W1 Blood Clotting and Drugs for Hemostasis 1 (Damuni).pdf

Blood Clotting Presented by Dr. Zahi Damuni Professor of Biochemistry 1 1 • Office hours: typically, online via https://teach.webex.com/meet/zahi.damuni • Email for questions or to set up a one-on-one meeting: [email protected] • Reading: Simmons, Gerhard Meisenberg, W. Principles of Medical...

Blood Clotting Presented by Dr. Zahi Damuni Professor of Biochemistry 1 1 • Office hours: typically, online via https://teach.webex.com/meet/zahi.damuni • Email for questions or to set up a one-on-one meeting: [email protected] • Reading: Simmons, Gerhard Meisenberg, W. Principles of Medical Biochemistry (3rd Edition), Chapter 15 2 2 Learning Objectives 1. Know the sequential mechanisms involved in normal hemostasis and summarize the process by which the vessel wall regulates hemostatsis and thrombosis. 2. Describe the role of platelets in blood clotting. 3. Know the pathway of blood coagulation and its regulation. 4. List the physiological and therapeutic inhibitors of blood coagulation and their mode of action. 5. Describe the tests used in the laboratory to identify coagulation disorders. 6. Know the main components of the fibrinolytic system and anti-fibrin drugs. 7. Define the role of vitamin K in blood coagulation and the factors that need the vitamin K cycle. 3 3 Hemostasis Hemostasis is the process of blood clotting. I. Occurs when small blood vessel (capillary) is damaged II. Clot seals the blood vessel until it regenerates III. Occurs in just 3-6 minutes 4 4 Process of Hemostasis Three major events occur all beginning the moment the vessel is damaged: 1. Vasoconstriction 2. Platelet plug formation 3. Coagulation of blood Coagulation takes longer, and is completed after vasoconstriction and platelet plug formation occur 5 5 Process of Hemostasis This is a simplified overview of the clotting cascade. 6 Thromboplastin is the combination of both phospholipids and tissue factor III, both of which are needed in the activation of the extrinsic pathway. In contrast, partial thromboplastin is just phospholipids, and no tissue factor III. Platelet factor 3 is the same as tissue factor. Tissue factor, also called platelet tissue factor, factor III, or CD142. Prothrombin activator is a complex of a dozen blood coagulation factors that function to catalyze prothrombin into thrombin. The prothrombin activator complex is activated by a cascade of reactions in response to damage in a blood vessel. 6 Hemostatic Disorders – Blood Clots A thrombus is a blood clot that forms in an unbroken vessel. A large thrombus may block blood flow, causing tissue death. An embolus is a blood clot that forms then breaks away and floats freely in the blood vessels. An embolus may then lodge in a capillary and block blood flow. Coronary thrombosis Cerebral embolism Pulmonary embolism 7 Myocardial infarction (coronary thrombosis or heart attack) results from the complete occlusion (blockage) of one or more coronary arteries. It arises when atherosclerotic plaques rupture, causing platelet activation, adhesion and aggregation with subsequent thrombus formation within the coronary circulation. A pulmonary embolism (PE) is a blood clot that develops in a blood vessel in the body (often in the leg). It then travels to a lung artery where it suddenly blocks blood flow. 7 Hemostatic Disorders – Blood Clots Causes of thrombus • Injury to blood vessel or build-up of fatty plaques → Both create rough surfaces inside vessel, which may activate platelets ● Poor blood circulation → Clotting factors may accumulate Immobility increases the risk of deep vein thrombus in legs! 8 Deep vein thrombosis (DVT) is a medical condition that occurs when a blood clot forms in a deep vein. These clots usually develop in the lower leg, thigh, or pelvis, but they can also occur in the arm. 8 Hemostatic Disorders – Hemophilia Causes • Lack of Factor VIII ( Hemophilia A) or Factor IX (Hemophilia B) • Recessive sex-linked trait (more common in men) Symptoms • Prolonged bleeding even from minor injuries • Excessive bruising • Bruised and swollen joints • Excessive clumsiness and falling Treatment • Intravenous injection of clotting factors • Donated plasma • Synthetic clotting factors 9 Hemophilia C is a rare genetic disorder caused by missing or defective blot clotting protein called Factor XI. The disease was first recognized in 1953 in patients who experienced severe bleeding after dental extractions and, to this day, it is still not very well-known. Hemophilia C (also known as plasma thromboplastin