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Physiology of haemostasis
BLOOD
Physiology of haemostasis
Learning objectives After reading this article, you should be able to: C define haemostasis and identify the key components C explain the role of platelets and their mechanism in haemostasis C describe the principle of the cell-based model of coagulation C identify the parameters used in thromboelastography
James Sira Lorna Eyre
Abstract Haemostasis is a complex and sophisticated process that requires the interplay of multiple physiological pathways. Cellular and molecular mechanisms interact to seal damaged blood vessels with localized clot formation preventing significant bleeding. Once vascular integrity is restored, clot breakdown occurs and normal haemostasis is reinstated. Thrombohaemorrhagic imbalance may occur in the perioperative period or during critical illness, leading to an increased risk of thrombosis, bleeding or in some instances both. Therefore an understanding of the normal physiological processes is important for the anaesthetist as: (i) it allows us to identify targets for the therapeutic modulation of bleeding and thrombosis; (ii) many commonly encountered medications alter the normal haemostatic pathways and it is important to recognize their effects; and (iii) it enables enhanced understanding of the dynamic tests of haemostasis and clotting.
blood and the endothelial cells. Glycoproteins and proteoglycans form the bulk of the glycocalyx, and it is the former that act as adhesion molecules contributing to the coagulation, fibrinolytic and haemostatic systems. Under normal circumstances blood components pass unhindered through the circulatory system. The vascular barrier provides a non-thrombogenic surface due to the production of platelet inhibitors, coagulation inhibitors and fibrinolysis activators (Table 1). One of the most important substances produced is heparin sulphate that acts as a cofactor for the activation of antithrombin and thrombomodulin, both of which inhibit coagulation.1 In contrast, the subendothelial layer is highly thrombogenic and contains collagen, von Willebrand factor (VWF) and other proteins such as laminin, thrombospondin and vitronectin that are involved in platelet adhesion.2 When the vascular endothelial layer is interrupted, for example, by trauma or inflammation, VWF is released, collagen is exposed and tissue factor (TF) is expressed on the surface of endothelial cells. This switch to a prothrombotic and proinflammatory state sees the endothelium orchestrate vasoconstriction, platelet and leucocyte activation and adhesion, promotion of thrombin formation, coagulation and fibrin deposition at the vascular wall.
Keywords Blood coagulation; blood platelets; endothelium; glycocalyx; haemostasis; thromboelastography; vascular Royal College of Anaesthetists CPD Matrix: 1A01, 2A05
Haemostasis (from the Greek: aima, blood þ stasis, halting) is defined as the arrest of bleeding and requires the rapid interaction of a number of closely regulated processes. These culminate in the production of a localized clot at the site of vessel injury usually over the course of seconds to minutes. Disruption of the vascular endothelium triggers this interplay of physiological processes, which include formation of an initial platelet plug (primary haemostasis), activation of coagulation to form a fibrin mesh (secondary haemostasis), fibrinolysis and vessel repair.
Examples of vascular endothelial antithrombotic mediators
Vascular endothelium The vascular endothelium has traditionally been described as a fine cellular monolayer lining the circulatory system, which together with the basement membrane forms the intima. It is now accepted that a sugareprotein glycocalyx is also an integral part of all healthy vascular endothelium. The glycocalyx can be considered as a complex gel that is produced by and coats the endothelium. It occupies a critical position between flowing
Action
Prostacyclin Nitric oxide ADPase
Inhibition platelet activation
Thrombomodulin Heparin sulphate Inhibition of coagulation Tissue factor pathway inhibitor Tissue-plasminogen activator
Modulation of fibrinolysis
Examples of vascular endothelial pro-thrombotic mechanisms
James Sira MBChB BSc (Hons) FRCA is a Specialist Registrar at St James’s University Hospital, Leeds Teaching Hospitals NHS Trust, Leeds, UK. Conflicts of interest: none declared.
Mediator
Action
Tissue factor
Released following endothelial damage Platelet adhesion and aggregation Inhibits fibrinolysis
Von Willebrand factor Plasminogen activator inhibitor-1
Lorna Eyre BSc (Hons) FRCA DICM is a Consultant in Critical Care and Anaesthesia at St James’s University Hospital, Leeds Teaching Hospitals NHS Trust, Leeds, UK. Conflicts of interest: none declared.
