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Coagulation in Liver Disease Maureane Hoffman, MD, PhD1,2 1 Pathology and Laboratory Medicine Service, Durham Veterans Affairs
Medical Center, Durham, North Carolina 2 Department of Pathology, Duke University Medical Center, Durham, North Carolina
Address for correspondence Maureane Hoffman, MD, PhD, Pathology and Laboratory Medicine Service (113), Durham Veterans Affairs Medical Center, 508 Fulton St, Durham, NC 27705 (e-mail: [email protected]).
Abstract
Keywords
► ► ► ►
cirrhosis thrombin generation prothrombin time fibrinolysis
The liver plays a key role in hemostasis as the site of synthesis of many of the proteins involved in the coagulation, antithrombotic and fibrinolytic systems that interact to both establish hemostasis, and preventing thrombosis. The common laboratory tests, prothrombin time (PT) and activated partial thromboplastin time (aPTT), evolved from studies of plasma clotting in test tubes. Such studies laid the basis for the coagulation cascade model of hemostasis. However, thought has evolved to place a greater emphasis on the active roles of cells in localizing and regulating hemostasis. The PT and aPTT do not reflect the roles of cellular elements in hemostasis, nor do they reflect the crucial roles of antithrombotic and fibrinolytic systems. Thus, though the PT may indeed reflect the synthetic capacity of the liver, it does not accurately reflect the risk of bleeding or thrombosis in patients with liver failure.
Hemostasis is profoundly altered by liver disorders. Most proand anticoagulant factors, as well as the pro- and antifibrinolytic factors, are synthesized by hepatocytes. Most factors are also cleared by the liver. In addition, reduced hepatic production of thrombopoietin and hypersplenism due to portal hypertension can lead to decreased platelet counts. Though the field is making progress in understanding the nature of the changes, we do not have good clinical laboratory tests to assess the ability of the coagulation system to maintain hemostasis and prevent thrombosis in patients with liver dysfunction. The goals of this review are (1) to discuss the functioning of the hemostatic system in the body, and how it is altered in the setting of hepatic dysfunction; (2) highlight the limitations of the laboratory tests available to assess hemostatic function.
Mechanisms of Hemostasis Coagulation Testing and the Coagulation Cascade In the 1930s two groups developed the first versions of the prothrombin time (PT) test. The PT was developed as an assay for the level of prothrombin based on the prevailing model of coagulation at that time. This was
published online June 6, 2015
Issue Theme Hemostatic Dysfunction in Liver Diseases; Guest Editors: Ton Lisman, PhD, and Hau C. Kwaan, MD, FRCP.
Thromboplastin þ Prothrombin þ Calcium ¼ Thrombin The idea was that if a constant amount of calcium and an excess of thromboplastin were added to plasma, the only variable would be the level of prothrombin. Thus, the rate of thrombin production under these conditions would be a function of the prothrombin level. It is notable that Quick describes the development of the PT test,1 stating that he first observed a delayed clotting time on plasma from a jaundiced subject. Of course the bleeding tendency in patients with liver disorders was well recognized at that time. He found the clotting time to be normal in plasma from hemophiliacs, but prolonged in samples from most jaundiced patients. On this basis he suggested the hypothesis in 1935 that “the hemorrhagic tendency in jaundice is brought about by a diminution of prothrombin.” The group led by Brinkhous nearly simultaneously developed a two-stage version of the PT test, and similarly concluded that the hemostatic defect in acute liver injury was due to prothrombin deficiency.2 Their conclusions and the pleasingly simple model of hemostasis on which they were based fell by the wayside as more plasma coagulation factors were discovered: FVIII in 1936,3 FV in 1947,4 FVII in 1951,5 FIX in 1952,6,7 and FXI in 1953.8
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0035-1550435. ISSN 0094-6176.
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The forerunner of the modern partial thromboplastin time (PTT) was developed in the 1950s as a means to assay the deficient factor in hemophilia.9 This additional test was necessary because the Quick PT was not abnormal in hemophilia. In the PT test a “thromboplastin” reagent and calcium are added to citrated platelet poor plasma. The time required for the sample to clot is then measured. It had been discovered years earlier that, while tissue thromboplastins clotted hemophilic plasma rapidly, diluted thromboplastin could give longer clotting times with hemophilic than normal plasma. In 1953 Langdell et al developed this concept into a simple quantitative assay, which could be used to make the diagnosis of hemophilia and monitor therapy.9 This PTT was later improved by using an activator (kaolin) to speed up the clotting process and give more reproducible results. This innovation was termed the activated partial thromboplastin time (aPTT).10 Both the PT and PTT were developed before the “cascade” model of coagulation was proposed, and results obtained using these assays contributed to the development of this model. The “cascade” model was proposed nearly simultaneously in two publications in 1964.11,12 This model views coagulation as a sequential series of proteolytic steps in which activation of one clotting factor leads to the activation of another, ultimately resulting in the activation of enough thrombin to convert soluble fibrinogen to an insoluble fibrin clot. It was a significant conceptual advance to recognize that a series of proteases could act as a biological amplifier. The original models evolved into the now-familiar Y-shaped scheme that we call the “coagulation cascade” today. The “intrinsic” and “extrinsic” pathways are initiated by factor XII (FXII) and FVIIa/tissue factor (TF), respectively, and converge on a “common” pathway at the level of the FXa/FVa (prothrombinase) complex. This “cascade” was not proposed as a literal model of hemostasis in vivo, but primarily as a scheme of how the many coagulation factors interact biochemically. To facilitate biochemical studies, platelets were replaced by phospholipid vesicles.13 In addition, the anticoagulant pathways that are critical to localization and control of thrombin formation in vivo are not addressed by the cascade model. Because the cascade model was based on the same types of studies as the PT and aPTT, it should not be surprising that the cascade model is an excellent tool for interpreting results of the PT and aPTT tests. On the other hand, the limitations of the cascade model are shared by these related laboratory assays. A major limitation of the cascade model is that clotting times in the PT and aPTT do not correlate well with the risk of bleeding in vivo. For example, even though deficiencies of each of the factors in the intrinsic pathway can have equally long aPTT values, they have dramatically different risks of hemorrhage. Deficiencies of factor XII are not associated with significant hemorrhage and deficiencies of factor XI might or might not be associated with hemorrhage, but deficiencies of factors VIII and IX (hemophilia A and B) are consistently associated with hemorrhage. A particularly important limitation of the cascade model is that it cannot explain why a normally functioning “extrinsic” pathway cannot compensate Seminars in Thrombosis & Hemostasis
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for a defect in the “intrinsic” pathway, as in hemophilia. Thus, the intrinsic and extrinsic systems cannot operate as independent and redundant pathways as implied by this model.
A Cell-Based Model of Hemostasis In the time since it was first proposed, many have recognized that the “cascade” has serious failings as a model of normal and abnormal coagulation. It was also recognized from the earliest studies of coagulation that cells are important participants in the process14,15 and that normal hemostasis is not possible in the absence of cell-associated TF and platelets. Substituting phospholipid vesicles in the place of cells in coagulation tests fails to capture the active participation of cells in hemostasis in vivo. A cell-based model of coagulation, in which hemostasis occurs in a step-wise process on cell surfaces, incorporates the important roles of cells and anticoagulants that the cascade model does not.16 A summary of the cell-based model of hemostasis is shown in ►Fig. 1.
