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Endogenous Anticoagulants
Topics in Compan An Med 27 (2012) 81-87
Topical Review
Endogenous Anticoagulants Amy Kubier, DVM, DACVIM, and Mauria O’Brien, DVM, DACVECC
A B S T R A C T Keywords: antithrombin coagulation protein C protein S tissue factor pathway inhibitor
Blood coagulation is a complex and highly coordinated process that is constantly altered and impacted by procoagulant and anticoagulant “players.” It is vital that these components work in concert to maintain a balance to keep coagulation in check. Several important endogenous anticoagulants will be discussed in this review including tissue factor pathway inhibitor, antithrombin, protein C, and protein S in origin, structure, mechanism of action, effects of deficiency, and current knowledge in veterinary medicine.
University of Illinois Urbana-Champaign, Champaign, IL, USA Address reprint requests to: Amy Kubier, DVM, DACVIM, 1008 W Hazelwood Dr, Urbana, IL 61802.
䉷 2012 Published by Elsevier Inc.
E-mail: [email protected]
Blood coagulation is a complex, highly coordinated process that is continuously modulated by procoagulant and anticoagulant “players.” It is vital that these components work in concert to maintain a balance to keep coagulation in check. In certain disease states, particularly those with a massive inflammatory response, the scales are tipped toward a procoagulant state and the production of anticoagulant mediators is downregulated. The goal of this review is to discuss several important endogenous anticoagulants including tissue factor pathway inhibitor (TFPI), antithrombin (AT), protein C (PC), and protein S (PS) in origin, structure, mechanism of action, effects of deficiency, and current knowledge and applicability in veterinary medicine. Tissue Factor Pathway Inhibitor Background Tissue factor (TF) is a transmembrane protein that acts as a receptor for plasma factor VII (FVII) and its activated form, FVIIa. Perivascular cells constitutively express TF, underlying the importance of its location to provide rapid activation of coagulation after vascular injury. TF is also constitutively expressed in the heart, kidney, brain, lungs, and placenta; these tissue-specific locations impart hemostatic protection in these highly vascular and vital organs.1 When a vascular injury occurs, the adventitial cells expressing TF are exposed, allowing circulating FVIIa to bind to the uncovered TF. The TF-FVIIa complex then activates, through a positive feedback mechanism, FVII to FVIIa. Additionally, the TF-FVIIa complex activates small amounts of FIX and FX. This is the initiation step in the cell-based model of coagulation and acts as the primary initiator of coagulation in vivo.2 More information regarding the cell-based model of coagulation can be found in the article in this issue. TFPI has a dual inhibitory function: it is the primary inhibitor of the TF-VIIa complex as well as an inhibitor of FXa.1,2 Coagulation must be initiated for TFPI to function.3
proximately 10% of TFPI is bound to plasma lipoproteins1,5,6 and a very small proportion circulates freely.7 Activated platelets and heparin lead to the release of TFPI from intracellular stores.8 Estimations of endogenous TFPI levels have been based on the amount of TFPI released after heparin injection.7 Structure and Mechanism of Action TFPI is a Kunitz-type protease inhibitor containing Kunitz domains (K1-3) that inhibit the function of protein-degrading enzymes. In a 2-stage process, the second Kunitz domain, K2, binds to and directly inhibits FXa. The first domain, K1, then binds to a TF-FVIIa complex and inhibits it and prevents any further activation of FX. The formation of this quaternary structure is essential to the inhibitory actions of TFPI on TF-FVIIa. Although K3 is involved in lipoprotein binding and contains a heparin-binding site, it does not seem to function as a protease inhibitor1,6 but is essential for optimal FXa inhibition. The specific role of K3 in the inhibition of coagulation appears to be through its relationship with protein S4 (Fig. 1). The binding of TFPI to FXa is reversible and independent of calcium, whereas the binding of the TFPI-FXa complex to TF-VIIa is irreversible and requires calcium. Inactivation of TF-FVIIa by TFPI is both calcium- and FXa-dependent.9 It should be noted that TFPI does not stop coagulation, but it limits further generation of FIXa and FXa by the TF-FVIIa complex.10 TFPI can also stimulate monocytes to internalize and degrade TF-FVIIa complexes expressed on the cell surface.11 As stated earlier, heparin, both unfractionated and low molecular weight,12 increases the levels of TFPI by inducing synthesis and causing secretion of TFPI by endothelial cells as well as displacing the bound portion11,13 The inhibitory effects of TFPI are significantly enhanced in the presence of heparin.14 As heparin is cleared from circulation, the effects of the heparin on TFPI cease and TFPI becomes endothelial bound again.15
Location TFPI is primarily produced and expressed on the luminal surface of endothelial cells,1,4 although megakaryocytes, monocytes, lung fibroblasts, and synovial cells are able to express low levels of TFPI. Ap-
Measurement of TFPI TFPI can be measured by commercially available enzyme-linked immunosorbent assays (ELISA) and functional endpoint assays. The
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Figure 1. The functions of TFPI. The TF-VIIa complex is able to activate FIX to FIXa and stimulate the intrinsic pathway as well as activate FX to Xa (top center). TFPI binds to the tertiary complex of TF-VIIa-Xa, which is produced during the activation of Xa (center left). This binding forms the quaternary complex, with K1 binding to and inactivating VIIa and K2 binding to and inactivating Xa (bottom). TFPI is also able to bind via K2 to Xa and PS via K3, forming an inhibitory complex (center right). TFPI, Tissue factor pathway inhibitor; TF, tissue factor; K1, K2, K3, Kunitz domains 1, 2, and 3. (Adapted from Broze GJ, Jr, Girard TJ. Tissue factor pathway inhibitor: structure-function. Front Biosci 17:262280, 2012.)
