General Overview of Hemostatic Process (Blood Coagulation)

Lecture 6 and 7 General overview of hemostatic process (Blood coagulation) Understand the process and stages (cascade) of blood coagulation and its significance. List and understand the role of factors involved in blood coagulation. Understand the role of serine proteases in the cascade of blood coagulation. Understand the intrinsic and extrinsic Pathways of blood clot Understand the cause of excessive bleeding Understand bleeding time, clotting time and prothrombin time 63 Hemostasis • It is the process of forming clots (coagulation) in the walls of damaged blood vessels and preventing blood loss while maintaining blood in a fluid state within the vascular system • Damage exposes collagen fibers to blood, producing: a.Vasoconstriction reduces blood flow, result from (1) local myogenic spasm, (2)local autacoid factors (3) nervous reflexes b.Formation of platelet plug (primary hemostasis): small cut sealed by a platelet plug rather than by a blood clot c.Formation of a blood clot (Secondary hemostasis ): damaged endothelial cover of blood vessels d.Formation of fibrin protein web to close the hole in the vessel permanently 64 Intact endothelium protective mechanisms: • Separate the blood from collagen • Secretes prostacyclin (PGI2) and nitric oxide, which: 1)Vasodilate 2)Inhibit platelet aggregation • Secrets CD39 Enzyme, which breaks down ADP into AMP and Pi to inhibit platelet aggregation further • Damaged endothelium exposes collagen . 65 Damaged endothelium exposes collagen 1)Platelets bind to collagen. 2)von Willebrand’s factor (VWF) binds to both collagen and platelets helps anchor the platelets 3)Platelets recruit more platelets and form a platelet plug by secreting: a) ADP (sticky platelets) b) Serotonin (vasoconstriction) c) Thromboxane A (sticky platelets and vasoconstriction) 4) Activated platelets also activate plasma clotting factors 66 1. Formation of platelet plug A. Platelets adhesion • Triggered by exposure to Collagen • The glycolprotein (GP) receptors expressed on platelets surface: GP Ib-V-IX complex form a bridging interaction with the exposed collagen that is mediated by vWF The vWF recruits platelets to exposed collagen to prevent platelets from interacting with circulating vWF in undamaged areas GP Ia/IIa (also known as integrin 2 1): Bind to collagen GP VI: Bind to collagen 67 B. Platelet Activation Triggered by Collagen mediated platelet receptor binding, especially interaction with GP VI Thrombin (produced from the initial response of the extrinsic pathway) bind to a class of G-protein-couple Receptor called Protease activating receptor (PAR1,PAR3, PAR4) • The thrombin induced signaling leads to increase platelet activation and leukocytes adhesion • Platelets are activated by second messenger signaling leads to: Activation of GP IIb/IIIa Granule secretion Shape change - They lose regular, discoid shape in response to increase intracellular Ca - They become highly irregular with multiple filopodial extensions - Their surface area increase - They become sticky to neighboring platelets 68 C. Platelet Aggregation • It is mediated by a specific platelet receptor that binds to fibrinogen or fibrin. • Only activated platelets have functional glycoproteins (GPs) IIb/IIIa prevent unwanted platelet aggregation). 3 integrins) that bind to fibrin or fibrinogen (to • To promote platelet-to-platelet aggregation, fibrin and fibrinogen have multiple binding sites for the receptor one fibrin or fibrinogen molecule can bind to a GP IIb/IIIa on one platelet and at the same time bind to another GP IIb/IIIa on a second platelet. • A single platelet contains many copies (about 50,000) of the GP IIb/IIIa receptor each platelet can interact with many other platelets, thereby creating aggregated platelets The final common pathway of platelet aggregation. 69 2. Initiation of coagulation (red clot formation) Coagulation process is initiated by: 1. 2. 