Blood Clotting Lecture 33-34 PDF

STRUCTURE AND FUNCTION Blood Clotting LECTURE 33-34 REFERENCES Boron & Boulpaep (2017) Kierszenbaum (2020) 3rd edition 5th edition C...

STRUCTURE AND FUNCTION Blood Clotting LECTURE 33-34 REFERENCES Boron & Boulpaep (2017) Kierszenbaum (2020) 3rd edition 5th edition Ch. 18 Ch. 6  HSB L.612  x8406 [email protected] LEARNING OBJECTIVES This portion of the course presents the student with the pathways for blood clotting. A Define blood hemostasis and provide an overview of the major events that occur during this process. B Describe the role of platelets in the pathway and describe; a. Platelet adhesion and von Willebrand factor b. Platelet activation c. Platelet aggregation C Describe the molecular pathway and the roles of the clotting factors. D Describe the following pathways a. Extrinsic pathway b. Intrinsic pathway c. Common pathway E Explain the importance of vitamin K-dependent g carboxylation of certain glutamate residues of factors VII, IX, X, II and proteins C and S F Describe the prevention of clotting and the action mechanisms of the following anticoagulants: a. Tissue factor pathway inhibitor (TFPI) b. Heparin c. Antithrombin d. Thrombomodulin e. Proteins C and S G Define fibrinolysis and define the roles of t-PA, urokinase, plasminogen, plasminogen activator inhibitor, and protein S H Describe the action of antiplatelet, clot busters and fibrinolytic therapies. I Describe disseminated intravascular coagulation. Hemorrhage is a discharge of blood (bleeding) out of the intravascular space which could occur internally (into the surrounding tissue/space) or externally (out of the body). Hemostasis refers to the termination of bleeding from a break in blood vessel. It consists of initial vasoconstriction followed by coagulation of blood. Coagulation (clotting) is a multi‐step process that involved both cellular and molecular mechanisms resulting in the formation of blood clot. Thrombosis is triggered when the endothelial lining of blood vessels is injured leading to coagulation at the site of injury. Thrombolysis describes the breakdown (dissolution) of clot while Fibrinolysis refers more specifically to the process of breaking down fibrin, hence fibrinolysis is a part of thrombolysis process. Three main players in coagulation include blood vessels, platelets, and liver. Each of them synthesizes, secretes, and expresses several components of the coagulation pathways. Blood vessels also facilitate hemostasis by the constriction of smooth muscle cells in its tunica media. In the first step of hemostasis, the damaged vessel produces endothelin, a potent vasoconstrictor, leading to vasoconstriction and a decrease in blood flow to the site of injury. This is followed by a molecular and a cellular responses that result in the formation of the hemostatic plug. Coagulation is a multi‐step process that result in the formation of blood clot. It can be divided into two phases. 1. Platelet plug formation is the initial response of platelets to a site of injured blood vessel. This cellular response is initiated by exposure of collagen and other factors, especially von Willebrand factor (vWF), which cause adhesion of platelets to the site of injury. Platelets then release several factors that activate and attract more platelets to the site of injury. The activation causes platelets to change shape and aggregate forming a platelet plug, blocking the breakage in the vascular wall. 2. Clot formation follows platelet plug formation. In this molecular response, soluble fibrinogen is converted into fibrin through activation of coagulation enzymatic cascades. Fibrin then organizes into a fibrin mesh that stabilizes and strengthens the platelet aggregate forming a hemostatic plug (in hemorrhage; in thrombosis it is called thrombus). The repair of blood vessel wall and surrounding tissue follows. The coagulatory response is kept localized by anticoagulants released from intact endothelium in other areas. Once the injury is repaired, thrombolysis occurs which involves enzymatic breakdown of fibrin (fibrinolysis) leading to removal of the clot. Platelet plug is formed by a multi‐step process that includes Adhesion, Activation, and Aggregation of platelets. Platelets are small anucleated cellular fragments produced by megakaryocytes. There are normally 200,000‐400,000 platelets per a μL of blood with a turnover rate of roughly 10 days. Platelet surface is covered by several glycoproteins that are involved in coagulation, particularly glycoprotein Ia/IIa (GP Ia/IIa, also integrin α2β1), glycoprotein Ib (GP Ib) and glycoprotein IIb/IIIa (GP IIb/IIIa, also integrin α2b/β3). Platelet also contains different types of granules. Specifically, α granules store various proteins involved in homeostasis, δ granules store vasoactive mediators, and λ granules store hydrolytic enzymes involved in thrombolysis. von Willebrand factor (vWF) is a protein containing 2,050 amino acid residues. The vWF gene is located on the short arm of chromosome 12. vWF is produced exclusively by megakaryocytes and endothelial cells and stored inside the storage