antecedent (PTA) deficiency or Rosenthal syndrome) is a mild form of hemophilia affecting both sexes, due to factor XI deficiency. It predominantly occurs in Ashkenazi Jews. 9 Endothelium vs. Subendothelium Endothelial cells – line the vessels. Are thromboresistant in nature. They express thrombomodulin and heparin sulfate to keep inappropriate thrombi from forming. They also release tissue plasminogen activator and urokinase to shut off the coagulation cascade in the presence of Factor IIa (thrombin). Subendothelium – beneath the endothelium. Are thrombogenic in nature. Express collagen, and tissue factor to kick off the coagulation cascade. Contain binding sites for von Willebrand Factor (vWF). Subendothelium Endothelium Source: http://facstaff.gpc.edu/~jaliff/vein1.gif 10 10 Formation of the Platelet Plug: A. Platelets adhere to exposed subendothelium. The binding is mediated by von Willebrand factor (vWF). Direct binding to tissue fibronectin or other tissue components occur as well. B. Platelets become activated after binding to the subendothelial tissue and exposure of thrombin (T), resulting in shape change and degranulation. Functional fibrinogen receptors are assembled on the cell surface. C. Continued thrombin exposure together with some of the released mediators (ADP, thromboxane, serotonin) activates more and more platelets. The platelets are glued together by fibrinogen and the platelet plug forms. The action of thrombin on fibrinogen forms insoluble fibrin, and the platelets become enmeshed in the fibrin clot. 5HT, 5-Hydroxytryptamine; PDGF, Plateletderived Growth Factor. 11 11 The Adhesive Nature of Platelets 12 12 Prostacyclin (PGI2) generated by the vascular wall is a potent vasodilator, and the most potent endogenous inhibitor of platelet aggregation so far discovered. Prostacyclin inhibits platelet aggregation by increasing cyclic AMP levels. 13 Arachidonic acid is a polyunsaturated omega-6 fatty acid. Thromboxane A2 (TXA2) is a short-lived, lipid mediator synthesized by platelets from arachidonic acid and released from the phospholipid membrane upon platelet activation. Its main role is in amplification of platelet activation and recruitment of additional platelets to the site of injury. Thromboxane B2 is an inactive metabolite/product of thromboxane A2. 6 keto PGF1 alpha is the inactive hydrolysis product of PGI2 prostacyclin. 13 Hemostasis: Coagulation & Clot Stabilization • • • • • Prothrombin Ca++ Fibrinogen Fibrin Polymerization Figure 16-13: The Coagulation Cascade 14 Factor VIII (FVIII) functions as a co-factor in the blood coagulation cascade for the proteolytic activation of factor X by factor IXa. Deficiency of FVIII causes hemophilia A, the most commonly inherited bleeding disorder. 14 Extrinsic v Intrinsic Pathways • The extrinsic pathway is activated by external trauma that causes blood to escape from the vascular system. • The intrinsic pathway is activated by trauma inside the vascular system, and is activated by platelets, exposed endothelium, chemicals, or collagen. • Both the intrinsic and extrinsic pathways are needed for efficient clotting in the body. In a test tube, fibrin can form along either pathway. But, in the body, the pathways are intertwined in such a way that if some step is missing on either the extrinsic or intrinsic side, a clot won’t be able to form properly or as efficiently. 15 15 In our bodies, the clotting cascade is kicked off by tissue factor “exposure.” Tissue factor is not floating around in the blood normally – or at least, it isn’t normally “visible” to the blood (it might be in little membrane fragments, but it’s not active until needed). When formation of a clot is needed, tissue factor appears, and together with factor VIIa (which is present in the blood), converts factor X to Xa (which then converts prothrombin to thrombin, which converts fibrinogen to fibrin). So: clotting initially begins along the extrinsic pathway. As as soon as a little Xa is produced, that Xa along with the aptly-named tissue factor pathway inhibitor turns off the extrinsic pathway! A little thrombin is formed, though, before the pathway gets turned off – and that thrombin kicks off the intrinsic pathway (the other side of the cascade, with factors VIII and IX). Fibrin formation then proceeds along this pathway until it’s no longer needed. The bottom line: both the intrinsic and extrinsic pathways are needed to form fibrin in vivo. If factors VIII or IX are deficient, very little fibrin is formed efficiently! 