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Mediator
Table 1
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Ó 2016 Elsevier Ltd. All rights reserved.
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The endothelium is also responsible for triggering and regulating fibrinolysis through the synthesis of tissue-type plasminogen activator and its inhibitor plasminogen activator inhibitor PAI-1.3 These mechanisms constitute the initial step towards vascular repair.
Adhesion Platelets initially bind to collagen exposed by damage to the endothelium via glycoprotein (GP) receptor complexes. Of these, GP1b/V/IX and GPVI expressed on their surface are the most important. They initiate platelet aggregation and thrombus formation by primary interactions with von Willebrand factor and collagen,4 respectively, and are also involved in coagulation and leucocyte engagement. Cell adhesion ligands such as VWF and thrombospondin act as a bridge strengthening the association between platelet surface receptors and endothelial collagen. In the case of VWF it specifically binds to the GPIb component of the GP1b/V/IX glycoprotein complex5 and in addition contributes significantly to plateleteplatelet interactions. These tethering glycoproteins on the surface of platelets bring about the firm binding of a platelet monolayer to the exposed collagen.
Platelets Platelets play a pivotal role in primary haemostasis and alongside the vessel wall and adhesive proteins, lead to the formation of an initial ‘platelet plug’. There are normally between 150 and 400 billion platelets per litre of blood in a healthy adult, produced by megakaryocytes in the bone marrow. Their lifespan ranges from 8 to 14 days.4 At the site of vessel injury, platelets recognize disruption of endothelial cells lining the blood vessels and the exposed underlying fibrous matrix. They subsequently form a core of thrombi through a process of adhesion, activation, secretion of the contents of intracellular storage organelles, and aggregation detailed below (Figure 1). In addition, activated platelets express phospholipids which promote localized coagulation and generation of thrombin and fibrin.
Activation As platelets adhere to the subendothelial components of the damaged vascular endothelium, activation occurs from a number of stimuli including some produced by the platelet itself. One of the major downstream consequences of ligand engagement of GPIb/GPVI by VWF/collagen is the secretion of platelet dense granule contents, ADP and thromboxane A2 (TxA2). These induce G-protein receptor mediated secondary platelet activation and amplify signals leading ultimately to activation of the platelet specific integrin, glycoprotein GPIIb-IIIa.4 It is worth noting that the final pathway for all agonists is the activation of the GPIIb eIIIa integrin which serves as the main receptor for platelet adhesion and aggregation.5 Other stimuli for activation include collagen and thrombin. Thrombin is a particularly effective activator of platelets. It forms following exposure of tissue factor (TF) to plasma coagulation factors and appears on cellular surfaces including those of platelets. Activation of platelets results in a change in shape from discoid to spherical increasing plateleteplatelet interactions, it also results in an increase in granule secretion and platelet aggregation.
Role of platelets Adhesion GPIb receptor
vWF
Endoth End otheli oth elium eli um Endothelium
Expose Exp osed ose d sube ssubendothelial ubendo ube ndothe ndo thelia the liall matr lia m atrix atr ix Exposed matrix Release GTP/GDP ATP/ADP Serotonin
Fibrinogen Fibronectin vWF
Secretion After adhesion, degranulation from both types of platelet storage granules (A-granules and dense bodies) occurs. The release of calcium is of particular importance in the activation of platelet surface phospholipids that then provide a surface for the assembly of various coagulation factors. A-granules contain platelet-specific proteins such as platelet-derived growth factor, chemokines, adhesive molecules and coagulation proteins such as factor V and protein S. Dense bodies contain non-metabolic adenines (e.g. ADP, GTP), divalent cations (Mg2þ, Ca2þ) and serotonin.