total TFPI ELISA measures binary and quaternary TFPI-Xa complexes, intact and truncated forms of TFPI as well as complexes of TFPI with TF and FVIIa. The activity assay uses thawed anticoagulated whole blood in which the cells are lysed and cellular debris removed. The supernatant is then incubated with TF-FVIIa and FX. Residual TF-VIIa activity is based on measuring FXa activity and TFPI is measured from this with a standard curve.1 Interpretation of results is method specific, and levels cannot be compared between different types of tests.16 TFPI in Disease There are no known deficiencies of TFPI, suggesting it is essential for life.12 Increased levels of TFPI have been observed in certain cancers, sepsis, uremia, unregulated diabetes, and in patients with hyperlipidemia.17-21 Depending on the disease process, the measurement of TFPI may be used as a biomarker and the trends of increase or decline used as a prognostic indicator.20 In septic humans and those with disseminated intravascular coagulation (DIC), an increase in mortality is noted if the production of TF is not adequately balanced by the production of TFPI.22 Several types of human neoplastic cells are known to alter the expression of TFPI,23-25 contributing to a poor prognosis. A recent study determined that in certain human breast cancer models, TFPI was downregulated.26 Also noted was that solid tumors, versus hematological cancers, trend toward higher levels of TFPI. This has been speculated to be potentially protective against microthrombosis and organ failure in certain cancers.20 There are limitations when evaluating studies on the role of TFPI in disease states because there is a wide range of TFPI among normal individuals, and many of the studies on TFPI and risk of thrombosis are retrospective or case-controlled and do not address whether the low levels of TFPI are the cause or the effect. There remains an insufficient number of prospective studies evaluating the role of TFPI in the development of venous thromboembolism.1
Therapeutic Applications of TFPI Given the dominant role of TF in certain disease states it would seem intuitive that TFPI could be used therapeutically as an anticoagulant as well as an anti-inflammatory agent. People with low circulating levels of TFPI have been shown to be at increased risk for venous thromboembolism.27 A study determined that TFPI was increased after oral supplementation with a fatty acid in people with chronic atherosclerotic disease, a disease characterized by high levels of TF. This increase in TFPI could promote its use as a mild antithrombotic agent to help to dampen the TF pathway.28 Recombinant TFPI (rTFPI) has been examined for its role as an antithrombotic agent, and it was determined that local administration (injections of rTFPI near a created wound) prevented thrombosis and was equivocal to heparin and dextran.29 Several studies have evaluated recombinant TFPI supplementation in animal models.30,31 Human studies focus importance on the levels of TFPI secondary to heparin therapy and use those levels as a marker for postoperative bleeding.32,33 The use of rTFPI showed great promise in animal models of sepsis and DIC. A study evaluating intravenous rTFPI determined that the rTFPI prevented thrombosis and progression into DIC in a rabbit model.34 Administration of rTFPI reduced mortality in baboons and rabbits with Escherichia coli–induced septic shock.35,36 Unlike animal studies using rTFPI, the use of TFPI in humans currently shows no benefit as a therapeutic agent in sepsis. Initially phase I and II studies were promising37 and rTFPI was safe in humans, but when taken to a phase III level, there was no survival benefit to its use in sepsis.38 Antithrombin Background AT, previously termed antithrombin III, is a plasma-derived glycoprotein. AT is a serpin (serine protease inhibitor) and shares approximately 30% homology in amino acid sequence with other serine pro-
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Figure 2. Antithrombin’s actions with unfractionated heparins and low-molecular-weight heparins. When antithrombin binds to UFH at a specific pentasaccharide sequence, it causes a conformation change in AT at its reactive site. This change accelerates AT interaction with FXa. Both heparin forms are able to interact with and inactivate FXa, but only the long chains (at least 18 saccharide units) of UFH are capable of forming a heparin-AT-thrombin complex, making UFH a stronger inhibitor of thrombin than LMWH. UFH, Unfractionated heparins; LMWH, low-molecular-weight heparins; AT, antithrombin; FXa, activated factor X. (Adapted from Weitz JI. Low-molecular-weight heparins. N Engl J Med 337:688-698, 1997.)
tease inhibitors.39 AT’s activity is 2-fold in that it is known to act as a potent endogenous anticoagulant, but newer studies have also shown that AT also has powerful anti-inflammatory properties.40-42 Structure AT is produced by the liver and consists of 432 amino acids and contains 3 disulfide bonds and 4 potential glycosylation sites.43 Plasma AT exists as 2 isoforms, ␣ and . The -isoform lacks 1 of 4 carbohydrate side-chains that are present on the ␣-isoform. The -isoform constitutes about 5% to 10% of AT in plasma and has a higher affinity for heparin and heparan sulfate glycosaminoglycans (HSG) than does the ␣-isoform. The -isoform of AT appears to be concentrated in the vessel wall, whereas the ␣-isoform exists predominately in plasma.44 Mechanism of Action AT influences the activity of many coagulation factors. In addition to the inhibition of thrombin (FIIa), AT also inhibits FIXa, FXa, FXIa, and FXIIa. In the presence of heparin, FVIIa is also inhibited.40,45-47 Endogenous heparins are termed heparans. Heparans are glycosaminoglycans expressed on endothelial cell surfaces. Heparans bind to and activate AT via allosteric activation. Synthetic heparins and heparan sulfates possess repeating sequences of a specific pentasaccharide sequence that binds to AT with high affinity.48 AT binds to its target and irreversibly inhibits the protease, and the reticuloendothelial system of the liver then removes the complex.46,49 Heparin is an important but not essential cofactor for AT. Without heparin or HSG, AT inhibits coagulation very slowly and methodically.40 In the presence of heparin/HSG, AT’s activity is increased 1000-fold.49 Heparin/HSG binds to AT in a distinct manner. A conformational change in AT is induced when heparin binds to AT by a
specific pentasaccharide sequence. Heparin must specifically attach to thrombin while simultaneously bind to AT for the inactivation of thrombin. This is possible with unfractionated heparins (UFH), which have complex, long-chain structures with multiple pentasaccharide sequences. This expanded binding capacity is not needed for AT to inactivate other serine proteases such as FXa. This explains why lowmolecular-weight heparins (LMWHs) fail to directly inhibit thrombin yet retain their intrinsic anticoagulant activity against other coagulation factors50,51 (Fig. 2). LMWHs are small, with an average weight of 5000 Da, whereas UFH weigh up to 30,000 Da. LMWHs and UFH both exert their anticoagulant activity by activating AT. The activation of AT is mediated by binding through a unique pentasaccharide sequence that is present on one third of the chains of all UFH, but only 15% to 25% of LMWH chains.52 LMWHs produce a more predictable anticoagulant response than UFH.53 The plasma half-life of LMWH is 2 to 4 times longer than UFH.52 Some of this behavior is attributable to a decreased likelihood of LMWHs to bind to plasma proteins, endothelial cells, and macrophages like UFH do.54 The inhibition of FXa by LMWH persists longer than their inhibitory activity against thrombin.55 LMWHs cause less bleeding than UFH and with its more predictable behavior does not require laboratory monitoring.56 In vivo and in vitro studies have demonstrated that AT has potent anti-inflammatory effects.42,57-59 It is well known that thrombin is a central procoagulant factor with many inflammatory side effects.60 By inhibiting thrombin, the pro-inflammatory response is attenuated. AT is an inhibitor of the protease FXa, which has several pro-inflammatory effects including stimulation and production of pro-inflammatory cytokines (IL-8, IL-6), monocyte chemotactic protein-1, E-selectin, and vascular cell adhesion molecule.61 By successfully inhibiting leukocyte adhesion, this results in reduced or diminished neutrophil rolling and adhesion to the endothelium. The exact mechanism of this
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action is under investigation, but it is suspected to be secondary to downregulation of P-selectin by AT in the lipopolysaccharide-stimulated endothelium.42,62 More information regarding AT’s role in inflammation can be found in the inflammation and coagulation article in this issue.