3. Trauma to the vascular wall and adjacent tissues Blood trauma Blood contact with damaged endothelial cells or with collagen and other tissue elements outside the blood vessel Red blood cells Fibrin Plasma Clotting Factors • The clot is formed by the activation of the blood-clotting factors proteins • They are proteolytic enzymes in inactive forms when they are activated cause cascading reactions of the clotting process. • Most of the clotting factors are designated by Roman numerals. To indicate the activated form of the factor, a small letter “a” is added after the Roman numeral, such as Factor VIIIa to indicate the activated state of Factor VIII • Serine proteases factors: (#6) (Thrombin, FVIIa, IXa, Xa, XIa and XIIa) • Cofactors: (#4) (Tissue Factor, Ca ion, F-Va and VIIIa) 70 Clotting factors: Formation of Fibrin Fibrinogen is converted to fibrin via one of two pathways: 2 1 Extrinsic pathway 1. Extrinsic pathway: Damaged tissue release a chemical that initiates the formation of fibrin Initiated by tissue factor thromboplastin (factor III) secreted by the subendothelial cells and leukocyte after external damage. This is a more direct pathway (shortcut). Common pathway Activator: tissue factor X VII Intrinsic pathway 3 Activators: collagen, glass, and others X activated V complex (V, X activated, calcium, phospholipids) VII activated Prothrombin XII XII activated XI activated XI Thrombin IX VII complex (VII, tissue factor, calcium, phospholipids) IX activated VIII complex (VIII, IX activated, calcium, phospholipids) Fibrinogen Fibrin XIII 71 Fibrin polymer Continue…. 1. Begins with a traumatized vascular wall or traumatized extravascular tissues that come in contact with the blood 2. Tissue factor initiates the extrinsic pathway result in 3. Release of tissue factor (thromboplastin): 4. Activation of Factor X by Factor VII and tissue factor: Factor VII in the presence of Ca ions, acts enzymatically on Factor X to form activated Factor X (Xa). 5. Factor V interact with Xa to form prothrombin activator complex in the presence of Ca and tissue phospholipids. prothrombin is split to form thrombin. required for reproduction or display. 2 1 Extrinsic pathway X VII activated Intrinsic pathway Common pathway Activator: tissue factor VII 3 Activators: collagen, glass, and others X activated XII XII activated V complex (V, X activated, calcium, XI activated XI phospholipids) Prothrombin Thrombin VII complex (VII, tissue factor, calcium, phospholipids) Fibrinogen Fibrin IX IX activated VIII complex (VIII, IX activated, calcium, phospholipids) Fibrin polymer XIII 72 2. Intrinsic pathway: - 2 1 Blood left in test tube will clot without the addition of any chemicals Activated by exposure to collagen Factor XII activates a cascade of other blood factors Extrinsic pathway Common pathway Activator: tissue factor X VII Intrinsic pathway 3 Activators: collagen, glass, and others X activated V complex (V, X activated, calcium, phospholipids) VII activated Prothrombin XII XII activated XI activated XI Thrombin IX VII complex (VII, tissue factor, calcium, phospholipids) IX activated VIII complex (VIII, IX activated, calcium, phospholipids) Fibrinogen Fibrin Fibrin polymer XIII 73 Continue…. 1. Begins with trauma to the blood or exposure of the blood to collagen from a traumatized blood vessel wall 2. The contact of Factor XII and platelets with collagen in the vascular wall initiates the intrinsic pathway cause 3. The activation of Factor XII Factor XIIa 4. The release of Platelet phospholipids that contain the lipoprotein called platelet factor 3 (plays a role in subsequent clotting reactions) 5. Activation of Factor XI: The activated Factor XII acts enzymatically to activate Factor XI. This reaction also requires high-molecular-weight kininogen and is accelerated by prekallikrein. 6. Activation of Factor IX by activated Factor XI. The activated Factor XI then acts to activate Factor IX 7. Activation of Factor X. The activated Factor IX with activated Factor VIII and with the platelet phospholipids and Factor III from the traumatized platelets, activates Factor X. 8. Factor X action to form prothrombin activator. Activated Factor X combines with Factor V and platelet or tissue phospholipids to form prothrombin activator complex which in turn initiates the cleavage of prothrombin to form thrombin 9. The prothrombin activator, in the presence of Ca causes the conversion of prothrombin to thrombin. 10. The thrombin converts fibrinogen (soluble) into fibrin fibers (insoluble) that trap platelets, blood cells, and plasma to form the clot. Permission required for reproduction or display. 1 Extrinsic pathway3 2 Intrinsic pathway Common pathway Activator: tissue factor Activators: collagen, glass, and others X VII VII activated X activated XII XII activated V complex (V, X activated, calcium, XI activated XI phospholipids) Prothrombin Thrombin IX IX activated VII complex (VII, tissue factor, calcium, phospholipids) FibrinogenFibrin VIII complex (VIII, IX activated, calcium, phospholipids) Fibrin polymer XIII prothrombin activator prothrombin ——————> thrombin complex Inactive form Active form 74 3. Common pathway: - 2 1 • Both intrinsic and extrinsic pathways lead to activate Factor X activated factor Xa Prothrombin Thrombin Fibrinogen Fibrin • This is mediated by Ca and phospholipids (from the platelets) Extrinsic pathway Common pathway Activator: tissue factor X VII Intrinsic pathway 3 Activators: collagen, glass, and others X activated V complex (V, X activated, calcium, phospholipids) VII activated Prothrombin XII activated XII XI activated XI Thrombin IX VII complex (VII, tissue factor, calcium, phospholipids) IX activated VIII complex (VIII, IX activated, calcium, phospholipids) Fibrinogen Fibrin Fibrin polymer XIII 75 4. Formation of Fibrin polymer 1. The formation of fibrin involves the release of two pairs of polypeptides from each fibrinogen molecule. 