16 Tissue factor pathway inhibitor 1 is a single-chain polypeptide which can reversibly inhibit Factor Xa. While Xa is inhibited, the Xa-TFPI complex can subsequently also inhibit the FVIIa-tissue factor complex. 16 Von Willebrand factor and Factor VIII Factor VIII is bound to VWF while inactive in circulation; factor VIII degrades rapidly when not bound to VWF. Factor VIII is released from VWF by the action of thrombin. In the absence of VWF, factor VIII has a half-life of 1-2 hours; when carried by intact VWF, factor VIII has a half-life of 8-12 hours. VWF binds to collagen, e.g., when collagen is exposed beneath endothelial cells due to damage occurring to the blood vessel. This results in the release of factor VIII which then functions as a co-factor in the blood coagulation cascade for the proteolytic activation of factor X by factor IXa. 17 17 Factors Involved In Blood Clotting • • • • • • • • • • • • • Factor I – Fibrinogen Factor II – Prothrombin Factor III - Tissue Thromboplastin (Tissue Factor) Factor IV - Ionized Calcium ( Ca++ ) Factor V - Labile Factor or Proaccelerin Factor VI – Unassigned Factor VII - Stable Factor or Pro-convertin Factor VIII - Antihemophilic Factor Factor IX - Christmas Factor, Plasma Thromboplastin Component Factor X - Stuart-Prower Factor Factor XI - Plasma Thromboplastin Antecedent Factor XII - Hageman Factor Factor XIII - Fibrin-stabilizing Factor 18 18 Formation of Fibrin Polymer 19 19 Transglutaminase Reaction Fibrin Dimer Fibrin Polymer 20 Transglutaminase catalyzes acyl transfer reactions, deamidation, and crosslinking (polymerization) between protein intra- or interchain glutamine (acyl donor) and lysine (acyl acceptor) peptide residues. 20 Pharmacologic Considerations • PT (prothrombin time) – measures the function of the extrinsic pathway and the common pathway. • aPTT (partial thromboplastin time) – measures the function of the intrinsic pathway and the common pathway. In vitro extension by heparin. • Vitamin-K dependent coagulation components – Factors X, IX, VII, II, proteins C, S (mnemonic: 1972 [10, 9, 7, 2]). Deficiency of Vit K • Warfarin (Coumadin) – inhibits vitamin-K epoxide reductase and effective levels of vitamin-K dependent coagulation components. Will extend the PT and aPTT. • Heparin (used as drug) – purified from animals. Increases the activity of Antithrombin III. Will increase the aPTT in vitro. • Thromboxane A2 (TXA2) – synthesis of TXA2 is initiated by activated platelets. TXA2 increases platelet activation and aggregation. Its synthesis is inhibited by aspirin. 21 Thromboplastin is the combination of both phospholipids and tissue factor III, both of which are needed in the activation of the extrinsic pathway. In contrast, partial thromboplastin is just phospholipids, and no tissue factor III. 21 Reference range: 10-13 secs Reference range: 25-35 sec. 22 22 Bleeding Time • Bleeding time (BT): A normal bleeding time indicates adequate platelet hemostatic function. An incision is made under the forearm (standard size with an automatic device) and the amount of time it takes for the bleeding to stop is recorded. • Reference range: 3-9 minutes 23 23 Dissolving The Clot & Anticoagulants Figure 16-14: Coagulation and Fibrinolysis 24 24 Fibrinolysis As soon as the injury is healed, clot dissolution starts to restore the normal flow of blood 2. Plasminogen is converted to the active form Plasmin by 2 distinct Plasminogen Activators (PAs): a) Tissue plasminogen activator (t-PA) from injured endothelial cells b) Urokinase from kidney endothelial cells and plasma 1. 25 25 Breakdown of Fibrin 26 ✓ TPA and urokinase activate plasminogen ✓ Plasmin breaks down the fibrin clot into di-fibrinopeptides ✓ D-dimer (or D dimer) is a fibrin degradation product (or FDP), a small protein fragment present in the blood after a blood clot is degraded by fibrinolysis. It is so named because it contains two D fragments of the fibrin protein joined by a crosslink. 26 Plasminogen Activators 1. Tissue Plasminogen Activator (tPA) 2. Urokinase 3. Streptokinase 27 27 Physiological Inhibitors of Coagulation 1. Antithrombin III - activated by heparin and heparan 2. Activated Thrombomodulin - Activates Protein C, which in turn inhibits Factors Va and VIIIa 28 28 Anticoagulant Activity of Thrombomodulin 29 Protein S is a vitamin K-dependent plasma glycoprotein synthesized in the liver. In the circulation, Protein S exists in two forms: a free form and a complex form bound to complement protein C4b-binding protein (C4BP). Protein C is a zymogen, i.e., an inactive enzyme. The activated form plays an important role in regulating anticoagulation, inflammation, and cell death and maintaining the permeability of blood vessel walls in humans and other animals. Thrombomodulin (TM), a high affinity thrombin receptor present on endothelial cell membrane, plays an important role as a natural anticoagulant. It acts as a cofactor of thrombin-catalyzed activation of protein C and inhibits the procoagulant functions of thrombin. 