vesicles in the respective cells: Weibel‐Palade bodies (endothelial cells), α granules (platelets). There are two main functions of vWF. It mediates binding of platelet receptor GP Ib‐IX‐V to collagen. It also acts as a binding protein for factor VIII which helps prevent factor VIII degradation in the circulation. Platelets normally do not adhere to themselves, other blood cells, or the endothelial cells. When a break in blood vessel occurs, platelets adhere to collagen at the site of injury via GP Ib‐IX‐V (mediated by vWF), GP VI, and GP Ia/IIa. Platelet binding to collagen (also thrombin or ADP) results in a dramatic change in shape of the platelets and exocytosis the contents of their storage (α and δ) granules (called release reaction or platelet activation). The activation of platelet also amplify the platelet responses by the release of several mediators that can further activate more platelets (positive feedback), such as Ca2+, ADP, thromboxane A2 (TXA2), etc., resulting in an amplification of the coagulatory response. Platelet activation (by binding of collagen, thrombin, ADP, etc.) induced activation of phospholipase A2 (PLA2) in the cytoplasm by increasing cytoplasmic calcium levels through a IP3‐mediated pathway.. PLA2 releases arachidonic acid (AA) from the platelet membrane phospholipids, allowing AA to be converted by cyclooxygenases (COXs) into TXA2. TXA2 binds to its specific receptor (TP) on platelets and further increases platelet activation and aggregation TXA2 also has a vasoconstrictor effect. Non‐steroidal anti‐inflammatory drugs (NSAIDs), such as aspirin, irreversibly inhibits COXs and prevents coagulation (anti‐platelet drugs). Platelet activation causes a flip‐flop inversion of negatively charged phospholipids (phosphatidylserine and phosphatidylinositol) to the outside leaflet of the plasma membrane (both in platelets and subendothelium), providing a more negative surface to aid in localization of the molecular pathway. Lastly platelet activation results in a conformational change (activation) of GP IIb/IIIa on the surface which changes the GP affinity to fibrinogen and vWF. This conformational change is crucial to the aggregation of platelets in the next step. Fibrinogen and vWF bind to activated GP IIb/IIIa on adjacent activated platelets linking them to each other. Each platelet express several copies of GP IIB/IIIa so they can be linked to multiple other platelets, forming a platelet aggregate (platelet plug). Clot formation strengthens the platelet plug formed by aggregated platelets into a hemostatic plug. Clot formation relies on the molecular response involving cascades of enzymatic activations a.k.a. the coagulation pathways. The coagulation pathways consist of three pathways: intrinsic, extrinsic, and common pathways. The extrinsic pathway is activated by exposure to the subendothelium while the intrinsic pathway is activated by exposure to a negatively charged surface. Both extrinsic and intrinsic pathways converge on the common pathway which eventually leads to conversion of fibrinogen to fibrin. Several proteins are involved in the control of blood coagulation. Most of them have more than one name. Twelve of the factors are assigned a Roman numeral for their names: factors I‐V and factors VII‐XIII (there is no factor VI). Most of them also have two forms, activated form is usually designated by the letter ‘a’ after its respec ve name e.g. factor V → factor Va. Factors I‐IV are commonly referred to by their common names (see table). Proteins involved in coagulation can be generally categorized into three groups: zymogens, cofactors, and other proteins. Zymogens are precursors that when activated, usually through enzymatic cleavage, form active enzymes. Cofactors do not have a catalytic function but are required for their corresponding enzymes to function properly. Other proteins related to coagulation serve various functions including structural (i.e. fibrinogen, vWF) and regulatory (e.g. TFPI, AT III, etc.) proteins. Vitamin K is required for the syntheses of six of the proteins (II, VII, IX, X, protein C, and protein S). The extrinsic pathway involves tissue factor (TF, factor III), factor VII, factor X, and Ca2+. It is stimulated by exposure of TF in the subendothelium (and on activated monocytes) upon vascular damage. TF activates factor VII and also serves as a cofactor for factor VIIa. TF along with factor VIIa and Ca2+ form a complex, TF‐VIIa‐Ca2+ complex (also called extrinsic tenase). Extrinsic tenase can then cleave factor X into factor Xa which starts the common pathway. The intrinsic pathway involves factors VIII‐XII, prekallikrein (PK), high‐molecular‐weight kininogen (HMWK), Ca2+, and negatively charged phospholipid (phosphatidylserine) on a cell membrane. The intrinsic pathway is triggered by contact with a negatively charged surface (activated platelet membrane, glass, other molecules resulting from cell damage such as extracellular DNA, RNA, etc.). Upon contact with a negatively charged surface, factor XII is converted to factor XIIa with the help of HMWK which also help binding of the factor to the charged surface. Factor XIIa activates factor XI to factor XIa by proteolysis (HMWK also helps binding of factor XIa to the charged surface). Factor XIa cleaves and activate factor IX. Factor IXa together with factor VIIIa (activated by thrombin) and Ca2+ form a trimolecular complex called tenase on the charged surface that can activate factor X. Factor Xa from either extrinsic or intrinsic pathways enters the common pathway by forming a complex, called prothrombinase, with Ca2+ and factor Va (which is also activated by thrombin). Prothrombinase converts prothrombin to thrombin. Thrombin (α thrombin) is the major serine protease that has several roles in the coagulation cascade. 