Measurement AT can be measured with an ELISA or immunologic (quantitative) or functional (qualitative) chromogenic assays and is typically done to evaluate congenital or acquired deficits.63 The ELISA detects AT antigen. With the chromogenic assays, plasma is incubated with an excess of the relevant substrate in the presence of heparin. The heparin binds to and causes a conformational change in the structure of AT, significantly increasing its inhibitory activity. A chromogenic substrate specific for the protease is then added and any residual protease causes the cleavage of the substrate and a color change. The absorbance is inversely proportional to the AT activity concentration in the plasma sample.64 AT measurement can be performed in veterinary species but is used in a research or an academic setting and is not considered a routine test in practice.65-68
AT in Disease Congenital AT deficiency occurs in humans and is classified into either type I or type II.69 Type I AT deficiency is characterized by a decrease in both AT activity and concentration, and type II deficiency is characterized by normal levels but reduced AT activity.70 Acquired AT deficiency has been documented in humans and animals and can result from diverse disorders including liver dysfunction, sepsis, kidney disease, and secondary to major surgery or cardiopulmonary bypass.71 In veterinary medicine, AT deficits have been documented with disease processes that may contribute to a hypercoagulable state. The best-characterized diseases affecting canine patients with AT deficiency include protein-losing nephropathy (PLN) and protein-losing enteropathy (PLE). Dogs with PLN and renal failure had significantly lower AT levels compared with normal dogs. In addition to reduced AT levels, other coagulation abnormalities were discovered that could also contribute to a hypercoagulable state.66 AT concentrations were borderline low in dogs with PLE compared with healthy dogs and were thought to contribute to thromboembolic complications.67 Dogs with naturally occurring sepsis were also noted to have significantly lower AT levels.72
Therapeutic Applications of AT AT administration has been evaluated as a therapeutic option in patients with sepsis to reduce the severity of DIC or to decrease the inflammatory response. Multiple studies have been evaluated with mixed results; some studies indicated AT could reduce the mortality associated with sepsis,80,81 whereas more recent studies revealed there was no effect of AT treatment versus placebo.82,83 Evaluation of the KyberSept study84 revealed that those patients who received high-dose AT without concomitant heparin had decreased mortality compared with those that received both.85 According to Surviving Sepsis Campaign guidelines, high-dose AT therapy is not recommended at this time.86 Protein C Background PC is a vitamin K– dependent protein synthesized predominantly in the liver as a single polypeptide chain that circulates as a zymogen.87 It undergoes modifications, both co-translational and posttranslational, before it expresses anticoagulant activity. Once activated, PC, termed activated PC (APC), plays a fundamental role in balancing coagulation through the inactivation of FVa and FVIIIa.88 PC has a short half-life, approximately 6 hours, which is shorter than other vitamin K– dependent factors. Because of the short halflife, it is rapidly depleted with warfarin administration, resulting in an initial and transient hypercoagulable state.89 Heparin and APC display anticoagulant synergy in plasma. The mechanisms include heparin enhancement of AT-dependent inhibition of FV, inactivation of FVa by APC, and proteolytic inactivation of factor V by APC.90 Structure PC in plasma exists primarily as a 2-chain molecule containing both a light and heavy chain linked by a disulfide bond. Initially, PC is secreted as a single chain molecule but is cleaved by a protease into the 2-chain form.91 Conversion of PC to APC is catalyzed by the thrombin-thrombomodulin (TM) complex that resides on the endothelium.92 Alternatively, thrombin by itself can function as a catalyst to convert PC to APC, although much less efficiently than when combined with TM.93 APC has a specific receptor, endothelial protein C receptor (EPCR), which is located on the endothelial cells of large blood vessels. EPCR enhances the activation of PC by binding the protein on cell surfaces and presenting it to the thrombin-TM complex.94 Mechanism of Action
AT as a Prognostic Indicator During severe inflammatory diseases like sepsis, AT levels are markedly decreased secondary to impaired synthesis, degradation by elastase from activated neutrophils, and consumption secondary to ongoing thrombin generation.73 Pro-inflammatory cytokines also cause reduced synthesis of HSG on endothelial cells contributing to reduced AT function.74 Lower AT levels in patients with sepsis worsen survival prediction indexes.75 Lower AT levels have also had prognostic significance in neoplastic diseases and DIC.76,77 AT levels have been used as a prognostic indicator in veterinary medicine. A study by Kuzi et al revealed that when evaluating dogs with immune-mediated hemolytic anemia, pancreatitis, hepatopathy, or neoplasia, AT levels ⬍ 60% normal were associated with an increased mortality.65 AT has been evaluated in horses with colic, and it was found that low AT levels are a negative prognostic indicator.78,79 Dogs with naturally occurring sepsis and low AT levels that did not increase over time had a worse prognosis than those with increases in AT levels.72