Step 1: Initiation of Coagulation on TF-Bearing Cells When a vessel is disrupted, platelets escape from the vessel, bind to collagen and other extracellular matrix components at the site of injury, and are partially activated. This process forms a platelet plug that provides primary hemostasis. However, long-term hemostasis requires that the platelet plug be stabilized by a meshwork of fibrin formed by the action of thrombin. The process of thrombin generation is initiated when TFbearing cells are exposed to blood at a site of injury. TF is a transmembrane protein that acts as a receptor and cofactor for FVII. It is normally expressed only on cells outside the vasculature. TF is highly expressed by pericytes surrounding arterioles and venules, and by adventitial cells around larger vessels.17,18 This distribution has been referred to as a “hemostatic envelope” to stop bleeding in the event of vascular injury. Once bound to TF, zymogen FVII is rapidly activated to FVIIa.19 At sites around vessels a significant proportion of the TF has bound FVII/FVIIa even in the absence of any injury.20 The FVIIa/TF complex catalyzes activation of both FX and FIX.21 The FXa formed on the TF-bearing cell interacts with its cofactor FVa to generate a small amount of thrombin on the TF cells.22 Most coagulation factors can leave the vasculature, percolate through the extravascular space, and collect in the lymph.23 Therefore, it is likely that low levels of FIXa, FXa, and thrombin are produced on TF-bearing cells at all times.24 However, these activated factors are normally separated from other components of the coagulation system by the vessel wall. Platelets and FVIII bound to von Willebrand factor (VWF) are so large that they only enter the extravascular compartment when an injury disrupts the vessel wall. Activated platelets release a platelet-specific form of FV that is partially activated,25 and can immediately form prothrombinase complexes with FXa that has been activated on the TF-bearing cell. At that point the small amounts of thrombin produced on TF-bearing cells can interact with platelets and FVIII/VWF to initiate the hemostatic process that ultimately enmeshes the initial platelet plug in a stable fibrin clot (secondary hemostasis).
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Fig. 1 The phases in a cell-based model of coagulation. Initiation occurs on the TF-bearing cell as FX activated by FVIIa/TF combines with its cofactor, FVa, to activate small amounts of thrombin. This small amount of thrombin is important during the amplification phase, as it participates multiple positive feedback loops by activating cofactors, FXI and platelets. The propagation phase takes place on the platelet surface and is responsible for producing the large burst of thrombin that is required for effective hemostasis. The prothrombin time (PT) test assesses the levels of procoagulants involved in Initiation of coagulation. The activated partial thromboplastin time (aPTT) assay assesses the levels of the procoagulants involved in platelet surface thrombin generation in the propagation phase. vWF, von Willebrand factor.
Step 2: Amplification of the Procoagulant Signal by Thrombin Generated on the TF-Bearing Cell
Step 3: Propagation of Thrombin Generation on the Platelet Surface
During the amplification step, the small amounts of thrombin formed on TF-bearing cells promote maximal platelet activation26 and also activate additional coagulation cofactors on the platelet surface. Although this small amount of thrombin may not be sufficient to produce a stable fibrin clot, it plays a key role by “priming” the clotting system for a subsequent burst of platelet surface thrombin generation by activating FV, FVIII, and FXI on the platelet surface.27–29 Thrombin activation of FXI on platelet surfaces explains why FXII is not needed for normal hemostasis. FIXa activated both on the TF-bearing cell and by platelet-surface FXIa binds to FVIIIa on the platelet surface to assemble FIXa/ FVIIIa (“tenase”) complexes.
The burst of thrombin generation needed for effective hemostasis is produced on platelet surfaces during the propagation phase of coagulation. Once the platelet “tenase” complex is assembled, FX from the plasma is activated to FXa on the platelet surface. FXa then associates with FVa to form “prothrombinase” complexes and produce a burst of thrombin generation. Even though the cell-based model depicts the hemostatic process as occurring in discrete steps, these should be viewed as an overlapping continuum of events. For example, thrombin produced on the platelet surface early in the propagation phase may initially cleave substrates on the platelet surface and continue to amplify the procoagulant response, in addition to leaving the platelet to promote fibrin assembly.
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The cell-based model of coagulation shows us that the “extrinsic” and “intrinsic” pathways are not redundant. The TF (“extrinsic”) pathway operates on TF-bearing cells to initiate and amplify coagulation. By contrast, components of the “intrinsic” pathway act on activated platelet surfaces to propagate large-scale thrombin generation. Thus, the PT assay tests the levels of procoagulants involved in the initiation phase of coagulation, whereas the aPTT tests the levels of procoagulants involved in platelet-surface–mediated thrombin generation during the propagation phase. Neither assay gives a complete picture of hemostatic function and includes the contributions of cellular components.
Protease Inhibitors Localize Coagulation to the Appropriate Surfaces Our discussion of the cell-based model highlights another feature of hemostasis that is not obvious in the cascade model: the importance of the coagulation protease inhibitors. The “intrinsic” and “extrinsic” pathways are needed to produce activated FX on the two different cell surfaces because the presence of the coagulation protease inhibitors antithrombin (AT) and tissue factor pathway inhibitor (TFPI) localizes FXa activity to the surface on which it is formed. AT is primarily synthesized by hepatic parenchymal cells,30 though TFPI is not.31 FXa on a surface is relatively protected from inhibition by AT and TFPI.32 However, FXa in solution is rapidly inhibited, with a half-life counted in seconds to minutes.33 Thus, FXa that leaves the cellular surface on which it was formed is likely to be inactivated before it can reach a different cell surface. In addition, AT can bind to heparan sulfates on endothelial cell surfaces.34 Plasma TFPI can bind to endothelial cells, and another form of TFPI, TFPI-β, is tethered to endothelial surfaces by a glycosyl phosphatidylinositol linkage.35 Localization to the endothelial surface allows these protease inhibitors to specifically play an antithrombotic role by inhibiting activated factors that diffuse to or are inappropriately formed on the endothelium. A deficiency of AT is associated with a significant thrombotic tendency.36 TFPI deficiency has not been recognized in humans, and the knockout is lethal during embryonic development in mice.37 Thus, coagulation inhibitors are a critical control mechanism, limiting and localizing FXa activity and, thereby, thrombin generation to a site of injury.
Protein C/S/Thrombomodulin System Protects against Thrombosis Proteins C and S are vitamin K–dependent factors synthesized by hepatic parenchymal cells.38 Protein C is the precursor of a protease, activated protein C (APC),39 whereas protein S is a nonenzymatic cofactor that enhances the activity of APC. Though APC is often called an “anticoagulant,” it does not normally act in the fluid phase and has limited ability to inactivate FVa on platelet surfaces.40 Protein C from the plasma binds to a specific endothelial protein C receptor (EPCR), which enhances its activation and localizes its antithrombotic activity to endothelial surfaces.41 Thrombomodulin (TM) is a cell surface receptor for thrombin that is present on healthy endothelial cells.42 When Seminars in Thrombosis & Hemostasis
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thrombin escapes from a site of injury onto nearby intact endothelial cells, it is bound by TM. The thrombin/TM complex can no longer carry out procoagulant functions, such as clotting fibrinogen or activating platelets.43 Instead, the thrombin/TM complex activates protein C that has been localized to the platelet surface by EPCR. The APC can then bind the cofactor protein S. The activated protein C/protein S complex cleaves and inactivates FVa on the endothelial surface.44 Because FVa is an essential cofactor for activation of prothrombin by FXa, inactivation of FVa disables thrombin production on endothelial surfaces. Thus, activated protein C/protein S does not normally “turn off” thrombin generation during hemostasis, but prevents the spread of thrombin generation from the site of injury throughout the vascular tree.45 This is an important function, as propagation of thrombin generation on healthy endothelium can lead to thrombosis. Both PC46 and PS47 deficiencies lead to a prothrombotic state. APC also has important anti-inflammatory and cytoprotective activities that are independent of its effect on the coagulation system.48 APC was tested as a potential therapy for severe sepsis.49 In this setting, in which APC was infused intravenously, it acted as a systemic anticoagulant, and bleeding was a significant side effect. It also appears that excess activation of protein C following trauma can contribute to a systemic coagulopathy.50 Thus, the protein C/S system normally exerts an antithrombotic effect on endothelial surfaces. When APC is not properly localized, but is present in the systemic circulation due to pathology or pharmacologic manipulation, it can act (inappropriately) as an anticoagulant. More recently, it has become clear that PS not only enhances the activity of APC, but also has additional protein C–independent effects. It enhances inhibition of FXa by TFPI,51,52 and also interacts directly with FVa to inhibit prothrombinase complex activity.53 These activities contribute significantly to the antithrombotic effects of PS and their roles in normal and pathologic coagulation will no doubt become more clear in the near future.