Aggregation GPIIb/IIIa receptor
Fi Fibrinogen
Aggregation Aggregation leads to the formation of a haemostatic thrombus. TxA2 produced by activated platelets along with ADP enlarge this platelet aggregate in an attempt to seal off any vascular injury. Following platelet activation, the integrin GPIIb/IIIa undergoes a conformational change rendering it capable of binding various extracellular ligands including fibrinogen and VWF. In addition to this GPIIb/IIIa is also capable of interacting with the
AD ADP throm thrombin GDP- guanosine 5’-diphosphate GTP- guanosine 5’-triphosphate ADP- adenosine 5’-diphosphate ATP- adenosine 5’-triphosphate vWF- von Willebrand factor
Figure 1
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Ó 2016 Elsevier Ltd. All rights reserved.
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initiated. TF binds to circulating factor VIIa and, in the presence of factor V, converts factor IX to factor IXa and factor X to factor Xa (Figure 2). Factor Xa then binds to prothrombin to generate a small amount of thrombin which alone is insufficient to generate fibrin. Thrombin generation through this reaction is not robust and can be effectively terminated by TF pathway inhibitor.
platelet cytoskeleton, controlling changes in shape, signalling and contraction of the formed thrombus, thus mediating platelet spread and clot retraction. Prostacyclin inhibits platelet activation and aggregation and the balance between this and TxA2 leads to localized platelet aggregation thus preventing extension of the clot thereby maintaining the vessel lumen patency.
Amplification Since the amount of thrombin generated is insufficient to convert fibrinogen to fibrin, numerous positive feedback loops exist. This thrombin triggered positive feedback occurs chiefly on the platelet surface, allowing further activation of platelets and of clotting factors. Throughout this process the platelets are covered in activated clotting factors in readiness for propagation. Thrombin generated in the initiation phase further activates factor V and factor VIII which serve as cofactors in its propagation2 (Figure 2).
Coagulation Coagulation results in the conversion of soluble fibrinogen to insoluble fibrin strands, which strengthen the aggregated platelets (secondary haemostasis). Most of the procoagulants and anticoagulants are produced by the liver except factors III, IV and VIII.2 The classical description of an extrinsic and intrinsic pathway that converge on activation of factor X is now defunct. It may assist in understanding in vitro coagulation tests (e.g. aPTT and PT), but fails to incorporate the central role of cell-based surfaces in in vivo coagulation processes. Current evidence supports the understanding that the intrinsic pathway is not a parallel pathway but indeed augments thrombin generation primarily initiated by the extrinsic pathway. Newer models describe coagulation with the following steps.
Propagation Continuous thrombin generation is ensured by the action of two complexes: factor VIIIa complexed to factor IXa (intrinsic tenase) and factor Va to factor Xa (prothrombinase). This again occurs on the surface of platelets and leads to the appropriately localized formation of much greater amounts of thrombin (the thrombin ‘burst’).
Initiation When blood is exposed to cells that express TF on their surface, for example, in vessel wall subendothelial tissue, coagulation is
Stabilization The thrombin generated results in the formation of fibrin from fibrinogen and activation of factor XIII (fibrin stabilizing factor). This covalently links soluble fibrin monomers to form a stable polymer and provide strength and stability to fibrin incorporated in the platelet plug. Thrombin also activates thrombinactivatable-fibrinolysis-inhibitor, which protects the clot from fibrinolysis. Coagulation is regulated and localized by several anticoagulant mechanisms. The most important of these includes antithrombin, which inhibits thrombin, and factors IXa, Xa, XIa and XIIa. Others include TF Pathway Inhibitor which inhibits the TF-VIIa complex and thrombin, and activated protein C (APC), which binds to thrombomodulin and cleaves factor Va and factor VIIIa. More recently the role of the cofactor protein Z in the inhibition of factor Xa through the protein z-dependent protease inhibitor has been reported.2
Cell-based theory of coagulation Wound, vessel injury
Tissue factor + Factor VIIa Tissue factor pathway inhibitor Factor IX
Factor XIa Factor XI APC + Thrombomodulin
Factor IXa + Factor VIIIa
Factor X Factor Xa + Factor Va
Antithrombin
Prothrombin Thrombin
Fibrinogen
Fibrin
Fibrinolysis The fibrinolytic system is a parallel system activated along with the coagulation cascade and serves to localize and limit clot formation. Fibrinolysis is an enzymatic process mediated by plasmin that dissolves the fibrin clot into fibrin degradation products (FDPs). The inactive precursor of plasmin, plasminogen, is activated by tissue plasminogen activator (t-Pa) and urokinase released into the blood by the damaged endothelium. Plasmin activity is tightly regulated by its inhibitor, A-2 antiplasmin, preventing widespread fibrinolysis. The fibrinolytic system is also regulated by plasminogen activator inhibitor which inhibits t-PA, urokinase and thrombin activatable fibrinolysis inhibitor (TAFI).