APC catalyzes the inactivation of FVa and FVIIIa in the presence of calcium, phospholipids, and its cofactor, PS.95 This significantly dampens the production of thrombin.87 Binding of APC to EPCR speeds up the activation of PC 20-fold, thus inhibiting coagulation at a more rapid pace. EPCR are located on endothelial cells of large blood vessels, thus minimizing clot formation in these larger blood vessels.94 PC is also known for inactivation of the fibrinolytic inhibitor, plasminogen activator inhibitor-1.96 Measurement Clotting-based and colorimetric (chromogenic substrate-based) assays have been developed to evaluate PC in plasma. The majority of these assays involve an activation step in which the zymogen is converted into a functional enzyme, followed by a step that measures PC activity.97 PC tests have been validated for use in veterinary species, but they are modifications of human assays and used in research or academic settings.98,99
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PC in Disease There are 2 main types of PC deficiency, type I and type II. With type I PC deficiency, there is a quantitative defect of PC. Type II deficiency is a qualitative defect in the function of PC.100 In patients with sepsis, the levels of APC are found to be low and shift the system into a hypercoagulable state.101 In dogs, low PC levels are found with hepatobiliary diseases,99,102 sepsis,72 congestive heart failure,103 and aflatoxicosis.104 The PC pathway has been found to be defective in human patients with chronic inflammatory conditions like inflammatory bowel disease,105 asthma,106 vascular inflammation and stroke,107 glomerulonephritis,108 and rheumatoid arthritis.109 Therapeutic Applications of APC APC has been extensively evaluated for its effectiveness as a treatment in sepsis because of the anticoagulant and anti-inflammatory properties in both animal models and human trials. One of the first studies in human medicine using recombinant human-activated PC (rhAPC), drotrecogin alfa, revealed that treatment in patients with sepsis decreased the risk of death by 19.4%.110 After these promising results, the use of rhAPC as an adjunctive treatment for humans with sepsis was promoted through the Surviving Sepsis Campaign.86 The Surviving Sepsis Campaign was sponsored by the manufacturer of drotrecogin alfa (Xigris) and this connection plagued the product with controversy from the start.111 The initial study that showed promise could not be successfully replicated, and in October 2011, Xigris was withdrawn from the market and is no longer recommended as a therapeutic option for sepsis.112,113 Besides a lack of outcome improvement, the major complication associated with the use of rhAPC was bleeding. Ecchymoses was the most frequently reported form of bleeding, but in 2.4% of all enrolled participants, serious internal hemorrhage occurred that required blood transfusions.114 Protein S Background and Structure PS is a vitamin K– dependent plasma glycoprotein synthesized by the endothelium and liver and stored in endothelial cells and platelet alpha granules.115 Unlike other vitamin K– dependent clotting factors, PS is non-enzymatic and acts as an antithrombotic cofactor in an APCdependent and APC-independent manner.116 The APC-independent function involves its action with TFPI.117,118 PS is a 635-amino acid protein that circulates in plasma; approximately 40% of PS is free, whereas 60% exists in a high-affinity complex with C4b-binding protein (C4BP), which is a complement regulatory factor.119
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cofactor for APC-FVa inactivation. PS-C4BP can still mediate inactivation of FVIIIa but does so with less intensity than free PS. Therefore, the interaction of PS with C4BP may alter the activity of PS rather than inhibit it.119 Protein S and TFPI Early experiments involving PS showed that it possessed anticoagulant activity independent of PC. These studies went on to show that in fact PS acted as a cofactor to TFPI. PS decreases the rate-limiting step of FXa-TFPI complex formation 10-fold, hastening FXa inhibition.92 The Kunitz-3 domain on TFPI is suspected to be the binding site for PS. The PS-C4BP complex has incomplete activity as a TFPI cofactor (approximately 60% as effective as PS alone).119 Measurement PS can be measured in plasma, but the measurement is complicated by the fact that it exists in 2 forms: free and as a complex with PS-C4BP.92 Clot-based activity assay tests are based on the ability of PS to act as a cofactor for APC. Other tests include a free PS antigen assay (ELISA and latex particle agglutination) and a total PS antigen assay.115 As stated above, PS-C4BP has incomplete activity as a cofactor, and the free PS is considered to have more biological activity. Therefore, it is important to measure the allocation of these 2 forms, and this can be achieved by crossed immunoelectrophoresis.123 PS in Disease Natural PS deficiency is uncommon.124 There are 3 types of congenital PS deficiencies in humans. In type I, there is a low amount of activity, total, and free PS. Type II deficiency is associated with a functional abnormality with a normal amount of PS, and in type III, there is low free PS but normal total PS.125 PS deficiency is an autosomal-dominant disorder. Homozygous deficiency is potentially fatal after birth if untreated,126 whereas heterozygous deficiency of PS is associated with increased risk of venous thrombosis and stroke.127 In severe sepsis, a deficit of PS levels occurs secondary to a decrease in production, an increase in consumption, or utilization and inhibition.128 Therapeutic Applications of PS PS is currently not used as a therapeutic intervention in either human or veterinary medicine. Individuals with PS deficiency are treated supportively for thromboembolic disease or ischemic stroke if this occurs.129
Mechanism of Action
Conclusions
PS’s ultimate role as an antithrombotic is to downregulate thrombin formation.115 Smaller roles involve complement regulation, fibrinolysis, and other defense mechanisms.116,120
The endogenous anticoagulants are a varied group of proteins that act to limit or minimize coagulation either alone or in concert with each other. Endogenous anticoagulants can act as markers for mortality and have been investigated as therapeutic interventions for sepsis and other disease states. The scientific exploration into the role and use of endogenous anticoagulants will hopefully increase in the future in veterinary medicine.