The Fibrinolytic System Removes Thrombi and Aids Healing Neither the cascade nor the cell-based model addresses the role of the fibrinolytic system. The fibrinolytic system has two critical activities that interface with the hemostatic system. First, it degrades clots that form within the intact vasculature, and thereby protects against thrombosis. Second, it removes the hemostatic clot during wound healing and tissue repair. Like the hemostatic system, the activities of the fibrinolytic system are normally localized to an appropriate surface and are not expressed systemically. This is mediated by binding of fibrinolytic components to cellular receptors and to fibrin. Proteins of the fibrinolytic system also have a variety of biological activities that are not directly linked to degradation of fibrin, which are beyond the scope of this review. Many, but not all, of the components of the fibrinolytic system are synthesized in the liver. The key fibrinolytic enzyme is plasmin. It is formed when the circulating zymogen, plasminogen, and is cleaved by one of the two types of
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plasminogen activators: tissue plasminogen activator (tPA) or urinary plasminogen activator (uPA or urokinase). Plasminogen is synthesized in the liver, but its activators are not. Endothelial cells synthesize tPA and secrete it in response to hypoxia/ischemia and thrombin. A variety of cells produce uPA. Both types of plasminogen activator are inhibited by plasminogen activator inhibitor-1 (PAI-1), which is synthesized by hepatocytes, endothelial cells, adipocytes, and others.54 Excess synthesis of PAI-1 by adipocytes is thought to contribute to the thrombotic tendency associated with obesity and the metabolic syndrome.55 Conversely, the absence of PAI-1 in humans has been associated with lifethreatening hemorrhage.56 The formation and activity of plasmin is regulated by a surface, but in this case that surface is the fibrin polymer. tPA initiates fibrinolysis by activating plasminogen while both are bound to fibrin. Thus, the formation of fibrin by the action of the coagulation system begins the process that can lead to its degradation.
How Is Hemostasis Altered in Liver Disease? Because most pro- and anticoagulant proteins are synthesized by hepatic parenchymal cells, it is easy to understand how advanced liver disease can disrupt hemostatic function. All the procoagulant factors except FVIII are reduced in hepatic insufficiency. By contrast, the level of FVIII/VWF is increased, often very dramatically, in cirrhosis.57 All of the components of the “extrinsic” pathway are produced by hepatocytes. As observed by Quick in 1935, the PT is prolonged in patients with liver disease58 and is still useful as a measure of liver synthetic function. However, not only are the procoagulant factors reduced in hepatic insufficiency, but also the level of AT and proteins C and S59,60—a situation that is not reflected in the PT or aPTT. This is made clear from experimental work showing that the level of AT61 has a large effect on the amount and pattern of thrombin generated. Specifically, the ratio of prothrombin to AT is a critical determinant of the rate and amount of thrombin generated.62 Because both prothrombin and AT synthesis are similarly reduced in liver disorders, thrombin generation is not reduced (and may even be increased) even though the PT is prolonged in these patients.63,64 Semiautomated assays of thrombin generation in platelet rich or platelet poor plasma have been gaining traction as potentially better ways of assessing the true hemostatic potential of a wide range of patients, including those with liver dysfunction.65 Though this approach holds promise, thrombin generation assays are still not well standardized,66,67 and are too cumbersome and time-consuming for routine clinical use. In general, that portion of the hemostatic process leading to thrombin generation is “rebalanced” in most patients with liver disorders so that a decreased level of procoagulants is roughly balanced by a decreased level of AT. Thus, the risk of bleeding due to impaired thrombin generation is much lower than has previously been assumed based on the PT values.68,69 Of course, the levels of coagulation proteins are
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not the only factors that play a role in hemostasis. Patients with cirrhosis also suffer from defects of platelet function and number that can contribute to a bleeding tendency.70 However, the platelet defects also seem to be balanced by the dramatic increase in the levels of FVIII/VWF commonly seen in cirrhosis, which can increase platelet adhesion and allow localization of sufficient numbers of platelets for hemostasis.71 Thus, the hemostatic balance in liver disease can be thought of not as intrinsically pro- or anticoagulant, but rather as a state in which there is a reduced ability to maintain this balance. The normal plasma levels of the coagulation factors are well above those needed for adequate function. It seems likely that this “excess” provides a margin of safety in accommodating physiologic and pathophysiologic events that consume factors or otherwise stress the system. In the absence of a significant perturbation, patients with liver failure do not necessarily have a hemorrhagic tendency.72 However, when the system is stressed, for example, by infection,73 the limited “margin of safety” makes the system fragile and more easily tipped into either a state of hemorrhage or thrombosis/disseminated intravascular coagulation (DIC). In contrast to the relative balance of the hemostatic system, a key antithrombotic mechanism is impaired in patients with liver disease, due to reduced hepatic synthesis of proteins C and S.74 A low protein C level does not generally change the results of thrombin generation assays. Rather, it allows thrombin generation to proceed on endothelial surfaces in vivo and leads to a prothrombotic tendency in liver disease. Finally, the fibrinolytic pathway can be quite deranged in patients with liver disorders. However, unlike the procoagulant, anticoagulant, and antithrombotic pathways, the degree of fibrinolytic dysfunction does not seem to be well correlated with the degree of liver dysfunction. Some, but not all, patients with liver disease have evidence of systemic activation of fibrinolysis, and those are more likely to suffer bleeding complications.75 The degree of systemic fibrinolysis has been associated with the level of circulating tPA activity76 and with the level of its inhibitor, PAI-177—neither of which is primarily produced by hepatocytes. Others have suggested that the increased fibrinolysis is secondary to ongoing activation of coagulation.78
Are There Better Laboratory Tests? It should be clear from the preceding discussion that the coagulation “cascade” model and the common clinical coagulation tests do not reflect the complexity of hemostasis/ thrombosis/fibrinolysis in vivo. These “screening” coagulation tests are sensitive to a deficiency of one or more of the soluble coagulation factors, and the PT does reflect the reduced hepatic synthetic capability in liver disorders. They do not reflect the roles played by inhibitors or fibrinolysis, and do not generally reflect the risk of bleeding in liver disorders. Modification of the PT-INR method to calibrate it specifically for liver patients79 would improve interlaboratory variability of results, but would not overcome the Seminars in Thrombosis & Hemostasis
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inherent limitations of this test. Several new tests have been proposed to better assess overall hemostatic function in liver disease. As previously noted, thrombin generation assays hold promise but have issues that currently limit their clinical utility. Viscoelastic assays (thromboelastography) would also seem to hold promise, especially for assessing the degree of fibrinolytic activation. However, they have not yet been validated for predicting bleeding or thrombosis in patients with liver disorders. This issue is discussed in a later article in this volume.
