This effect is transient.
- 4. 2. PRIMARY HEMOSTASIS/PLATELET PLUG FORMATION Endothelial injury exposes highly thrombogenic sub-endothelial extracellular matrix (ECM). Platelets adhere to ECM & become activated. Platelets undergo shape change & release secretory granules, which recruit additional platelets (aggregation) to form haemostatic plug k/a primary haemostasis.
- 5. 3. SECONDARY HEMOSTASIS/COAGULATION Tissue factor, pro-coagulant factor synthesized by endothelium is exposed at the site of injury. Activate coagulation cascade, resulting in activation of thrombin. Thrombin converts circulating soluble fibrinogen to insoluble fibrin, causing local fibrin deposition. This is k/a secondary hemostasis.
- 6. 4. CLOT STABILIZATION AND RESORPTION Polymerised fibrin & platelet aggregates form solid permanent plug to prevent any further haemorrhage. At this stage, counter-regulatory mechanisms e.g. t-PA are set into motion to limit the haemostatic plug at the site of injury.
- 7. HAEMOSTASIS contd… Normal haemostasis involves the interaction of 1. Blood vessel wall (endothelium) 2. Platelets 3. Coagulation factors
- 8. ROLE OF ENDOTHELIUM Maintains normal flow of blood by 1. Anti-platelet effect, 2. Anti-coagulant effect, and 3. Fibrinolytic properties.
- 9. 1. ANTI-PLATELET EFFECTS Prevents spontaneous platelet aggregation Produces : 1. Antithrombin. 2. Von Willebrand's factor (vWF). 3. Vasodilators like NO and prostacyclin (PGI2) which inhibits platelet aggregation.
- 10. 2. ANTI-COAGULANT EFFECTS Mediated by: • Heparin-like molecules. • Thrombomodulin. • Tissue factor pathway inhibitor (TFPI). Heparin-like molecules interact with antithrombin III to inactivate thrombin , factor Xa & several other coagulation factors
- 11. ANTI-COAGULANT EFFECTS CONTD.. Thrombomodulin binds thrombin activate protein C proteolytic cleavage of factors Va & VIIIa Inhibits clotting
- 12. ANTI-COAGULANT EFFECTS CONTD.. • Endothelium is also a major source for tissue factor pathway inhibitor, that complexes & inhibits activated tissue factor- factor VIIa and factor Xa molecules.
- 13. 3. FIBRINOLYTIC EFFECTS • Endothelium synthesizes tissue-type Plasminogen Activator (t-PA), promoting fibrinolytic activity to clear fibrin deposits from endothelial surfaces.
- 15. ROLE OF PLATELETS: •After vascular injury, platelets encounter ECM constituents & undergo 3 general reactions: 1) Platelet adhesion 2) Secretion (Release Reaction) 3) Platelet aggregation
- 16. 1. PLATELET ADHESION • Platelets adhere to extra-cellular matrix (ECM) via vWF which acts as bridge b/w plt. Surface receptors (e.g. GP 1b) & exposed collagen.
- 18. 2. SECRETION Release of contents of α and δ-granules. Dense body contents are especially important because: I. Calcium is required in the coagulation Cascade. II. ADP is a potent mediator of plt. Aggregation and “Recruitment”
- 20. 3. PLATELET AGGREGATION Important Stimulus for plt. aggregation: I. ADP II. TXA2 III. Thrombin
- 21. ROLE OF COAGULATION SYSTEM: 1. Constitutes 3rd component of the haemostatic process & is a major contributor to thrombosis. 2. Results in formation of thrombin. 3. Each reaction step involves an enzyme (an activated coagulation factor), a substrate (an inactive proenzyme form of a coagulation factor), and a cofactor (a reaction accelerator). 4. Blood coagulation pathway has been divided into extrinsic & intrinsic pathways, converging where factor X is activated.
- 22. There are major 13 factors which are involved in the coagulation cascade. All these factors are blood proteins or their derivatives. Even if one of the factor is defective, the whole clotting process is impaired leading to hemorrhage. These factors are F-I to F-XIII.