Protein S and APC PS increases the reaction rate of proteolysis by APC of FVa by approximately 20-fold and inhibits FXa-dependent protection of FVa to the actions of APC.92 PS also augments the APC-mediated inactivation of FVIIIa.119 In order for PS to exert activity as a cofactor for APC, PS must bind to phospholipid membranes via its glargine (Gla) domain as is required by other vitamin K– dependent factors.121 Previous literature has reported that adding C4BP to plasma will counteract the PS-dependent prolongation of clotting time in the presence of APC, and secondary to this, it was thought that the cofactor activity for APC was lost upon binding of PS to C4BP.122 More recent literature has shown that the PS-C4BP complex is still an active
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36. Camerota AJ, Creasey AA, Patla V, Larkin VA, Fink MP: Delayed treatment with recombinant human tissue factor pathway inhibitor improves survival in rabbits with gram-negative peritonitis. J Infect Dis 177:668 – 676, 1998 37. Abraham E: Tissue factor inhibition and clinical trial results of tissue factor pathway inhibitor in sepsis. Crit Care Med 28:S31–S33, 2000 38. Abraham E, Reinhart K, Opal S, et al: Efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial. JAMA 290:238 –247, 2003 39. Roemisch J, Gray E, Hoffmann JN, Wiedermann CJ: Antithrombin: a new look at the actions of a serine protease inhibitor. Blood Coagul Fibrinolysis 13:657– 670, 2002 40. Damus PS, Hicks M, Rosenberg RD: Anticoagulant action of heparin. Nature 246: 355–357, 1973 41. Okajima K, Uchiba M: The anti-inflammatory properties of antithrombin III: new therapeutic implications. Semin Thromb Hemost 24:27–32, 1998 42. 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Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity. Blood 85:121–129, 1995 48. Opal SM, Kessler CM, Roemisch J, Knaub S: Antithrombin, heparin, and heparan sulfate. Crit Care Med 30:S325–S331, 2002 49. Olds RJ, Lane DA, Mille B, Chowdhury V, Thein SL: Antithrombin: the principal inhibitor of thrombin. Semin Thromb Hemost 20:353–372, 1994 50. Opal SM: Therapeutic rationale for antithrombin III in sepsis. Crit Care Med 28: S34 –S37, 2000 51. Marcum JA, Rosenberg RD: Anticoagulantly active heparin-like molecules from vascular tissue. Biochemistry 23:1730 –1737, 1984 52. Harenberg J: Pharmacology of low molecular weight heparins. Semin Thromb Hemost 16(suppl):12–18, 1990 53. Handeland GF, Abildgaard U, Holm HA, Arnesen KE: Dose adjusted heparin treatment of deep venous thrombosis: a comparison of unfractionated and low molecular weight heparin. Eur J Clin Pharmacol 39:107–112, 1990 54. Young E, Wells P, Holloway S, Weitz J, Hirsh J: Ex-vivo and in-vitro evidence that low molecular weight heparins exhibit less binding to plasma proteins than unfractionated heparin. Thromb Haemost 71:300 –304, 1994 55. Bjornsson TD, Wolfram KM, Kitchell BB: Heparin kinetics determined by three assay methods. Clin Pharmacol Ther 31:104 –113, 1982 56. Weitz JI: Low-molecular-weight heparins. N Engl J Med 337:688 – 698, 1997 57. Gray E, Souter P, Roemisch J, Poole S: Antithrombin inhibits in vitro lipopolysaccharide induced procoagulant activity and cytokine production. Shock 13:147, 2000 58. Kaneider NC, Reinisch CM, Dunzendorfer S, R×misch J, Wiedermann CJ, Wiederman CJ: Syndecan-4 mediates antithrombin-induced chemotaxis of human peripheral blood lymphocytes and monocytes. J Cell Sci 115:227–236, 2002 59. Souter PJ, Thomas S, Hubbard AR, Poole S, R×misch J, Gray E: Antithrombin inhibits lipopolysaccharide-induced tissue factor and interleukin-6 production by mononuclear cells, human umbilical vein endothelial cells, and whole blood. Crit Care Med 29:134 –139, 2001 60. Coughlin SR: Protease-activated receptors in vascular biology. Thromb Haemost 86:298 –307, 2001 61. Senden NH, Jeunhomme TM, Heemskerk JW, et al: Factor Xa induces cytokine production and expression of adhesion molecules by human umbilical vein endothelial cells. J Immunol 161:4318 – 4324, 1998 62. Hoffmann JN, Vollmar B, Inthorn D, Schildberg FW, Menger MD: The thrombin antagonist hirudin fails to inhibit endotoxin-induced leukocyte/endothelial cell interaction and microvascular perfusion failure. Shock 14:528 –534, 2000 63. Goodnight SH, Schaeffer JL, Sheth K: Measurement of antithrombin III in normal and pathologic states using chromogenic substrate S-2238. Comparison with immunoelectrophoretic and factor Xa inhibition assays. Am J Clin Pathol 73:639 – 647, 1980 64. Axelsson F: Antithrombin, In Chromogenix. Mõlndal, Sweden, Chromogenix, 1995, pp 1-27 65. Kuzi S, Segev G, Haruvi E, Aroch I: Plasma antithrombin activity as a diagnostic and prognostic indicator in dogs: a retrospective study of 149 dogs. J Vet Intern Med 24:587–596, 2010 66. Donahue S, Brooks M, Otto C: Examination of hemostatic parameters to detect hypercoagulability in dogs with severe protein losing nephropathy. J Vet Emerg Crit Care 21:346 –355, 2011 67. Goodwin LV, Goggs R, Chan DL, Allenspach K: Hypercoagulability in dogs with protein-losing enteropathy. J Vet Intern Med 25:273–277, 2011 68. Eralp O, Yilmaz Z, Failing K, Moritz A, Bauer N: Effect of experimental endotoxemia on thrombelastography parameters, secondary and tertiary hemostasis in dogs. J Vet Intern Med 25:524 –531, 2011