3 Patek AJ, Stetson RP. Hemophilia. I. The abnormal coagulation of
Conclusion and Summary
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The coagulation “cascade” model is essential to interpreting the results of screening coagulation tests. However, it does not accurately model how hemostasis occurs in vivo. More modern models of hemostasis incorporate the active roles of cellular participants in directing and controlling the process. Different cell types express different pro- and anticoagulant properties. They localize different plasma protein components in a manner that tends to limit the activity of the coagulation reactions to cell surfaces at a site of injury. In hepatic insufficiency, a reduction in the levels of the pro- and anticoagulant proteins produced in the liver does not impair thrombin generation until levels are quite low. However, the ability of the coagulation system to tolerate or recover from an insult is markedly impaired in liver disease allowing the system to be more easily tipped into a state favoring either hemorrhage or thrombosis. The protein C/S/TM system that normally protects against thrombosis is compromised by reduced synthesis of proteins C and S in liver failure. The risk of thrombosis is further exacerbated by the reactive increase in FVIII/VWF seen in most patients. The development of systemic fibrinolysis predisposes to coagulopathic bleeding in significant proportion patients with liver failure. It may be secondary to ongoing activation of the coagulation process and is associated with elevated levels of tPA combined with depletion of PAI-1. There are currently no readily available assays of fibrinolytic potential and activation of fibrinolysis. This is an area in which advances could be very useful in predicting and managing bleeding in the setting of liver failure. We may find encouragement in Quick’s words as he reminisced about trying to master the subject of blood coagulation in 1934: “Evening after evening I struggled to find my way through the maze of conflicting theories. To say that the current literature of that period was bewildering is to under-state the facts.” Perhaps we have made at least a little progress in the intervening 80 years.
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cell surface complex does not contribute to the basal activation of the coagulation mechanism in vivo. Blood 1992;79(8):2039–2047 Ayombil F, Abdalla S, Tracy PB, Bouchard BA. Proteolysis of plasmaderived factor V following its endocytosis by megakaryocytes forms the platelet-derived factor V/Va pool. J Thromb Haemost 2013;11(8):1532–1539 Hung DT, Vu TK, Wheaton VI, Ishii K, Coughlin SR. Cloned platelet thrombin receptor is necessary for thrombin-induced platelet activation. J Clin Invest 1992;89(4):1350–1353 Hoffman M, Monroe DM, Roberts HR. Cellular interactions in hemostasis. Haemostasis 1996;26(Suppl 1):12–16 Oliver JA, Monroe DM, Roberts HR, Hoffman M. Thrombin activates factor XI on activated platelets in the absence of factor XII. Arterioscler Thromb Vasc Biol 1999;19(1):170–177 Baglia FA, Walsh PN. Prothrombin is a cofactor for the binding of factor XI to the platelet surface and for platelet-mediated factor XI activation by thrombin. Biochemistry 1998;37(8):2271–2281 Léon M, Aiach M, Coezy E, Guennec JY, Fiessinger JN. Antithrombin III synthesis in rat liver parenchymal cells. Thromb Res 1983; 30(4):369–375 Bajaj MS, Kuppuswamy MN, Saito H, Spitzer SG, Bajaj SP. Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis. Proc Natl Acad Sci U S A 1990; 87(22):8869–8873 Franssen J, Salemink I, Willems GM, Wun TC, Hemker HC, Lindhout T. Prothrombinase is protected from inactivation by tissue factor pathway inhibitor: competition between prothrombin and inhibitor. Biochem J 1997;323(Pt 1):33–37 Lu G, Broze GJJ Jr, Krishnaswamy S. Formation of factors IXa and Xa by the extrinsic pathway: differential regulation by tissue factor pathway inhibitor and antithrombin III. J Biol Chem 2004;279(17): 17241–17249 de Agostini AI, Watkins SC, Slayter HS, Youssoufian H, Rosenberg RD. Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta. J Cell Biol 1990; 111(3):1293–1304 Zhang J, Piro O, Lu L, Broze GJ Jr. Glycosyl phosphatidylinositol anchorage of tissue factor pathway inhibitor. Circulation 2003; 108(5):623–627 Hirsh J, Piovella F, Pini M. Congenital antithrombin III deficiency. Incidence and clinical features. Am J Med 1989;87(3B):34S–38S Huang ZF, Higuchi D, Lasky N, Broze GJ Jr. Tissue factor pathway inhibitor gene disruption produces intrauterine lethality in mice. Blood 1997;90(3):944–951 Fair DS, Marlar RA. Biosynthesis and secretion of factor VII, protein C, protein S, and the Protein C inhibitor from a human hepatoma cell line. Blood 1986;67(1):64–70 Esmon CT, Stenflo J, Suttie JW. A new vitamin K-dependent protein. A phospholipid-binding zymogen of a serine esterase. J Biol Chem 1976;251(10):3052–3056 Camire RM, Kalafatis M, Simioni P, Girolami A, Tracy PB. Plateletderived factor Va/Va Leiden cofactor activities are sustained on the surface of activated platelets despite the presence of activated protein C. Blood 1998;91(8):2818–2829 Esmon CT. The endothelial protein C receptor. Curr Opin Hematol 2006;13(5):382–385 Cadroy Y, Diquélou A, Dupouy D, et al. The thrombomodulin/ protein C/protein S anticoagulant pathway modulates the thrombogenic properties of the normal resting and stimulated endothelium. Arterioscler Thromb Vasc Biol 1997;17(3):520–527 Esmon CT, Esmon NL, Harris KW. Complex formation between thrombin and thrombomodulin inhibits both thrombin-catalyzed fibrin formation and factor V activation. J Biol Chem 1982;257(14): 7944–7947
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Activated protein C cleaves factor Va more efficiently on endothelium than on platelet surfaces. Blood 2002;100(2):539–546 Allaart CF, Poort SR, Rosendaal FR, Reitsma PH, Bertina RM, Briët E. Increased risk of venous thrombosis in carriers of hereditary protein C deficiency defect. Lancet 1993;341(8838):134–138 Comp PC, Nixon RR, Cooper MR, Esmon CT. Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 1984;74(6):2082–2088 Bouwens EA, Stavenuiter F, Mosnier LO. Mechanisms of anticoagulant and cytoprotective actions of the protein C pathway. J Thromb Haemost 2013;11(Suppl 1):242–253 Bernard GR, Vincent JL, Laterre PF, et al; Recombinant human protein C Worldwide Evaluation in Severe Sepsis (PROWESS) study group. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001;344(10):699–709 Chesebro BB, Rahn P, Carles M, et al. Increase in activated protein C mediates acute traumatic coagulopathy in mice. Shock 2009; 32(6):659–665 Ndonwi M, Broze G Jr. Protein S enhances the tissue factor pathway inhibitor inhibition of factor Xa but not its inhibition of factor VIIatissue factor. J Thromb Haemost 2008;6(6):1044–1046 Hackeng TM, Rosing J. Protein S as cofactor for TFPI. Arterioscler Thromb Vasc Biol 2009;29(12):2015–2020 Wood JP, Bunce MW, Maroney SA, Tracy PB, Camire RM, Mast AE. Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation. Proc Natl Acad Sci U S A 2013;110(44):17838–17843 Loskutoff DJ, Sawdey M, Keeton M, Schneiderman J. Regulation of PAI-1 gene expression in vivo. Thromb Haemost 1993;70(1): 135–137 Palomo I, Alarcón M, Moore-Carrasco R, Argilés JM. Hemostasis alterations in metabolic syndrome [review]. Int J Mol Med 2006; 18(5):969–974 Iwaki T, Tanaka A, Miyawaki Y, et al. Life-threatening hemorrhage and prolonged wound healing are remarkable phenotypes manifested by complete plasminogen activator inhibitor-1 deficiency in humans. J Thromb Haemost 2011;9(6):1200–1206 Jennings I, Calne RY, Baglin TP. Predictive value of von Willebrand factor to ristocetin cofactor ratio and thrombin-antithrombin complex levels for hepatic vessel thrombosis and graft rejection after liver transplantation. Transplantation 1994;57(7): 1046–1051 Quick A, Stanley-Brown M, Bancroft F. A study of the coagulation defect in hemophilia and in jaundice. Am J Med Sci 1935; 190:501–511 De Caterina M, Tarantino G, Farina C, et al. Haemostasis unbalance in Pugh-scored liver cirrhosis: characteristic changes of plasma levels of protein C versus protein S. Haemostasis 1993;23(4): 229–235 Raya-Sánchez JM, González-Reimers E, Rodríguez-Martín JM, et al. Coagulation inhibitors in alcoholic liver cirrhosis. Alcohol 1998; 15(1):19–23 Butenas S, van’t Veer C, Mann KG. “Normal” thrombin generation. Blood 1999;94(7):2169–2178 Horne MK III, Merryman PK, Cullinane AM, Nghiem K, Alexander HR. The impact of major surgery on blood coagulation factors and thrombin generation. Am J Hematol 2007;82(9):815–820 Tripodi A, Chantarangkul V, Primignani M, et al. Thrombin generation in plasma from patients with cirrhosis supplemented with normal plasma: considerations on the efficacy of treatment with fresh-frozen plasma. Intern Emerg Med 2012;7(2):139–144 Gatt A, Riddell A, Calvaruso V, Tuddenham EG, Makris M, Burroughs AK. Enhanced thrombin generation in patients with cirrhosis-induced coagulopathy. J Thromb Haemost 2010;8(9):1994–2000
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thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb 2003;33(1):4–15 Dargaud Y, Luddington R, Gray E, et al. Standardisation of thrombin generation test—which reference plasma for TGT? An international multicentre study. Thromb Res 2010;125(4):353–356 Dargaud Y, Wolberg AS, Luddington R, et al. Evaluation of a standardized protocol for thrombin generation measurement using the calibrated automated thrombogram: an international multicentre study. Thromb Res 2012;130(6):929–934 Caldwell SH, Hoffman M, Lisman T, et al; Coagulation in Liver Disease Group. Coagulation disorders and hemostasis in liver disease: pathophysiology and critical assessment of current management. Hepatology 2006;44(4):1039–1046 Tripodi A, Mannucci PM. The coagulopathy of chronic liver disease. N Engl J Med 2011;365(2):147–156 Tripodi A, Primignani M, Chantarangkul V, et al. Thrombin generation in patients with cirrhosis: the role of platelets. Hepatology 2006;44(2):440–445 Lisman T, Bongers TN, Adelmeijer J, et al. Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity. Hepatology 2006;44(1):53–61 Ben-Ari Z, Osman E, Hutton RA, Burroughs AK. Disseminated intravascular coagulation in liver cirrhosis: fact or fiction? Am J Gastroenterol 1999;94(10):2977–2982
disseminated intravascular coagulation and endotoxemia in patients with liver cirrhosis. Gastroenterology 1995;109(2): 531–539 Tripodi A, Primignani M, Chantarangkul V, et al. An imbalance of pro- vs anti-coagulation factors in plasma from patients with cirrhosis. Gastroenterology 2009;137(6):2105–2111 Francis RB Jr, Feinstein DI. Clinical significance of accelerated fibrinolysis in liver disease. Haemostasis 1984;14(6): 460–465 Hersch SL, Kunelis T, Francis RB Jr. The pathogenesis of accelerated fibrinolysis in liver cirrhosis: a critical role for tissue plasminogen activator inhibitor. Blood 1987;69(5):1315–1319 Ferguson JW, Helmy A, Ludlam C, Webb DJ, Hayes PC, Newby DC. Hyperfibrinolysis in alcoholic cirrhosis: relative plasminogen activator inhibitor type 1 deficiency. Thromb Res 2008;121(5): 675–680 Violi F, Ferro D. Clotting activation and hyperfibrinolysis in cirrhosis: implication for bleeding and thrombosis. Semin Thromb Hemost 2013;39(4):426–433 Tripodi A, Chantarangkul V, Primignani M, et al. The international normalized ratio calibrated for cirrhosis (INR(liver)) normalizes prothrombin time results for model for endstage liver disease calculation. Hepatology 2007;46(2): 520–527
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Coagulation in Liver Disease Maureane Hoffman, MD, PhD1,2 1 Pathology and Laboratory Medicine Service, Durham Veterans Affairs
Medical Center, Durham, North Carolina 2 Department of Pathology, Duke University Medical Center, Durham, North Carolina
Address for correspondence Maureane Hoffman, MD, PhD, Pathology and Laboratory Medicine Service (113), Durham Veterans Affairs Medical Center, 508 Fulton St, Durham, NC 27705 (e-mail: [email protected]).
Abstract
Keywords
► ► ► ►
cirrhosis thrombin generation prothrombin time fibrinolysis
The liver plays a key role in hemostasis as the site of synthesis of many of the proteins involved in the coagulation, antithrombotic and fibrinolytic systems that interact to both establish hemostasis, and preventing thrombosis. The common laboratory tests, prothrombin time (PT) and activated partial thromboplastin time (aPTT), evolved from studies of plasma clotting in test tubes. Such studies laid the basis for the coagulation cascade model of hemostasis. However, thought has evolved to place a greater emphasis on the active roles of cells in localizing and regulating hemostasis. The PT and aPTT do not reflect the roles of cellular elements in hemostasis, nor do they reflect the crucial roles of antithrombotic and fibrinolytic systems. Thus, though the PT may indeed reflect the synthetic capacity of the liver, it does not accurately reflect the risk of bleeding or thrombosis in patients with liver failure.
Hemostasis is profoundly altered by liver disorders. Most proand anticoagulant factors, as well as the pro- and antifibrinolytic factors, are synthesized by hepatocytes. Most factors are also cleared by the liver. In addition, reduced hepatic production of thrombopoietin and hypersplenism due to portal hypertension can lead to decreased platelet counts. Though the field is making progress in understanding the nature of the changes, we do not have good clinical laboratory tests to assess the ability of the coagulation system to maintain hemostasis and prevent thrombosis in patients with liver dysfunction. The goals of this review are (1) to discuss the functioning of the hemostatic system in the body, and how it is altered in the setting of hepatic dysfunction; (2) highlight the limitations of the laboratory tests available to assess hemostatic function.