- 24. • There are 3 major stages in the coagulation cascade: • Stage 1: Formation of Prothrombinase Complex (Prothrombin Activator) • Prothrombinase is formed in two ways: • Extrinsic Pathway (also known as Tissue Factor Pathway) • Intrinsic Pathway (also known as Contact Activation Pathway) • Stage 2: Conversion of Prothrombin into Thrombin. • Stage 3: Conversion of Fibrinogen into Fibrin • Stage 2 & Stage 3 is collectively called as Final Common Pathway
- 27. Extrinsic Pathway • In this pathway, the formation of prothrombinase complex is initiated by the tissue thromboplastin Mechanism: •It begins with trauma to blood vessel or tissues outside the blood vessel. It releases F-VII and tissue phosholipids.F-VII comes in contact with F- III (TF or Thromboplastin) expressed on TF-bearing cells (stromal fibroblasts & leukocytes) forming an activated complex (TF-VIIa) •TF-VIIa activates F-X in presence of Ca++ and tissue phospholipids •F-Xa acts on F-V and activates it •F-Xa complexes with tissue phospholipids, F-Va, Ca++ and forms a complex called prothrombinase complex or prothrombin
- 28. INTRINSIC PATHWAY ; • In this pathway, the formation of prothrombinase complex is initiated by platelets which are within the blood itself .(starts with S.E.C.) • MECHANISM: Begins with the formation of the primary complex on collagen by HMWK, prekallikrein and F-XII • Prekallikrein is converted to kallikrein and F-XII gets activated. • Damaged platelets adhere to the wet surface of blood vessel and release platelet phospholipids. • F-XIIa acts enzymatically on F-XI (Plasma Thromboplastin Antecedent) and activates it • F-XIa acts enzymatically on F-IX and activates it in presence of Ca++ • F-IXa activates F-VIII (Anti Haemophilic Factor) • F-VIIIa and F-IXa activate F-X • F-Xa acts enzymatically on F-V (Proaccelerin) and activates it in presence of Ca++ • F-Va,F-Xa, Phospholipid and Ca++ form a complex called prothrombin complex
- 29. • • In the presence of prothrombin activator or prothrombinase complex and calcium prothrombin is converted to thrombin • •Thrombin then activates other components of the coagulation cascade, including F-V and F- VIII (which activates F-XI,which in turn activates F- IX)and activates and releases F-VIII from being bound to vWF • •F-VIIIa is the co-factor of F-IXa,and together they form the "tenase" complex, which activates F-X and so the cycle continues.("Tenase" is a contraction of "ten" and the suffix "-ase" used for enzymes)
- 30. • • Thrombin converts fibrinogen (plasma protein produced by the liver) to fibrin • • Thrombin also activates F-XIII (Fibrin Stabilizing Factor) which in presence of Ca++ stabilizes the fibrin polymer through covalent bonding of fibrin monomers
- 31. • Once activated, the coagulation cascade must be restricted to the local site of vascular injury to prevent clotting of the entire vascular tree. • Clotting is also regulated by 3 types of natural anti-coagulants: 1. Anti-thrombin 2. Protein C & protein S 3. Tissue factor pathway inhibitor (TFPI) • Clotting cascade also sets into motion a fibrinolytic cascade that limits the size of final clot. This is accomplished by the generation of plasmin from plasminogen.
- 32. Factors limiting Coagulation Once initiated, coagulation must be restricted to the site of vascular injury to prevent deleterious consequences. 1. One limiting factor is simple dilution; blood flowing past the site of injury washes out activated coagulation factors, which are rapidly removed by the liver. 2. A second is the requirement for negatively charged phospholipids, which, as mentioned, are mainly provided by platelets that have been activated by contact with subendothelial matrix at sites of vascular injury. However, the most important counterregulatory mechanisms involve factors that are expressed by intact endothelium adjacent to the site of injury. Activation of the coagulation cascade also sets into motion a fibrinolytic cascade that limits the size of the clot and contributes to its later dissolution. Fibrinolysis is largely accomplished through the enzymatic activity of plasmin, which breaks down fibrin and interferes with its polymerization. An elevated level of breakdown products of fibrinogen (often called fibrin split products), most notably fibrin- derived D-dimers, are a useful clinical markers of several thrombotic states.
- 33. • Plasmin is generated by enzymatic catabolism of the inactive circulating precursor plasminogen, either by a factor XII–dependent pathway (possibly explaining the association of factor XII deficiency and thrombosis) or by plasminogen activators. The most important plasminogen activator is t-PA; it is synthesized principally by endothelium and is most active when bound to fibrin. This characteristic makes t-PA a useful therapeutic agent, since its fibrinolytic activity is largely confined to sites of recent thrombosis. Once activated, plasmin is in turn tightly controlled by counterregulatory factors such as α2-plasmin inhibitor, a plasma protein that binds and rapidly inhibits free plasmin.
- 35. Coagulation Monitoring – Conventional Tests ( details in separate session) * Platelets – Number and Function • Clotting studies= PT(PT-INR) ,APTT ,Thrombin time, Fibrinogen levels etc.