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69. Sas G, Pet× I, BÂnhegyi D, BlaskÔ G, DomjÂn G: Heterogeneity of the “classical” antithrombin III deficiency. Thromb Haemost 43:133–136, 1980 70. Sas G: Hereditary antithrombin III deficiency: biochemical aspects. Haematologica (Budap) 17:81– 86, 1984 71. Maclean PS, Tait RC: Hereditary and acquired antithrombin deficiency: epidemiology, pathogenesis and treatment options. Drugs 67:1429 –1440, 2007 72. de Laforcade AM, Rozanski EA, Freeman LM, Li W: Serial evaluation of protein C and antithrombin in dogs with sepsis. J Vet Intern Med 22:26 –30, 2008 73. Levi M, van der Poll T, Bller HR: Bidirectional relation between inflammation and coagulation. Circulation 109:2698 –2704, 2004 74. Kobayashi M, Shimada K, Ozawa T: Human recombinant interleukin-1 beta- and tumor necrosis factor alpha-mediated suppression of heparin-like compounds on cultured porcine aortic endothelial cells. J Cell Physiol 144:383–390, 1990 75. Hagag AA, Elmahdy HS, Ezzat AA: Prognostic value of plasma pro-adrenomedullin and antithrombin levels in neonatal sepsis. Indian Pediatr 48:471– 473, 2011 76. Iwako H, Tashiro H, Amano H, et al: Prognostic significance of antithrombin III levels for outcomes in patients with hepatocellular carcinoma after curative hepatectomy. Ann Surg Oncol 31 March 2012 [epub ahead of print] 77. Egi M, Morimatsu H, Wiedermann CJ, et al: Non-overt disseminated intravascular coagulation scoring for critically ill patients: the impact of antithrombin levels. Thromb Haemost 101:696 –705, 2009 78. Zbanyszek M, Procajło A, Stopyra A, Sobiech P, Rajski K: The coagulation system in horses with colic. Pol J Vet Sci 7:53–58, 2004 79. Darien B, Potema J, Moore J, Travis J: Antithrombin III activity in horses with colic: an analysis of 46 cases. Equine Vet J 23:211–214, 1991 80. Opal S: Antithrombin II in the treatment of severe sepsis: the results of a phase II multicenter clinical trial. J Anesth Intensive Lung 25:273–277, 2001 81. Warren BL, Eid A, Singer P, et al: Caring for the critically ill patient. High-dose antithrombin III in severe sepsis: a randomized controlled trial. JAMA 286:1869 – 1878, 2001 82. Knaub S, Keinecke J, Mescheder A, Heinrichs H: Antithrombin III in patients with severe sepsis—a clinical development plan. Ann Hematol 76:A47, 1998 83. Sakr Y, Reinhart K, Hagel S, Kientopf M, Brunkhorst F: Antithrombin levels, morbidity, and mortality in a surgical intensive care unit. Anesth Analg 105:715–723, 2007 84. Eid A, Wiedermann CJ, Kinasewitz GT: Early administration of high-dose antithrombin in severe sepsis: single center results from the KyberSept-trial. Anesth Analg 107:1633–1638, 2008 85. Kienast J, Juers M, Wiedermann CJ, et al: Treatment effects of high-dose antithrombin without concomitant heparin in patients with severe sepsis with or without disseminated intravascular coagulation. J Thromb Haemost 4:90 –97, 2006 86. Dellinger RP, Levy MM, Carlet JM, et al: Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock. Crit Care Med 2008(36):296 –327, 2008 87. Strickland DK, Kessler CM: Biochemical and functional properties of protein C and protein S. Clin Chim Acta 170:1–24, 1987 88. Castellino FJ, Ploplis VA: The protein C pathway and pathologic processes. J Thromb Haemost 7(suppl 1):140 –145, 2009 89. Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C: Deficiency of protein C in congenital thrombotic disease. J Clin Invest 68:1370 –1373, 1981 90. PetÅjÅ J, FernÂndez JA, Gruber A, Griffin JH: Anticoagulant synergism of heparin and activated protein C in vitro. Role of a novel anticoagulant mechanism of heparin, enhancement of inactivation of factor V by activated protein C. J Clin Invest 99:2655–2663, 1997 91. Foster D, Davie EW: Characterization of a cDNA coding for human protein C. Proc Natl Acad Sci U S A 81:4766 – 4770, 1984 92. 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 257:7944 –7947, 1982 93. Vehar G, Davie E: Preparation and properties of bovine factor VII (antihemophiliac factor). Biochemistry (Mosc) 19:401, 1980 94. Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W: Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 296:1880 – 1882, 2002 95. Kisiel W, Canfield WM, Ericsson LH, Davie EW: Anticoagulant properties of bovine plasma protein C following activation by thrombin. Biochemistry 16:5824 –5831, 1977 96. Taylor FB Jr., Lockhart MS: A new function for activated protein C: activated protein C prevents inhibition of plasminogen activators by releasate from mononuclear leukocytes—platelet suspensions stimulated by phorbol diester. Thromb Res 37:155–164, 1985 97. Hickton CM, Felding P, Ikeda K, Martinsson G, Nilsson IM: A functional assay of protein C in human plasma. Thromb Res 41:501–508, 1986 98. Fry MM, Snyder KR, Tobias KM, Williamson BG, Reed GA: Protein C activity in dogs: adaptation of a commercial human colorimetric assay and evaluation of effects of storage time and temperature. Vet Med Int 751849, 2011