Mechanisms of Hemostasis Coagulation Testing and the Coagulation Cascade In the 1930s two groups developed the first versions of the prothrombin time (PT) test. The PT was developed as an assay for the level of prothrombin based on the prevailing model of coagulation at that time. This was
published online June 6, 2015
Issue Theme Hemostatic Dysfunction in Liver Diseases; Guest Editors: Ton Lisman, PhD, and Hau C. Kwaan, MD, FRCP.
Thromboplastin þ Prothrombin þ Calcium ¼ Thrombin The idea was that if a constant amount of calcium and an excess of thromboplastin were added to plasma, the only variable would be the level of prothrombin. Thus, the rate of thrombin production under these conditions would be a function of the prothrombin level. It is notable that Quick describes the development of the PT test,1 stating that he first observed a delayed clotting time on plasma from a jaundiced subject. Of course the bleeding tendency in patients with liver disorders was well recognized at that time. He found the clotting time to be normal in plasma from hemophiliacs, but prolonged in samples from most jaundiced patients. On this basis he suggested the hypothesis in 1935 that “the hemorrhagic tendency in jaundice is brought about by a diminution of prothrombin.” The group led by Brinkhous nearly simultaneously developed a two-stage version of the PT test, and similarly concluded that the hemostatic defect in acute liver injury was due to prothrombin deficiency.2 Their conclusions and the pleasingly simple model of hemostasis on which they were based fell by the wayside as more plasma coagulation factors were discovered: FVIII in 1936,3 FV in 1947,4 FVII in 1951,5 FIX in 1952,6,7 and FXI in 1953.8
Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.
DOI http://dx.doi.org/ 10.1055/s-0035-1550435. ISSN 0094-6176.
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The forerunner of the modern partial thromboplastin time (PTT) was developed in the 1950s as a means to assay the deficient factor in hemophilia.9 This additional test was necessary because the Quick PT was not abnormal in hemophilia. In the PT test a “thromboplastin” reagent and calcium are added to citrated platelet poor plasma. The time required for the sample to clot is then measured. It had been discovered years earlier that, while tissue thromboplastins clotted hemophilic plasma rapidly, diluted thromboplastin could give longer clotting times with hemophilic than normal plasma. In 1953 Langdell et al developed this concept into a simple quantitative assay, which could be used to make the diagnosis of hemophilia and monitor therapy.9 This PTT was later improved by using an activator (kaolin) to speed up the clotting process and give more reproducible results. This innovation was termed the activated partial thromboplastin time (aPTT).10 Both the PT and PTT were developed before the “cascade” model of coagulation was proposed, and results obtained using these assays contributed to the development of this model. The “cascade” model was proposed nearly simultaneously in two publications in 1964.11,12 This model views coagulation as a sequential series of proteolytic steps in which activation of one clotting factor leads to the activation of another, ultimately resulting in the activation of enough thrombin to convert soluble fibrinogen to an insoluble fibrin clot. It was a significant conceptual advance to recognize that a series of proteases could act as a biological amplifier. The original models evolved into the now-familiar Y-shaped scheme that we call the “coagulation cascade” today. The “intrinsic” and “extrinsic” pathways are initiated by factor XII (FXII) and FVIIa/tissue factor (TF), respectively, and converge on a “common” pathway at the level of the FXa/FVa (prothrombinase) complex. This “cascade” was not proposed as a literal model of hemostasis in vivo, but primarily as a scheme of how the many coagulation factors interact biochemically. To facilitate biochemical studies, platelets were replaced by phospholipid vesicles.13 In addition, the anticoagulant pathways that are critical to localization and control of thrombin formation in vivo are not addressed by the cascade model. Because the cascade model was based on the same types of studies as the PT and aPTT, it should not be surprising that the cascade model is an excellent tool for interpreting results of the PT and aPTT tests. On the other hand, the limitations of the cascade model are shared by these related laboratory assays. A major limitation of the cascade model is that clotting times in the PT and aPTT do not correlate well with the risk of bleeding in vivo. For example, even though deficiencies of each of the factors in the intrinsic pathway can have equally long aPTT values, they have dramatically different risks of hemorrhage. Deficiencies of factor XII are not associated with significant hemorrhage and deficiencies of factor XI might or might not be associated with hemorrhage, but deficiencies of factors VIII and IX (hemophilia A and B) are consistently associated with hemorrhage. A particularly important limitation of the cascade model is that it cannot explain why a normally functioning “extrinsic” pathway cannot compensate Seminars in Thrombosis & Hemostasis
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for a defect in the “intrinsic” pathway, as in hemophilia. Thus, the intrinsic and extrinsic systems cannot operate as independent and redundant pathways as implied by this model.
A Cell-Based Model of Hemostasis In the time since it was first proposed, many have recognized that the “cascade” has serious failings as a model of normal and abnormal coagulation. It was also recognized from the earliest studies of coagulation that cells are important participants in the process14,15 and that normal hemostasis is not possible in the absence of cell-associated TF and platelets. Substituting phospholipid vesicles in the place of cells in coagulation tests fails to capture the active participation of cells in hemostasis in vivo. A cell-based model of coagulation, in which hemostasis occurs in a step-wise process on cell surfaces, incorporates the important roles of cells and anticoagulants that the cascade model does not.16 A summary of the cell-based model of hemostasis is shown in ►Fig. 1.
Step 1: Initiation of Coagulation on TF-Bearing Cells When a vessel is disrupted, platelets escape from the vessel, bind to collagen and other extracellular matrix components at the site of injury, and are partially activated. This process forms a platelet plug that provides primary hemostasis. However, long-term hemostasis requires that the platelet plug be stabilized by a meshwork of fibrin formed by the action of thrombin. The process of thrombin generation is initiated when TFbearing cells are exposed to blood at a site of injury. TF is a transmembrane protein that acts as a receptor and cofactor for FVII. It is normally expressed only on cells outside the vasculature. TF is highly expressed by pericytes surrounding arterioles and venules, and by adventitial cells around larger vessels.17,18 This distribution has been referred to as a “hemostatic envelope” to stop bleeding in the event of vascular injury. Once bound to TF, zymogen FVII is rapidly activated to FVIIa.19 At sites around vessels a significant proportion of the TF has bound FVII/FVIIa even in the absence of any injury.20 The FVIIa/TF complex catalyzes activation of both FX and FIX.21 The FXa formed on the TF-bearing cell interacts with its cofactor FVa to generate a small amount of thrombin on the TF cells.22 Most coagulation factors can leave the vasculature, percolate through the extravascular space, and collect in the lymph.23 Therefore, it is likely that low levels of FIXa, FXa, and thrombin are produced on TF-bearing cells at all times.24 However, these activated factors are normally separated from other components of the coagulation system by the vessel wall. Platelets and FVIII bound to von Willebrand factor (VWF) are so large that they only enter the extravascular compartment when an injury disrupts the vessel wall. Activated platelets release a platelet-specific form of FV that is partially activated,25 and can immediately form prothrombinase complexes with FXa that has been activated on the TF-bearing cell. At that point the small amounts of thrombin produced on TF-bearing cells can interact with platelets and FVIII/VWF to initiate the hemostatic process that ultimately enmeshes the initial platelet plug in a stable fibrin clot (secondary hemostasis).