1. It cleaves fibrinogen to produce fibrin. 2. It activates factor XIII so that factor XIIIa can help cross‐linking of fibrin to form a stable fibrin mesh. Note that factor XIIIa is the only transglutaminase (non protease) in the cascade. 3. It creates positive feedback loops by catalyzing the production of itself, factor VIIIa, and factor Va. 4. It binds to protease‐activated receptor‐1 (PAR‐1) on the platelets, activating them (enhancing both cellular and molecular responses). 5. It stimulates a release of several mediators from endothelial cells including prostaglandin I2 (PGI2/prostacyclin), nitric oxide (NO), vWF, ADP, and tissue plasminogen activator (t‐PA). The stable fibrin mesh helps strengthen the hemostatic plug formed by platelet aggregation. The clot will undergo further reduction in size by mechanisms that involve platelets, red blood cells, and several coagulation factors, which further close the break at the site of injury. Apart from what has been described, there are other cross‐talks between two pathways such as activation of factor VII by factors IXa and Xa, extrinsic tenase activation of factors IXa and XIa. The molecular response of the coagulation is dependent on the ability to form multi‐molecular complexes involved in the coagulation cascades. That ability is dependent on a specific domain in the N‐terminal called γ‐carboxyglutamic acid (Gla) domain. This domain (negatively charged) is necessary in allowing the protein to interact with Ca2+ and anchor it to the negatively charged membrane, facilitating the assembly of the enzyme complexes. The proteins that are vitamin K‐dependent includes factors VII, IX, X, prothrombin, proteins C and S. Note that activation of prothrombin into thrombin cleaves the Gla‐containing N‐terminal, releasing thrombin from the membrane so it is free to move around and exert its role in coagulation. The Gla residue is synthesized by a post‐translational modification of glutamic acid by a vitamin K‐ dependent process. Vitamin K is a group of several substances which work as a cofactor for γ‐glutamyl carboxylase enzyme in the endoplasmic reticulum of hepatocytes. This carboxylase reaction is crucial in production of coagulation factors VII, IX, X, prothrombin, proteins C and S. In addition to oxygen and carbondioxide, a reduced form of vitamin K (vitamin K hydroquinone, KH2) is required for each conversion of glutamic acid (Glu) to γ‐carboxyglutamic acid (Gla). The reaction adds a carboxyl group to the Glu and also oxidized vitamin K to its oxidized form, vitamin K epoxide (KO). In order for the process to keep going, recycling of KO back to KH2 is needed and is catalyzed by another enzyme, vitamin K epoxide reductase. Warfarin is a anticoagulant that can reversibly inhibit epoxide reductase. Warfarin is in a group of drug called vitamin K antagonists (although they just prevent vitamin K recycling and do not directly interfere with vitamin K function).  F REGULATION OF COAGULATION introduction Low levels of activated coagulation factors in plasma, rapid blood flow Endothelial cells produce and release several factors that interfere with all aspects of coagulation. Antiplatelet Prostacyclin (PGI2): dilates vessels, inhibits platelet activation Nitric oxide (NO): inhibits platelet adhesion and aggregation Anticoagulant Tissue factor pathway inhibitor (TFPI) Heparin Antithrombin Thrombomodulin F REGULATION OF COAGULATION tissue factor pathway inhibitor Tissue factor pathway inhibitor (TFPI) Has several isoforms Soluble in plasma but some are anchored to cell membrane Binds to TF-VIIa-Ca2+ complex (extrinsic tenase) and inhibits VIIa from converting prothrombin to thrombin Thrombomodulin (a transmembrane protein on endothelial cell membrane) binds to thrombin and modifies its action. The modified thrombin specificity switches from procoagulant molecules (V, VIII, XIII, prothrombin, fibrinogen) to protein C, an anticoagulant. Protein C is a vitamin K‐dependent zymogen synthesized in the liver. Protein C is activated through proteolysis by thrombin‐thrombomodulin complex (the activation is enhanced by endothelial protein C receptor) forming activated protein C (APC). APC, working with its cofactor protein S, cleaves and inactivates factors Va (in the prothrombinase complex) and VIIIa (in the intrinsic tenase complex) in a calcium‐ and membrane‐dependent manner via binding to activated platelet membrane