2. The remaining portion, fibrin monomer, then polymerizes with other monomer molecules to form fibrin. 3. The fibrin (loose mesh of interlacing strands) is converted by the formation of covalent cross-linkages to a dense, tight aggregate (stabilization) This reaction is catalyzed by 4. Activated factor XIII (fibrin-stabilizing factor) which causes more and more cross-linking bonds between adjacent fibrin fibers 5. This step requires Ca • Deficiency of XIII affects clot stability and increases bleeding tendency. • The rate-limiting factor in causing blood coagulation is the formation of prothrombin activator that convert prothrombin to thrombin • Plasmin digests fibrin Dissolution of clots • When clots are formed within blood vessels, activation of plasminogen to plasmin may lead to their removal. • Vitamin K is needed by the liver to make several of the needed clotting factors 76 Platelets role in the coagulation process • The platelets contribute directly to clot contraction by activating platelet 1. Thrombosthenin (A contractile protein from thrombocytes) 2. Actin, and myosin (contractile proteins in the platelets) cause strong contraction of the platelet spicules attached to the fibrin compress the fibrin meshwork into a smaller mass. 3. The contraction is activated and accelerated by thrombin and Ca 4. As the clot retracts, the edges of the broken blood vessel are pulled together, thus contributing further to hemostasis 77 The role of other proteins in the coagulation process • Prekallikrein (PK): protein that complexes with kininogen. PK is the precursor of plasma kallikrein, which is a serine protease that activates kinins. PK is cleaved to produce kallikrein by activated Factor XII • Kininogens: is a circulating high molecular weight plasma protein which participates in the initiation of blood coagulation, and in the generation of the vasodilator bradykinin via the kallikreinkinin system. • Bradykinin is the end-product of the contact activation system. This enzymatic cascade circulates in the plasma and consists of factor XII, plasma prekallikrein and high molecular weight kininogen cause vasodialation This system is linked to the intrinsic coagulation system via factor XI 78 Vitamin K role in the coagulation process • It is involved in the formation of gammacarboxy-glutamate residues which occurs during synthesis of clotting factors in the liver. • The Carboxy-glutamic acid (carboxy group is added to glutamic acid) present in the clotting factors introduces an affinity for Ca ions required for binding to platelet membrane phosphatidylserine (PS) • In the blood coagulation cascade, Vitamin K is required to introduce gamma-carboxylation of clotting factors II, VII, IX, X. • Non-carboxylated factors are “inactive” because they cannot bind Ca or to PS, therefore, cannot participate in the formation of the essential coagulation complexes that are required for fibrin formation. 79 Role of Calcium Ions in the Intrinsic and Extrinsic Pathways • Ca ions are required for promotion or acceleration of all the blood-clotting reactions. Therefore, in the absence of Ca ions, blood clotting by either pathway is inhibited • Clotting can be prevented with certain drugs: 1) Calcium chelators (sodium citrate, EDTA) 2) Heparin: blocks thrombin (The heparin molecule is negatively charged it has little or no anticoagulant properties, but when it combines with antithrombin III, the effectiveness of antithrombin III for removing thrombin increases by a hundredfold to a thousand fold, and thus it acts as an anticoagulant) 3) Coumarin: inhibits vitamin K 80 Summary of the coagulation process tenase:activation factor X • The activating complexes are together called tenase (the complex activates its substrate (inactive factor X) by cleaving it) • Extrinsic tenase complex is made up of tissue factor, factor VII, and Ca2+ as an activating ion. • Intrinsic tenase complex contains the active factor IX (IXa), its cofactor factor VIII (VIIIa), the substrate (factor X) 1. 2. 