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Fig. 1 The phases in a cell-based model of coagulation. Initiation occurs on the TF-bearing cell as FX activated by FVIIa/TF combines with its cofactor, FVa, to activate small amounts of thrombin. This small amount of thrombin is important during the amplification phase, as it participates multiple positive feedback loops by activating cofactors, FXI and platelets. The propagation phase takes place on the platelet surface and is responsible for producing the large burst of thrombin that is required for effective hemostasis. The prothrombin time (PT) test assesses the levels of procoagulants involved in Initiation of coagulation. The activated partial thromboplastin time (aPTT) assay assesses the levels of the procoagulants involved in platelet surface thrombin generation in the propagation phase. vWF, von Willebrand factor.
Step 2: Amplification of the Procoagulant Signal by Thrombin Generated on the TF-Bearing Cell
Step 3: Propagation of Thrombin Generation on the Platelet Surface
During the amplification step, the small amounts of thrombin formed on TF-bearing cells promote maximal platelet activation26 and also activate additional coagulation cofactors on the platelet surface. Although this small amount of thrombin may not be sufficient to produce a stable fibrin clot, it plays a key role by “priming” the clotting system for a subsequent burst of platelet surface thrombin generation by activating FV, FVIII, and FXI on the platelet surface.27–29 Thrombin activation of FXI on platelet surfaces explains why FXII is not needed for normal hemostasis. FIXa activated both on the TF-bearing cell and by platelet-surface FXIa binds to FVIIIa on the platelet surface to assemble FIXa/ FVIIIa (“tenase”) complexes.
The burst of thrombin generation needed for effective hemostasis is produced on platelet surfaces during the propagation phase of coagulation. Once the platelet “tenase” complex is assembled, FX from the plasma is activated to FXa on the platelet surface. FXa then associates with FVa to form “prothrombinase” complexes and produce a burst of thrombin generation. Even though the cell-based model depicts the hemostatic process as occurring in discrete steps, these should be viewed as an overlapping continuum of events. For example, thrombin produced on the platelet surface early in the propagation phase may initially cleave substrates on the platelet surface and continue to amplify the procoagulant response, in addition to leaving the platelet to promote fibrin assembly.
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The cell-based model of coagulation shows us that the “extrinsic” and “intrinsic” pathways are not redundant. The TF (“extrinsic”) pathway operates on TF-bearing cells to initiate and amplify coagulation. By contrast, components of the “intrinsic” pathway act on activated platelet surfaces to propagate large-scale thrombin generation. Thus, the PT assay tests the levels of procoagulants involved in the initiation phase of coagulation, whereas the aPTT tests the levels of procoagulants involved in platelet-surface–mediated thrombin generation during the propagation phase. Neither assay gives a complete picture of hemostatic function and includes the contributions of cellular components.
Protease Inhibitors Localize Coagulation to the Appropriate Surfaces Our discussion of the cell-based model highlights another feature of hemostasis that is not obvious in the cascade model: the importance of the coagulation protease inhibitors. The “intrinsic” and “extrinsic” pathways are needed to produce activated FX on the two different cell surfaces because the presence of the coagulation protease inhibitors antithrombin (AT) and tissue factor pathway inhibitor (TFPI) localizes FXa activity to the surface on which it is formed. AT is primarily synthesized by hepatic parenchymal cells,30 though TFPI is not.31 FXa on a surface is relatively protected from inhibition by AT and TFPI.32 However, FXa in solution is rapidly inhibited, with a half-life counted in seconds to minutes.33 Thus, FXa that leaves the cellular surface on which it was formed is likely to be inactivated before it can reach a different cell surface. In addition, AT can bind to heparan sulfates on endothelial cell surfaces.34 Plasma TFPI can bind to endothelial cells, and another form of TFPI, TFPI-β, is tethered to endothelial surfaces by a glycosyl phosphatidylinositol linkage.35 Localization to the endothelial surface allows these protease inhibitors to specifically play an antithrombotic role by inhibiting activated factors that diffuse to or are inappropriately formed on the endothelium. A deficiency of AT is associated with a significant thrombotic tendency.36 TFPI deficiency has not been recognized in humans, and the knockout is lethal during embryonic development in mice.37 Thus, coagulation inhibitors are a critical control mechanism, limiting and localizing FXa activity and, thereby, thrombin generation to a site of injury.
Protein C/S/Thrombomodulin System Protects against Thrombosis Proteins C and S are vitamin K–dependent factors synthesized by hepatic parenchymal cells.38 Protein C is the precursor of a protease, activated protein C (APC),39 whereas protein S is a nonenzymatic cofactor that enhances the activity of APC. Though APC is often called an “anticoagulant,” it does not normally act in the fluid phase and has limited ability to inactivate FVa on platelet surfaces.40 Protein C from the plasma binds to a specific endothelial protein C receptor (EPCR), which enhances its activation and localizes its antithrombotic activity to endothelial surfaces.41 Thrombomodulin (TM) is a cell surface receptor for thrombin that is present on healthy endothelial cells.42 When Seminars in Thrombosis & Hemostasis
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thrombin escapes from a site of injury onto nearby intact endothelial cells, it is bound by TM. The thrombin/TM complex can no longer carry out procoagulant functions, such as clotting fibrinogen or activating platelets.43 Instead, the thrombin/TM complex activates protein C that has been localized to the platelet surface by EPCR. The APC can then bind the cofactor protein S. The activated protein C/protein S complex cleaves and inactivates FVa on the endothelial surface.44 Because FVa is an essential cofactor for activation of prothrombin by FXa, inactivation of FVa disables thrombin production on endothelial surfaces. Thus, activated protein C/protein S does not normally “turn off” thrombin generation during hemostasis, but prevents the spread of thrombin generation from the site of injury throughout the vascular tree.45 This is an important function, as propagation of thrombin generation on healthy endothelium can lead to thrombosis. Both PC46 and PS47 deficiencies lead to a prothrombotic state. APC also has important anti-inflammatory and cytoprotective activities that are independent of its effect on the coagulation system.48 APC was tested as a potential therapy for severe sepsis.49 In this setting, in which APC was infused intravenously, it acted as a systemic anticoagulant, and bleeding was a significant side effect. It also appears that excess activation of protein C following trauma can contribute to a systemic coagulopathy.50 Thus, the protein C/S system normally exerts an antithrombotic effect on endothelial surfaces. When APC is not properly localized, but is present in the systemic circulation due to pathology or pharmacologic manipulation, it can act (inappropriately) as an anticoagulant. More recently, it has become clear that PS not only enhances the activity of APC, but also has additional protein C–independent effects. It enhances inhibition of FXa by TFPI,51,52 and also interacts directly with FVa to inhibit prothrombinase complex activity.53 These activities contribute significantly to the antithrombotic effects of PS and their roles in normal and pathologic coagulation will no doubt become more clear in the near future.
The Fibrinolytic System Removes Thrombi and Aids Healing Neither the cascade nor the cell-based model addresses the role of the fibrinolytic system. The fibrinolytic system has two critical activities that interface with the hemostatic system. First, it degrades clots that form within the intact vasculature, and thereby protects against thrombosis. Second, it removes the hemostatic clot during wound healing and tissue repair. Like the hemostatic system, the activities of the fibrinolytic system are normally localized to an appropriate surface and are not expressed systemically. This is mediated by binding of fibrinolytic components to cellular receptors and to fibrin. Proteins of the fibrinolytic system also have a variety of biological activities that are not directly linked to degradation of fibrin, which are beyond the scope of this review. Many, but not all, of the components of the fibrinolytic system are synthesized in the liver. The key fibrinolytic enzyme is plasmin. It is formed when the circulating zymogen, plasminogen, and is cleaved by one of the two types of
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plasminogen activators: tissue plasminogen activator (tPA) or urinary plasminogen activator (uPA or urokinase). Plasminogen is synthesized in the liver, but its activators are not. Endothelial cells synthesize tPA and secrete it in response to hypoxia/ischemia and thrombin. A variety of cells produce uPA. Both types of plasminogen activator are inhibited by plasminogen activator inhibitor-1 (PAI-1), which is synthesized by hepatocytes, endothelial cells, adipocytes, and others.54 Excess synthesis of PAI-1 by adipocytes is thought to contribute to the thrombotic tendency associated with obesity and the metabolic syndrome.55 Conversely, the absence of PAI-1 in humans has been associated with lifethreatening hemorrhage.56 The formation and activity of plasmin is regulated by a surface, but in this case that surface is the fibrin polymer. tPA initiates fibrinolysis by activating plasminogen while both are bound to fibrin. Thus, the formation of fibrin by the action of the coagulation system begins the process that can lead to its degradation.