F XIIIa
Stable blood clot
Tissue factor- factor VIIa act as the initiation step on exposure to damaged endothelium. The dotted arrows indicate amplification of cascade by thrombin. The process of coagulation is regulated by antithrombin, tissue factor pathway inhibitor and activated protein C (APC).
Figure 2
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Ó 2016 Elsevier Ltd. All rights reserved.
BLOOD
clot is formed and is thus increased by increased fibrinogen levels or platelet function, and decreased by anticoagulants, hypofibrinogenaemia and thrombocytopenia. MA (maximum amplitude): MA is the greatest diameter of the clot and a measure of its strength. It relies fundamentally on the interaction of fibrin and platelets, with the latter exerting the most influence. It is decreased by deficiencies or medications which affect either of these. LY30 (lysis 30 minutes after MA): This measures percent lysis at 30 minutes reflecting fibrinolysis. If significantly raised, primary and/or secondary hyperfibrinolysis must be considered. Figure 3
Conclusion Anaesthetists will frequently be involved in patients in whom, for a variety of reasons, a disturbance of haemostasis will be present. It is therefore essential that they have a basic understanding of the normal haemostatic pathways. Furthermore the move to more dynamic and physiological tests of coagulation necessitate that this knowledge can be applied to a variety of situations in order to improve patient care. A
Thromboelastography The acceptance of newer models of coagulation has led to an emphasis on the crucial role of platelets and hence an interest in testing whole blood for its viscoelastic properties more reflective of in vivo haemostasis. These tests cover the initial phase of fibrin formation, clot retraction and finally fibrinolysis. Thromboelastography (TEG) is one such method. Whole blood is added to activators and a pin attached to torsion wires immersed in the sample as it rotates. As the sample clots it provides a characteristic tracing from which parameters are derived (Figure 3). Specific component deficiencies may be deduced and the parameters and interpretation of abnormalities are as follows: R-time (reaction time): Time from initiation to a 2 mm amplitude. Prolonged by anticoagulants (warfarin, heparin), factor deficiencies, and severe hypofibrinogenaemia. If shortened, it indicates the presence of hypercoagulability. K-time (clot formation time): Time from R-time to a TEG amplitude of 20 mm representing clot formation kinetics. Prolonged by anticoagulants, hypofibrinogenaemia and thrombocytopenia. Reduced by increased fibrinogen levels or platelet function. Alpha-angle: This is the angle between the middle of the TEG and the K inclination. It indicates the rate at which
ANAESTHESIA AND INTENSIVE CARE MEDICINE 17:2
REFERENCES 1 Rasche H. Haemostasis and thrombosis: an overview. Eur Heart J Suppl 2001; 3(suppl Q): Q3e7. 2 Palta S, Saroa R, Palta A. Overview of the coagulation system. Indian J Anaesth 2014; 58: 515e23. 3 Van Hinsbergh V. The endothelium: vascular control of haemostasis. Eur J Obstet Gynecol Reprod Biol 2001; 95: 198e201. 4 Berndt M, Metharom P, Andrews K. Primary haemostasis: newer insights. Haemophilia 2014; 20: 15e22. 5 Davi G, Patrono D. Platelet activation and atherothrombosis. N Eng J Med 2007; 357: 2482e94. FURTHER READING Alphonsus C, Rodseth R. The endothelial glycocalyx: a review of the vascular barrier. Anaesthesia 2014; 69: 777e84. Galvez K, Cortes C. Thromboelastography: new concepts in haemostasis physiology and correlation with trauma associated coagulopathy. Colomb J Anesthesiol 2012; 40: 224e30.
82
Ó 2016 Elsevier Ltd. All rights reserved.