3. Initial Phase: formation of (TF-FVIIa): extrinsic tenase complex TF: tissue factor Amplification phase: activating platelets as well as FV, FVIII, and FXI F: factor Propagation phase: formation of (IXa-VIIa): intrinsic tenase complex and (Xa-Va): prothrombinase In the propagation phase, a burst of thrombin is generated, which is sufficient for the clotting of fibrinogen and formation of a fibrin meshwork. A thrombus is formed. Complex Proteinase Cofactor Substrate extrinsic tenase VIIa,TF,Ca, Phospholipid VIIa Tissue factor X intrinsic tenase IXa,VIIIA,Ca, Phospholipid IXa VIIIa X Va Prothrombin prothrombinase Xa,Va,Ca, Phospholipid Xa 81 Factors interfere with the clotting mechanism A. Anti-clotting mechanism • The Endothelial Surface Factors that prevent clotting in the normal vascular system are: (1) The smoothness of the endothelial cell surface, which prevents contact activation of the intrinsic clotting system. (2) The Glycocalyx layer (a mucopolysaccharide adsorbed to the surfaces of the endothelial cells) on the endothelium repels clotting factors and platelets, thereby preventing activation of clotting (3) Endothelium secret Nitric oxide (NO) which causes vasodilation and inhibits platelet aggregation (4) Endothelium secret prostaglandin which causes vasodilation and inhibits platelet aggregation (5) Anti-thrombin III (antithrombin-heparin cofactor) The intact endothelium adjacent to the damaged tissue actively secretes several anti-thrombotic substances including heparan sulfate, and thrombomodulin The thrombin that does not adsorb to the fibrin fibers combines with antithrombin III, which further blocks the effect of the thrombin on the fibrinogen inactivates the thrombin itself during the next 12 to 20 minutes Antithrombin III inactivates Factors Xa, IXa, XIa, XIIa, thrombin (IIa),VIIa, kallikrein and plasmin (6)Thrombomodulin: Most of the endothelial cells produce thrombomodulin (a thrombin-binding protein) on their surfaces. When thrombin binds to thrombomodulin it becomes an anticoagulant forming thrombomodulin-thrombin complex, which activates protein C (APC). APC inactivates factors V and VIII and increase the formation of plasmin. 82 B. Tissue factor pathway inhibitor (TFPI) • TFPI inhibits the extrinsic coagulation pathway it inhibits Factor Xa and Factor VIIa-tissue factor complex C. Fibrinolytic system • Plasminogen which is formed from in the liver when activated, becomes plasmin • Plasmin is a proteolytic enzyme that resembles trypsin (digestive enzyme of pancreatic secretion). • Plasmin degrades the fibrin mesh into soluble end products known as fibrin split products • plasmin cause lysis of a clot by destroying many of the clotting factors thereby causing hypo-coagulability of the blood. 83 Bleeding disorders • A bleeding disorder is a condition that affects the normal blood clotting process • In people with bleeding disorders: the clotting factors or platelets don’t work the way they should or are in short supply. • When the blood doesn’t clot, excessive bleeding can occur in the muscles, joints, or other parts of the body. • The majority of bleeding disorders are inherited Bleeding may caused by: 1. A low RBC count 2. Vit K deficiency 3. As a side effects from certain medications 4. Common bleeding disorders a) Hemophilia A and B conditions that occur when there are low levels of clotting factors in blood It causes heavy or unusual bleeding into the joints. (rare) b) Factor II, V, VII, X, or XII deficiencies c) Von Willebrand’s disease: the most common inherited bleeding disorder. It develops when the blood lacks von Willebrand factor, which helps the blood to clot. 84 Bleeding time, Clotting time and Prothrombin time • Bleeding times: the time needed after a cut in the tissue to stop bleeding (normally 1-3 min) • Clotting time: the time needed for the blood to make a clot (normally 5-8 min) • Prothrombin time (PT): measures the clotting tendency of the blood, it is an assay for evaluating the extrinsic pathway and common pathway of coagulation. • PT measures the blood-thinning medication warfarin (anticoagulant) dosage, liver damage and diseases (used to screen people waiting for liver transplants), Vit K status, Coagulation factors (I, II, V, VII, X) 85 Thrombin provides positive feedback 1. The intrinsic pathway by converting factor VIII to factor VIIIa and factor XI to factor XIa 2. The common pathway by converting factor V to factor Va; 3. Platelets by binding to thrombin receptors to amplify platelet activation -hard clot production by converting factor XIII to factor XIIIa. 4. Factor VII to factor VIIa 5. Factor XI to factor XIa 86