Editor's Notes
- #3: A process involving platelets, clotting factors, and endothelium that occurs at the site of vascular injury and results in the formation of a blood clot, which serves to prevent or limit the extent of bleeding
- #5: Disruption of the endothelium exposes subendothelial ,von Willebrand factor (vWF) and collagen, which promote platelet adherence and activation. Activation of platelets results in a dramatic shape change (from small rounded discs to flat plates with spiky protrusions that markedly increased surface area), as well as the release of secretory granules. Within minutes the secreted products recruit additional platelets, which undergo aggregation to form a primary hemostatic plug. Platelets bind via glycoprotein Ib (GpIb) receptors to von Willebrand factor (VWF) on exposed ECM and are activated, undergoing a shape change and granule release. Released ADP and thromboxane A2 (TXA2) induce additional platelet aggregation through platelet GpIIb-IIIa receptor binding to fibrinogen, and form the primary hemostatic plug.
- #6: Vascular injury exposes tissue factor at the site of injury. Tissue factor is a membrane-bound pro-coagulant glycoprotein that is normally expressed by subendothelial cells in the vessel wall, such as smooth muscle cells and fibroblasts. Tissue factor binds and activates factor VII (see later), setting in motion a cascade of reactions that culminates in thrombin generation. Thrombin cleaves circulating fibrinogen into insoluble fibrin, creating a fibrin mesh-work, and also is a potent activator of platelets, leading to additional platelet aggregation at the site of injury. This sequence, referred to as secondary hemostasis, consolidates the initial platelet plug.
- #7: Polymerized fibrin and platelet aggregates undergo contraction to form a solid, permanent plug that prevents further hemorrhage. At this stage, counter-regulatory mechanisms (e.g., tissue plasminogen activator, t-PA made by endothelial cells) are set into motion that limit clotting to the site of injury and eventually lead to clot resorption and tissue repair.
- #16: α-Granules have the adhesion molecule P-selectin on their membranes and contain proteins involved in coagulation, such as fibrinogen, coagulation factor V, and vWF, as well as protein factors that may be involved in wound healing, such as fibronectin, platelet factor 4 (a heparin-binding chemokine), platelet-derived growth factor (PDGF), and transforming growth factor-β. Dense (or δ) granules contain adenosine diphosphate (ADP) and adenosine triphosphate, ionized calcium, serotonin, and epinephrine.
- #17: Deficiency of vWF -> Von Will Brand Disease Deficiency of Gp1b -> Bernard Soulier Syndrome
- #19: Secretion (release reaction) of granule contents occurs along with changes in shape; these two events are often referred to together as platelet activation. Platelet activation is triggered by a number of factors, including the coagulation factor thrombin and ADP. Thrombin activates platelets through a special type of G-protein– coupled receptor referred to as a protease-activated receptor (PAR), which is switched on by a proteolytic cleavage carried out by thrombin. ADP is a component of dense-body granules; thus, platelet activation and ADP release begets additional rounds of platelet activation, a phenomenon referred to as recruitment. Activated platelets also produce the prostaglandin thromboxane A2 (TXA2), a potent inducer of platelet aggregation. Aspirin inhibits platelet aggregation and produces a mild bleeding defect by inhibiting cyclooxygenase, a platelet enzyme that is required for TXA2 synthesis. Although the phenomenon is less well characterized, it is also suspected that growth factors released from platelets contribute to the repair of the vessel wall following injury.
- #21: Platelet aggregation follows their activation. The conformational change in glycoprotein IIb/IIIa that occurs with platelet activation allows binding of fibrinogen, a large bivalent plasma polypeptide that forms bridges between adjacent platelets, leading to their aggregation. Predictably, inherited deficiency of GpIIb-IIIa results in a bleeding disorder called Glanzmann thrombasthenia. The initial wave of aggregation is reversible, but concurrent activation of thrombin stabilizes the platelet plug by causing further platelet activation and aggregation, and by promoting irreversible platelet contraction. Platelet contraction is dependent on the cytoskeleton and consolidates the aggregated platelets. In parallel, thrombin also converts fibrinogen into insoluble fibrin, cementing the platelets in place and creating the definitive secondary hemostatic plug. Entrapped red cells and leukocytes are also found in hemostatic plugs, in part due to adherence of leukocytes to P-selectin expressed on activated platelets.
- #36: prothrombin time -Tests the extrinsic and common coagulation pathway(TF,PT,WR)- Normal range of PT: around 10-14 seconds activated partial thromboplastin time (Aptt)- Kaolin cephalin clotting time (KccT), Tests the "intrinsic" and the common coagulation pathways- Normal range of APTT: Around 30-40 seconds thrombin clotting time (TCT)-measures the time taken for a clot to form in the plasma of a blood sample containing anticoagulant, after an excess of thrombin has been added-Normal range: 14 to 16 seconds Fibrinogen- adult: 200-400 mg/dl, newborn: 125-300mg/dl