How Is Hemostasis Altered in Liver Disease? Because most pro- and anticoagulant proteins are synthesized by hepatic parenchymal cells, it is easy to understand how advanced liver disease can disrupt hemostatic function. All the procoagulant factors except FVIII are reduced in hepatic insufficiency. By contrast, the level of FVIII/VWF is increased, often very dramatically, in cirrhosis.57 All of the components of the “extrinsic” pathway are produced by hepatocytes. As observed by Quick in 1935, the PT is prolonged in patients with liver disease58 and is still useful as a measure of liver synthetic function. However, not only are the procoagulant factors reduced in hepatic insufficiency, but also the level of AT and proteins C and S59,60—a situation that is not reflected in the PT or aPTT. This is made clear from experimental work showing that the level of AT61 has a large effect on the amount and pattern of thrombin generated. Specifically, the ratio of prothrombin to AT is a critical determinant of the rate and amount of thrombin generated.62 Because both prothrombin and AT synthesis are similarly reduced in liver disorders, thrombin generation is not reduced (and may even be increased) even though the PT is prolonged in these patients.63,64 Semiautomated assays of thrombin generation in platelet rich or platelet poor plasma have been gaining traction as potentially better ways of assessing the true hemostatic potential of a wide range of patients, including those with liver dysfunction.65 Though this approach holds promise, thrombin generation assays are still not well standardized,66,67 and are too cumbersome and time-consuming for routine clinical use. In general, that portion of the hemostatic process leading to thrombin generation is “rebalanced” in most patients with liver disorders so that a decreased level of procoagulants is roughly balanced by a decreased level of AT. Thus, the risk of bleeding due to impaired thrombin generation is much lower than has previously been assumed based on the PT values.68,69 Of course, the levels of coagulation proteins are
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not the only factors that play a role in hemostasis. Patients with cirrhosis also suffer from defects of platelet function and number that can contribute to a bleeding tendency.70 However, the platelet defects also seem to be balanced by the dramatic increase in the levels of FVIII/VWF commonly seen in cirrhosis, which can increase platelet adhesion and allow localization of sufficient numbers of platelets for hemostasis.71 Thus, the hemostatic balance in liver disease can be thought of not as intrinsically pro- or anticoagulant, but rather as a state in which there is a reduced ability to maintain this balance. The normal plasma levels of the coagulation factors are well above those needed for adequate function. It seems likely that this “excess” provides a margin of safety in accommodating physiologic and pathophysiologic events that consume factors or otherwise stress the system. In the absence of a significant perturbation, patients with liver failure do not necessarily have a hemorrhagic tendency.72 However, when the system is stressed, for example, by infection,73 the limited “margin of safety” makes the system fragile and more easily tipped into either a state of hemorrhage or thrombosis/disseminated intravascular coagulation (DIC). In contrast to the relative balance of the hemostatic system, a key antithrombotic mechanism is impaired in patients with liver disease, due to reduced hepatic synthesis of proteins C and S.74 A low protein C level does not generally change the results of thrombin generation assays. Rather, it allows thrombin generation to proceed on endothelial surfaces in vivo and leads to a prothrombotic tendency in liver disease. Finally, the fibrinolytic pathway can be quite deranged in patients with liver disorders. However, unlike the procoagulant, anticoagulant, and antithrombotic pathways, the degree of fibrinolytic dysfunction does not seem to be well correlated with the degree of liver dysfunction. Some, but not all, patients with liver disease have evidence of systemic activation of fibrinolysis, and those are more likely to suffer bleeding complications.75 The degree of systemic fibrinolysis has been associated with the level of circulating tPA activity76 and with the level of its inhibitor, PAI-177—neither of which is primarily produced by hepatocytes. Others have suggested that the increased fibrinolysis is secondary to ongoing activation of coagulation.78
Are There Better Laboratory Tests? It should be clear from the preceding discussion that the coagulation “cascade” model and the common clinical coagulation tests do not reflect the complexity of hemostasis/ thrombosis/fibrinolysis in vivo. These “screening” coagulation tests are sensitive to a deficiency of one or more of the soluble coagulation factors, and the PT does reflect the reduced hepatic synthetic capability in liver disorders. They do not reflect the roles played by inhibitors or fibrinolysis, and do not generally reflect the risk of bleeding in liver disorders. Modification of the PT-INR method to calibrate it specifically for liver patients79 would improve interlaboratory variability of results, but would not overcome the Seminars in Thrombosis & Hemostasis
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inherent limitations of this test. Several new tests have been proposed to better assess overall hemostatic function in liver disease. As previously noted, thrombin generation assays hold promise but have issues that currently limit their clinical utility. Viscoelastic assays (thromboelastography) would also seem to hold promise, especially for assessing the degree of fibrinolytic activation. However, they have not yet been validated for predicting bleeding or thrombosis in patients with liver disorders. This issue is discussed in a later article in this volume.
3 Patek AJ, Stetson RP. Hemophilia. I. The abnormal coagulation of
Conclusion and Summary
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The coagulation “cascade” model is essential to interpreting the results of screening coagulation tests. However, it does not accurately model how hemostasis occurs in vivo. More modern models of hemostasis incorporate the active roles of cellular participants in directing and controlling the process. Different cell types express different pro- and anticoagulant properties. They localize different plasma protein components in a manner that tends to limit the activity of the coagulation reactions to cell surfaces at a site of injury. In hepatic insufficiency, a reduction in the levels of the pro- and anticoagulant proteins produced in the liver does not impair thrombin generation until levels are quite low. However, the ability of the coagulation system to tolerate or recover from an insult is markedly impaired in liver disease allowing the system to be more easily tipped into a state favoring either hemorrhage or thrombosis. The protein C/S/TM system that normally protects against thrombosis is compromised by reduced synthesis of proteins C and S in liver failure. The risk of thrombosis is further exacerbated by the reactive increase in FVIII/VWF seen in most patients. The development of systemic fibrinolysis predisposes to coagulopathic bleeding in significant proportion patients with liver failure. It may be secondary to ongoing activation of the coagulation process and is associated with elevated levels of tPA combined with depletion of PAI-1. There are currently no readily available assays of fibrinolytic potential and activation of fibrinolysis. This is an area in which advances could be very useful in predicting and managing bleeding in the setting of liver failure. We may find encouragement in Quick’s words as he reminisced about trying to master the subject of blood coagulation in 1934: “Evening after evening I struggled to find my way through the maze of conflicting theories. To say that the current literature of that period was bewildering is to under-state the facts.” Perhaps we have made at least a little progress in the intervening 80 years.
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