29 Process of Hemostasis 30 30 Non-Physiological Inhibitors of Coagulation A. B. C. D. Vitamin K antagonists (in vivo) e.g., Warfarin EDTA (Ethylenediaminotetraacetic acid) Citrate Oxalate Calcium/divalent cation chelators 31 31 Vitamin K in Blood Coagulation 32 32 33 Warfarin inhibits both the epoxide reductase and the quinone reductase. 33 Vitamin K deficiency • Vitamin K deficiency – results in prolonged bleeding and coagulation time (PT and aPTT), even though normal blood calcium levels and normal liver function may be indicated. • The most common causes of vitamin K deficiency are insufficient dietary intake, inadequate absorption, and decreased storage of the vitamin due to liver disease, but it may also be caused by decreased production in the intestines. 34 34 Plasma D-Dimers • Cross-linked fragments containing D-Dimer epitopes • D-Dimer test is used to diagnose: Deep Vein Thrombosis Pulmonary Embolism Disseminated Intravascular Coagulation (DIC) • D-Dimer levels are elevated in these conditions 35 D-dimer (or D dimer) is a fibrin degradation product (or FDP), a small protein fragment present in the blood after a blood clot is degraded by fibrinolysis. It is so named because it contains two D fragments of the fibrin protein joined by a crosslink. Disseminated intravascular coagulation (DIC) is a rare but serious condition that causes abnormal blood clotting throughout the body's blood vessels. It is caused by another disease or condition, such as an infection or injury, that makes the body's normal blood clotting process become overactive. 35 Screening Tests Used in the Diagnosis of Coagulation Disorders Test Reference Range Abnormalities Indicated Common Cause of Disorder PT or International Normalized Ratio (INR) 11-13.5s (PT) 0.8-1.1 (INR) Extrinsic + Common Deficiency/Inhibition VII, X, V, II, I Liver disease, warfarin therapy, DIC (disseminated intravascular coagulation) APTT/PTT with Kaolin/Kaolin Cephalin Clotting Time 25-35 s Intrinsic + Common Deficiency/Inhibition XII, IX, VIII, X, V, II, I Liver disease, heparin therapy, VIII, IX, DIC Thrombin Time (TT) 14-19 s Deficiency/abnormal I, inhibition of II by heparin or FDPs DIC, Heparin therapy, fibrin clot therapy Fibrin Degradation Products (FDPs) or Fibrin Split Products (FSP) < 10 mg/mL (1.25 -10 mg/mL) Accelerated destruction of fibrinogen DIC (disseminated intravascular coagulation) Bleeding Time 3-9 min Abnormal platelet function Aspirin, uremia, von Willebrand disease 36 The international normalized ratio (INR) is a calculation based on results of a PT and is used to monitor individuals who are being treated with the blood-thinning medication (anticoagulant) warfarin (Coumadin®). The PT and INR are used to monitor the effectiveness of the anticoagulant warfarin. Kaolin clotting time (KCT) is a sensitive test to detect lupus anticoagulants. There is evidence that suggests it is the most sensitive test for detecting lupus anticoagulants. It can also detect factor VIII inhibitors but is sensitive to unfractionated heparin as well. FDP’s – fibrin degradation products. For patients on warfarin, the therapeutic range is 2.0 to 3.0. A normal thrombin time is about 14 to 19 seconds. A longer thrombin time can mean low fibrinogen, high fibrinogen, or fibrinogen that's not working normally. It can also be because of medicines that affect blood clotting, such as heparin or argatroban. 36 PT PTT Bleeding Time Platelet Count Thrombocytopenia (decreased platelet number) IgG CONDITION unaffected unaffected prolonged low Early liver failure prolonged unaffected unaffected unaffected End-stage liver failure prolonged prolonged prolonged decreased Uremia unaffected unaffected prolonged unaffected Afibrinogenemia prolonged prolonged prolonged unaffected Factor V & X deficiency prolonged prolonged unaffected unaffected Von Willebrand disease unaffected prolonged prolonged unaffected Hemophilia unaffected prolonged unaffected unaffected DIC prolonged prolonged prolonged low Vit. K deficiency/ Warfarin prolonged prolonged unaffected unaffected Aspirin unaffected unaffected prolonged unaffected TPA/Streptokinase prolonged prolonged prolonged unaffected 37 37