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99. Toulza O, Center SA, Brooks MB, Erb HN, Warner KL, Deal W: Evaluation of plasma protein C activity for detection of hepatobiliary disease and portosystemic shunting in dogs. J Am Vet Med Assoc 229:1761–1771, 2006 100. Bereczky Z, KovÂcs KB, Muszbek L: Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game. Clin Chem Lab Med 48(suppl 1):S53–S66, 2010 101. Kanji S, Devlin JW, Piekos KA, Racine E: Recombinant human activated protein C, drotrecogin alfa (activated): a novel therapy for severe sepsis. Pharmacotherapy 21:1389 –1402, 2001 102. Prins M, Schellens CJ, van Leeuwen MW, Rothuizen J, Teske E: Coagulation disorders in dogs with hepatic disease. Vet J 185:163–168, 2010 103. Tarnow I, Falk T, Tidholm A, et al: Hemostatic biomarkers in dogs with chronic congestive heart failure. J Vet Intern Med 21:451– 457, 2007 104. Dereszynski DM, Center SA, Randolph JF, et al: Clinical and clinicopathologic features of dogs that consumed foodborne hepatotoxic aflatoxins: 72 cases (200506). J Am Vet Med Assoc 232:1329 –1337, 2008 105. Scaldaferri F, Sans M, Vetrano S, et al: Crucial role of the protein C pathway in governing microvascular inflammation in inflammatory bowel disease. J Clin Invest 117:1951–1960, 2007 106. Finigan JH, Dudek SM, Singleton PA, et al: Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation. J Biol Chem 280:17286 –17293, 2005 107. Shibata M, Kumar SR, Amar A, et al: Anti-inflammatory, antithrombotic, and neuroprotective effects of activated protein C in a murine model of focal ischemic stroke. Circulation 103:1799 –1805, 2001 108. Isermann B, Vinnikov IA, Madhusudhan T, et al: Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. Nat Med 13:1349 –1358, 2007 109. Buisson-Legendre N, Smith S, March L, Jackson C: Elevation of activated protein C in synovial joints in rheumatoid arthritis and its correlation with matrix metalloproteinase 2. Arthritis Rheum 50:2151–2156, 2004 110. Bernard GR, Vincent JL, Laterre PF, et al: Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 344:699 –709, 2001 111. Barie PS: An opinion too far—the campaign against the Surviving Sepsis Campaign. Surg Infect (Larchmt) 7:485– 488, 2006 112. FDA Drug Safety Communication: voluntary market withdrawal of Xigris [drotrecogin alfa (activated)] due to failure to show a survival benefit. http://www.fda.gov/Drugs/DrugSafety/ucm277114.htm. Accessed July 30, 2012 113. Ranieri VM, Thompson BT, Barie PS, et al: Drotrecogin alfa (activated) in adults with septic shock. N Engl J Med 366:2055–2064, 2012 114. Marti-Carvajal AJ, Sola I, Lathyris D, Cardona AF: Human recombinant activated protein C for severe sepsis. Cochrane Database Syst Rev:CD004388, 2011 115. Marlar RA, Gausman JN: Protein S abnormalities: a diagnostic nightmare. Am J Hematol 86:418 – 421, 2011 116. Mosnier LO, Meijers JC, Bouma BN: The role of protein S in the activation of thrombin activatable fibrinolysis inhibitor (TAFI) and regulation of fibrinolysis. Thromb Haemost 86:1040 –1046, 2001 117. Peraramelli S, Rosing J, Hackeng TM: TFPI-dependent activities of protein S. Thromb Res 129(suppl 2):S23–S26, 2012 118. Castoldi E, Hackeng TM: Regulation of coagulation by protein S. Curr Opin Hematol 15:529 –536, 2008 119. Hackeng TM, Hessing M, van’t Veer C, et al: Protein S binding to human endothelial cells is required for expression of cofactor activity for activated protein C. J Biol Chem 268:3993– 4000, 1993 120. DahlbÅck B: The tale of protein S and C4b-binding protein, a story of affection. Thromb Haemost 98:90 –96, 2007 121. DahlbÅck B: Inhibition of protein Ca cofactor function of human and bovine protein S by C4b-binding protein. J Biol Chem 261:12022–12027, 1986 122. Van de Poel RH, Meijers JC, Bouma BN: C4b-binding protein inhibits the factor V-dependent but not the factor V-independent cofactor activity of protein S in the activated protein C-mediated inactivation of factor VIIIa. Thromb Haemost 85: 761–765, 2001 123. Comp PC, Doray D, Patton D, Esmon CT: An abnormal plasma distribution of protein S occurs in functional protein S deficiency. Blood 67:504 –508, 1986 124. ten Kate MK, van der Meer J: Protein S deficiency: a clinical perspective. Haemophilia 14:1222–1228, 2008 125. Rosing J, Maurissen LF, Tchaikovski SN, Tans G, Hackeng TM: Protein S is a cofactor for tissue factor pathway inhibitor. Thromb Res 122(suppl 1):S60 –S63, 2008 126. Pegelow CH, Ledford M, Young JN, Zilleruelo G: Severe protein S deficiency in a newborn. Pediatrics 89:674 – 676, 1992 127. Comp PC, Nixon RR, Cooper MR, Esmon CT: Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 74:2082–2088, 1984 128. Vervloet M, Thijs L, CE H: Derangements of coagulation and fibrinolysis in critically ill patients with sepsis and septic shock. Semin Thromb Hemost 24:33– 44, 1998 129. Lipe B, Ornstein DL: Deficiencies of natural anticoagulants, protein C, protein S, and antithrombin. Circulation 124:e365– e368, 2011
Topical Review
Endogenous Anticoagulants Amy Kubier, DVM, DACVIM, and Mauria O’Brien, DVM, DACVECC
A B S T R A C T Keywords: antithrombin coagulation protein C protein S tissue factor pathway inhibitor
Blood coagulation is a complex and highly coordinated process that is constantly altered and impacted by procoagulant and anticoagulant “players.” It is vital that these components work in concert to maintain a balance to keep coagulation in check. Several important endogenous anticoagulants will be discussed in this review including tissue factor pathway inhibitor, antithrombin, protein C, and protein S in origin, structure, mechanism of action, effects of deficiency, and current knowledge in veterinary medicine.
University of Illinois Urbana-Champaign, Champaign, IL, USA Address reprint requests to: Amy Kubier, DVM, DACVIM, 1008 W Hazelwood Dr, Urbana, IL 61802.
䉷 2012 Published by Elsevier Inc.