Physiology of haemostasis
Learning objectives After reading this article, you should be able to: C define haemostasis and identify the key components C explain the role of platelets and their mechanism in haemostasis C describe the principle of the cell-based model of coagulation C identify the parameters used in thromboelastography
James Sira Lorna Eyre
Abstract Haemostasis is a complex and sophisticated process that requires the interplay of multiple physiological pathways. Cellular and molecular mechanisms interact to seal damaged blood vessels with localized clot formation preventing significant bleeding. Once vascular integrity is restored, clot breakdown occurs and normal haemostasis is reinstated. Thrombohaemorrhagic imbalance may occur in the perioperative period or during critical illness, leading to an increased risk of thrombosis, bleeding or in some instances both. Therefore an understanding of the normal physiological processes is important for the anaesthetist as: (i) it allows us to identify targets for the therapeutic modulation of bleeding and thrombosis; (ii) many commonly encountered medications alter the normal haemostatic pathways and it is important to recognize their effects; and (iii) it enables enhanced understanding of the dynamic tests of haemostasis and clotting.
blood and the endothelial cells. Glycoproteins and proteoglycans form the bulk of the glycocalyx, and it is the former that act as adhesion molecules contributing to the coagulation, fibrinolytic and haemostatic systems. Under normal circumstances blood components pass unhindered through the circulatory system. The vascular barrier provides a non-thrombogenic surface due to the production of platelet inhibitors, coagulation inhibitors and fibrinolysis activators (Table 1). One of the most important substances produced is heparin sulphate that acts as a cofactor for the activation of antithrombin and thrombomodulin, both of which inhibit coagulation.1 In contrast, the subendothelial layer is highly thrombogenic and contains collagen, von Willebrand factor (VWF) and other proteins such as laminin, thrombospondin and vitronectin that are involved in platelet adhesion.2 When the vascular endothelial layer is interrupted, for example, by trauma or inflammation, VWF is released, collagen is exposed and tissue factor (TF) is expressed on the surface of endothelial cells. This switch to a prothrombotic and proinflammatory state sees the endothelium orchestrate vasoconstriction, platelet and leucocyte activation and adhesion, promotion of thrombin formation, coagulation and fibrin deposition at the vascular wall.
Keywords Blood coagulation; blood platelets; endothelium; glycocalyx; haemostasis; thromboelastography; vascular Royal College of Anaesthetists CPD Matrix: 1A01, 2A05
Haemostasis (from the Greek: aima, blood þ stasis, halting) is defined as the arrest of bleeding and requires the rapid interaction of a number of closely regulated processes. These culminate in the production of a localized clot at the site of vessel injury usually over the course of seconds to minutes. Disruption of the vascular endothelium triggers this interplay of physiological processes, which include formation of an initial platelet plug (primary haemostasis), activation of coagulation to form a fibrin mesh (secondary haemostasis), fibrinolysis and vessel repair.
Examples of vascular endothelial antithrombotic mediators
Vascular endothelium The vascular endothelium has traditionally been described as a fine cellular monolayer lining the circulatory system, which together with the basement membrane forms the intima. It is now accepted that a sugareprotein glycocalyx is also an integral part of all healthy vascular endothelium. The glycocalyx can be considered as a complex gel that is produced by and coats the endothelium. It occupies a critical position between flowing
Action
Prostacyclin Nitric oxide ADPase
Inhibition platelet activation
Thrombomodulin Heparin sulphate Inhibition of coagulation Tissue factor pathway inhibitor Tissue-plasminogen activator
Modulation of fibrinolysis
Examples of vascular endothelial pro-thrombotic mechanisms
James Sira MBChB BSc (Hons) FRCA is a Specialist Registrar at St James’s University Hospital, Leeds Teaching Hospitals NHS Trust, Leeds, UK. Conflicts of interest: none declared.
Mediator
Action
Tissue factor
Released following endothelial damage Platelet adhesion and aggregation Inhibits fibrinolysis
Von Willebrand factor Plasminogen activator inhibitor-1
Lorna Eyre BSc (Hons) FRCA DICM is a Consultant in Critical Care and Anaesthesia at St James’s University Hospital, Leeds Teaching Hospitals NHS Trust, Leeds, UK. Conflicts of interest: none declared.