through the Gla domain (the inactivated factors are called factors Vi and VIIIi, respectively). When the damaged tissue is repaired, clots had served its purpose and need to be removed. Fibrinolysis involves dissolution of clot by activation of a proteolytic enzyme zymogen plasminogen that is synthesized in the liver. Plasminogen can be cleaved by tissue plasminogen activator (t‐PA) or urokinase (UPA, urokinase‐type plasminogen activator) into plasmin. Plasmin is a serine protease and the major enzyme of fibrinolysis that breaks down fibrin into small fragments called fibrin degradation products resulting in the dissolution of clot. A specific fibrin degradation product that contains two crosslinked fragments of D domains called D‐dimer is used clinically as a marker for fibrin. t‐PA and UPA are also serine proteases. They are maintained in inactive state by binding to a group of serine protease inhibitor (serpin) called plasminogen activator inhibitor (PAI). PAI can be inactivated by activated protein C (APC) which is activated by modified thrombin (thrombomodulin‐thrombin complex). The modified thrombin also activates thrombin‐activatable fibrinolysis inhibitor (TAFI; activated form: TAFIa) which interferes with binding of both t‐PA and plasminogen to fibrin. Another regulatory mechanism is through α2‐antiplasmin (α2‐plasmin inhibitor, a serpin) direct inhibition of plasmin activity. α2‐antiplasmin is produced in liver and platelet (α granule). Thrombosis is involved in pathogenesis of multiple diseases. Thrombi can be categorized into two major types by their composition which also dictates how to effectively manage them. White thrombi usually occur as a result of vessel injury especially in areas with rapid flow velocity i.e. arteries. They are rich in platelet which makes them suitable for antiplatelet and fibrinolytic therapies. A common cause of this type is a ruptured atherosclerotic plaque which triggers a quick thrombus formation and occlusion of the affected artery, leading to ischemic injury of the organ e.g. myocardial ischemia, myocardial infarction, and stroke. Red thrombi are rich in fibrin (and red blood cells) and occur predominantly in the areas with slow blood flow or stasis i.e. veins. The primary therapeutic option for this type of thrombi would be anticoagulants. Thrombi formed this way usually associated with thromboembolic diseases such as in deep vein thrombosis and pulmonary embolism, and stroke. Antiplatelet therapies aim at prevent platelet actions: adhesion, activation, and aggregation. Preventing platelet adhesion: There is no commercially available drug in this category at this time. Possible mechanisms include GP Ib antagonist and GP Ia/IIa antagonists. Preventing platelet activation: Non‐steroidal anti‐inflammatory drugs (NSAIDs) such as aspirin inhibits cyclooxygenase from producing thromboxane A2. ADP receptor antagonists such as clopidogrel (Plavix®) inhibits the ADP receptor. PAR‐1 (protease activated receptor‐1) antagonists such as vorapaxar inhibits PAR‐1 receptor which is activated by thrombin. Preventing platelet aggregation: GP IIb/IIIa receptor antagonists such as abciximab blocks GP IIb/IIIa function which prevents platelet aggregation. Tissue plasminogen activator (t‐PA) and urokinase are used to prevent blood clot from growing larger (fibrinolytic therapies or clot busters). Their indications are limited to certain diseases (acute myocardial infarction and stroke), each of which only applicable in a short critical timeframe (12 hrs for acute myocardial infarction , 3 hrs for stroke). Warfarin (and coumadin) inhibits vitamin K recycling by inhibiting vitamin K epoxide reductase, preventing the γ‐carboxylation of glutamate residues on multiple coagulation proteins (prothrombin, factors VII, IX, and X). Heparin working together with antithrombin to inactivates thrombin. Other anticoagulants also target thrombin for their actions. Since coagulation requires several components, impaired coagulation due to one or more abnormal coagulation components can lead to bleeding disorders (hemorrhagic disorders, coagulopathies). A deficiency in factor VIII causes hemophilia A which is an inherited, X‐linked recessive disorder. Similarly, a deficiency in factor IX results in hemophilia B which is also an X‐linked recessive disorder. Vitamin K deficiency also can cause coagulopathy by decreasing the amount of vitamin K‐dependent proteins. A deficiency in von Willebrand factor (vWF) leads to von Willebrand disease which apart from lacking vWF, also have a decreased in factor VIII as well. Disseminated intravascular coagulation (DIC) is a syndrome characterized by widespread intravascular coagulation. Common causes of DIC are infection, cancer, trauma and major surgery, some pregnancy‐ related conditions Thrombi in organs → ischemia/infarc on → organ failure Deple on of coagula on factors → bleeding disorder Signs and symptoms vary depending on the organs affected Mortality rate 31‐86% Treatment: Treat the underlying causes Anticoagulant therapy: mainly heparin – still controversial Other supportive treatments