ANAESTHESIA AND INTENSIVE CARE MEDICINE 17:2
Mediator
Table 1
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Ó 2016 Elsevier Ltd. All rights reserved.
BLOOD
The endothelium is also responsible for triggering and regulating fibrinolysis through the synthesis of tissue-type plasminogen activator and its inhibitor plasminogen activator inhibitor PAI-1.3 These mechanisms constitute the initial step towards vascular repair.
Adhesion Platelets initially bind to collagen exposed by damage to the endothelium via glycoprotein (GP) receptor complexes. Of these, GP1b/V/IX and GPVI expressed on their surface are the most important. They initiate platelet aggregation and thrombus formation by primary interactions with von Willebrand factor and collagen,4 respectively, and are also involved in coagulation and leucocyte engagement. Cell adhesion ligands such as VWF and thrombospondin act as a bridge strengthening the association between platelet surface receptors and endothelial collagen. In the case of VWF it specifically binds to the GPIb component of the GP1b/V/IX glycoprotein complex5 and in addition contributes significantly to plateleteplatelet interactions. These tethering glycoproteins on the surface of platelets bring about the firm binding of a platelet monolayer to the exposed collagen.
Platelets Platelets play a pivotal role in primary haemostasis and alongside the vessel wall and adhesive proteins, lead to the formation of an initial ‘platelet plug’. There are normally between 150 and 400 billion platelets per litre of blood in a healthy adult, produced by megakaryocytes in the bone marrow. Their lifespan ranges from 8 to 14 days.4 At the site of vessel injury, platelets recognize disruption of endothelial cells lining the blood vessels and the exposed underlying fibrous matrix. They subsequently form a core of thrombi through a process of adhesion, activation, secretion of the contents of intracellular storage organelles, and aggregation detailed below (Figure 1). In addition, activated platelets express phospholipids which promote localized coagulation and generation of thrombin and fibrin.
Activation As platelets adhere to the subendothelial components of the damaged vascular endothelium, activation occurs from a number of stimuli including some produced by the platelet itself. One of the major downstream consequences of ligand engagement of GPIb/GPVI by VWF/collagen is the secretion of platelet dense granule contents, ADP and thromboxane A2 (TxA2). These induce G-protein receptor mediated secondary platelet activation and amplify signals leading ultimately to activation of the platelet specific integrin, glycoprotein GPIIb-IIIa.4 It is worth noting that the final pathway for all agonists is the activation of the GPIIb eIIIa integrin which serves as the main receptor for platelet adhesion and aggregation.5 Other stimuli for activation include collagen and thrombin. Thrombin is a particularly effective activator of platelets. It forms following exposure of tissue factor (TF) to plasma coagulation factors and appears on cellular surfaces including those of platelets. Activation of platelets results in a change in shape from discoid to spherical increasing plateleteplatelet interactions, it also results in an increase in granule secretion and platelet aggregation.
Role of platelets Adhesion GPIb receptor
vWF
Endoth End otheli oth elium eli um Endothelium
Expose Exp osed ose d sube ssubendothelial ubendo ube ndothe ndo thelia the liall matr lia m atrix atr ix Exposed matrix Release GTP/GDP ATP/ADP Serotonin
Fibrinogen Fibronectin vWF
Secretion After adhesion, degranulation from both types of platelet storage granules (A-granules and dense bodies) occurs. The release of calcium is of particular importance in the activation of platelet surface phospholipids that then provide a surface for the assembly of various coagulation factors. A-granules contain platelet-specific proteins such as platelet-derived growth factor, chemokines, adhesive molecules and coagulation proteins such as factor V and protein S. Dense bodies contain non-metabolic adenines (e.g. ADP, GTP), divalent cations (Mg2þ, Ca2þ) and serotonin.
Aggregation GPIIb/IIIa receptor
Fi Fibrinogen
Aggregation Aggregation leads to the formation of a haemostatic thrombus. TxA2 produced by activated platelets along with ADP enlarge this platelet aggregate in an attempt to seal off any vascular injury. Following platelet activation, the integrin GPIIb/IIIa undergoes a conformational change rendering it capable of binding various extracellular ligands including fibrinogen and VWF. In addition to this GPIIb/IIIa is also capable of interacting with the
AD ADP throm thrombin GDP- guanosine 5’-diphosphate GTP- guanosine 5’-triphosphate ADP- adenosine 5’-diphosphate ATP- adenosine 5’-triphosphate vWF- von Willebrand factor
Figure 1
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Ó 2016 Elsevier Ltd. All rights reserved.
BLOOD
initiated. TF binds to circulating factor VIIa and, in the presence of factor V, converts factor IX to factor IXa and factor X to factor Xa (Figure 2). Factor Xa then binds to prothrombin to generate a small amount of thrombin which alone is insufficient to generate fibrin. Thrombin generation through this reaction is not robust and can be effectively terminated by TF pathway inhibitor.
platelet cytoskeleton, controlling changes in shape, signalling and contraction of the formed thrombus, thus mediating platelet spread and clot retraction. Prostacyclin inhibits platelet activation and aggregation and the balance between this and TxA2 leads to localized platelet aggregation thus preventing extension of the clot thereby maintaining the vessel lumen patency.
Amplification Since the amount of thrombin generated is insufficient to convert fibrinogen to fibrin, numerous positive feedback loops exist. This thrombin triggered positive feedback occurs chiefly on the platelet surface, allowing further activation of platelets and of clotting factors. Throughout this process the platelets are covered in activated clotting factors in readiness for propagation. Thrombin generated in the initiation phase further activates factor V and factor VIII which serve as cofactors in its propagation2 (Figure 2).
Coagulation Coagulation results in the conversion of soluble fibrinogen to insoluble fibrin strands, which strengthen the aggregated platelets (secondary haemostasis). Most of the procoagulants and anticoagulants are produced by the liver except factors III, IV and VIII.2 The classical description of an extrinsic and intrinsic pathway that converge on activation of factor X is now defunct. It may assist in understanding in vitro coagulation tests (e.g. aPTT and PT), but fails to incorporate the central role of cell-based surfaces in in vivo coagulation processes. Current evidence supports the understanding that the intrinsic pathway is not a parallel pathway but indeed augments thrombin generation primarily initiated by the extrinsic pathway. Newer models describe coagulation with the following steps.
Propagation Continuous thrombin generation is ensured by the action of two complexes: factor VIIIa complexed to factor IXa (intrinsic tenase) and factor Va to factor Xa (prothrombinase). This again occurs on the surface of platelets and leads to the appropriately localized formation of much greater amounts of thrombin (the thrombin ‘burst’).
Initiation When blood is exposed to cells that express TF on their surface, for example, in vessel wall subendothelial tissue, coagulation is
Stabilization The thrombin generated results in the formation of fibrin from fibrinogen and activation of factor XIII (fibrin stabilizing factor). This covalently links soluble fibrin monomers to form a stable polymer and provide strength and stability to fibrin incorporated in the platelet plug. Thrombin also activates thrombinactivatable-fibrinolysis-inhibitor, which protects the clot from fibrinolysis. Coagulation is regulated and localized by several anticoagulant mechanisms. The most important of these includes antithrombin, which inhibits thrombin, and factors IXa, Xa, XIa and XIIa. Others include TF Pathway Inhibitor which inhibits the TF-VIIa complex and thrombin, and activated protein C (APC), which binds to thrombomodulin and cleaves factor Va and factor VIIIa. More recently the role of the cofactor protein Z in the inhibition of factor Xa through the protein z-dependent protease inhibitor has been reported.2
Cell-based theory of coagulation Wound, vessel injury
Tissue factor + Factor VIIa Tissue factor pathway inhibitor Factor IX
Factor XIa Factor XI APC + Thrombomodulin
Factor IXa + Factor VIIIa
Factor X Factor Xa + Factor Va
Antithrombin
Prothrombin Thrombin
Fibrinogen
Fibrin
Fibrinolysis The fibrinolytic system is a parallel system activated along with the coagulation cascade and serves to localize and limit clot formation. Fibrinolysis is an enzymatic process mediated by plasmin that dissolves the fibrin clot into fibrin degradation products (FDPs). The inactive precursor of plasmin, plasminogen, is activated by tissue plasminogen activator (t-Pa) and urokinase released into the blood by the damaged endothelium. Plasmin activity is tightly regulated by its inhibitor, A-2 antiplasmin, preventing widespread fibrinolysis. The fibrinolytic system is also regulated by plasminogen activator inhibitor which inhibits t-PA, urokinase and thrombin activatable fibrinolysis inhibitor (TAFI).
F XIIIa
Stable blood clot
Tissue factor- factor VIIa act as the initiation step on exposure to damaged endothelium. The dotted arrows indicate amplification of cascade by thrombin. The process of coagulation is regulated by antithrombin, tissue factor pathway inhibitor and activated protein C (APC).
Figure 2
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Ó 2016 Elsevier Ltd. All rights reserved.
BLOOD
clot is formed and is thus increased by increased fibrinogen levels or platelet function, and decreased by anticoagulants, hypofibrinogenaemia and thrombocytopenia. MA (maximum amplitude): MA is the greatest diameter of the clot and a measure of its strength. It relies fundamentally on the interaction of fibrin and platelets, with the latter exerting the most influence. It is decreased by deficiencies or medications which affect either of these. LY30 (lysis 30 minutes after MA): This measures percent lysis at 30 minutes reflecting fibrinolysis. If significantly raised, primary and/or secondary hyperfibrinolysis must be considered. Figure 3
Conclusion Anaesthetists will frequently be involved in patients in whom, for a variety of reasons, a disturbance of haemostasis will be present. It is therefore essential that they have a basic understanding of the normal haemostatic pathways. Furthermore the move to more dynamic and physiological tests of coagulation necessitate that this knowledge can be applied to a variety of situations in order to improve patient care. A
Thromboelastography The acceptance of newer models of coagulation has led to an emphasis on the crucial role of platelets and hence an interest in testing whole blood for its viscoelastic properties more reflective of in vivo haemostasis. These tests cover the initial phase of fibrin formation, clot retraction and finally fibrinolysis. Thromboelastography (TEG) is one such method. Whole blood is added to activators and a pin attached to torsion wires immersed in the sample as it rotates. As the sample clots it provides a characteristic tracing from which parameters are derived (Figure 3). Specific component deficiencies may be deduced and the parameters and interpretation of abnormalities are as follows: R-time (reaction time): Time from initiation to a 2 mm amplitude. Prolonged by anticoagulants (warfarin, heparin), factor deficiencies, and severe hypofibrinogenaemia. If shortened, it indicates the presence of hypercoagulability. K-time (clot formation time): Time from R-time to a TEG amplitude of 20 mm representing clot formation kinetics. Prolonged by anticoagulants, hypofibrinogenaemia and thrombocytopenia. Reduced by increased fibrinogen levels or platelet function. Alpha-angle: This is the angle between the middle of the TEG and the K inclination. It indicates the rate at which
ANAESTHESIA AND INTENSIVE CARE MEDICINE 17:2
REFERENCES 1 Rasche H. Haemostasis and thrombosis: an overview. Eur Heart J Suppl 2001; 3(suppl Q): Q3e7. 2 Palta S, Saroa R, Palta A. Overview of the coagulation system. Indian J Anaesth 2014; 58: 515e23. 3 Van Hinsbergh V. The endothelium: vascular control of haemostasis. Eur J Obstet Gynecol Reprod Biol 2001; 95: 198e201. 4 Berndt M, Metharom P, Andrews K. Primary haemostasis: newer insights. Haemophilia 2014; 20: 15e22. 5 Davi G, Patrono D. Platelet activation and atherothrombosis. N Eng J Med 2007; 357: 2482e94. FURTHER READING Alphonsus C, Rodseth R. The endothelial glycocalyx: a review of the vascular barrier. Anaesthesia 2014; 69: 777e84. Galvez K, Cortes C. Thromboelastography: new concepts in haemostasis physiology and correlation with trauma associated coagulopathy. Colomb J Anesthesiol 2012; 40: 224e30.
82
Ó 2016 Elsevier Ltd. All rights reserved.