E-mail: [email protected]
Blood coagulation is a complex, highly coordinated process that is continuously modulated by procoagulant and anticoagulant “players.” It is vital that these components work in concert to maintain a balance to keep coagulation in check. In certain disease states, particularly those with a massive inflammatory response, the scales are tipped toward a procoagulant state and the production of anticoagulant mediators is downregulated. The goal of this review is to discuss several important endogenous anticoagulants including tissue factor pathway inhibitor (TFPI), antithrombin (AT), protein C (PC), and protein S (PS) in origin, structure, mechanism of action, effects of deficiency, and current knowledge and applicability in veterinary medicine. Tissue Factor Pathway Inhibitor Background Tissue factor (TF) is a transmembrane protein that acts as a receptor for plasma factor VII (FVII) and its activated form, FVIIa. Perivascular cells constitutively express TF, underlying the importance of its location to provide rapid activation of coagulation after vascular injury. TF is also constitutively expressed in the heart, kidney, brain, lungs, and placenta; these tissue-specific locations impart hemostatic protection in these highly vascular and vital organs.1 When a vascular injury occurs, the adventitial cells expressing TF are exposed, allowing circulating FVIIa to bind to the uncovered TF. The TF-FVIIa complex then activates, through a positive feedback mechanism, FVII to FVIIa. Additionally, the TF-FVIIa complex activates small amounts of FIX and FX. This is the initiation step in the cell-based model of coagulation and acts as the primary initiator of coagulation in vivo.2 More information regarding the cell-based model of coagulation can be found in the article in this issue. TFPI has a dual inhibitory function: it is the primary inhibitor of the TF-VIIa complex as well as an inhibitor of FXa.1,2 Coagulation must be initiated for TFPI to function.3
proximately 10% of TFPI is bound to plasma lipoproteins1,5,6 and a very small proportion circulates freely.7 Activated platelets and heparin lead to the release of TFPI from intracellular stores.8 Estimations of endogenous TFPI levels have been based on the amount of TFPI released after heparin injection.7 Structure and Mechanism of Action TFPI is a Kunitz-type protease inhibitor containing Kunitz domains (K1-3) that inhibit the function of protein-degrading enzymes. In a 2-stage process, the second Kunitz domain, K2, binds to and directly inhibits FXa. The first domain, K1, then binds to a TF-FVIIa complex and inhibits it and prevents any further activation of FX. The formation of this quaternary structure is essential to the inhibitory actions of TFPI on TF-FVIIa. Although K3 is involved in lipoprotein binding and contains a heparin-binding site, it does not seem to function as a protease inhibitor1,6 but is essential for optimal FXa inhibition. The specific role of K3 in the inhibition of coagulation appears to be through its relationship with protein S4 (Fig. 1). The binding of TFPI to FXa is reversible and independent of calcium, whereas the binding of the TFPI-FXa complex to TF-VIIa is irreversible and requires calcium. Inactivation of TF-FVIIa by TFPI is both calcium- and FXa-dependent.9 It should be noted that TFPI does not stop coagulation, but it limits further generation of FIXa and FXa by the TF-FVIIa complex.10 TFPI can also stimulate monocytes to internalize and degrade TF-FVIIa complexes expressed on the cell surface.11 As stated earlier, heparin, both unfractionated and low molecular weight,12 increases the levels of TFPI by inducing synthesis and causing secretion of TFPI by endothelial cells as well as displacing the bound portion11,13 The inhibitory effects of TFPI are significantly enhanced in the presence of heparin.14 As heparin is cleared from circulation, the effects of the heparin on TFPI cease and TFPI becomes endothelial bound again.15
Location TFPI is primarily produced and expressed on the luminal surface of endothelial cells,1,4 although megakaryocytes, monocytes, lung fibroblasts, and synovial cells are able to express low levels of TFPI. Ap-
Measurement of TFPI TFPI can be measured by commercially available enzyme-linked immunosorbent assays (ELISA) and functional endpoint assays. The
0958-3947/$ – see front matter 䉷 2012 Topics in Companion Animal Medicine. Published by Elsevier Inc. http://dx.doi.org/10.1053/j.tcam.2012.07.003
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Figure 1. The functions of TFPI. The TF-VIIa complex is able to activate FIX to FIXa and stimulate the intrinsic pathway as well as activate FX to Xa (top center). TFPI binds to the tertiary complex of TF-VIIa-Xa, which is produced during the activation of Xa (center left). This binding forms the quaternary complex, with K1 binding to and inactivating VIIa and K2 binding to and inactivating Xa (bottom). TFPI is also able to bind via K2 to Xa and PS via K3, forming an inhibitory complex (center right). TFPI, Tissue factor pathway inhibitor; TF, tissue factor; K1, K2, K3, Kunitz domains 1, 2, and 3. (Adapted from Broze GJ, Jr, Girard TJ. Tissue factor pathway inhibitor: structure-function. Front Biosci 17:262280, 2012.)
total TFPI ELISA measures binary and quaternary TFPI-Xa complexes, intact and truncated forms of TFPI as well as complexes of TFPI with TF and FVIIa. The activity assay uses thawed anticoagulated whole blood in which the cells are lysed and cellular debris removed. The supernatant is then incubated with TF-FVIIa and FX. Residual TF-VIIa activity is based on measuring FXa activity and TFPI is measured from this with a standard curve.1 Interpretation of results is method specific, and levels cannot be compared between different types of tests.16 TFPI in Disease There are no known deficiencies of TFPI, suggesting it is essential for life.12 Increased levels of TFPI have been observed in certain cancers, sepsis, uremia, unregulated diabetes, and in patients with hyperlipidemia.17-21 Depending on the disease process, the measurement of TFPI may be used as a biomarker and the trends of increase or decline used as a prognostic indicator.20 In septic humans and those with disseminated intravascular coagulation (DIC), an increase in mortality is noted if the production of TF is not adequately balanced by the production of TFPI.22 Several types of human neoplastic cells are known to alter the expression of TFPI,23-25 contributing to a poor prognosis. A recent study determined that in certain human breast cancer models, TFPI was downregulated.26 Also noted was that solid tumors, versus hematological cancers, trend toward higher levels of TFPI. This has been speculated to be potentially protective against microthrombosis and organ failure in certain cancers.20 There are limitations when evaluating studies on the role of TFPI in disease states because there is a wide range of TFPI among normal individuals, and many of the studies on TFPI and risk of thrombosis are retrospective or case-controlled and do not address whether the low levels of TFPI are the cause or the effect. There remains an insufficient number of prospective studies evaluating the role of TFPI in the development of venous thromboembolism.1
Therapeutic Applications of TFPI Given the dominant role of TF in certain disease states it would seem intuitive that TFPI could be used therapeutically as an anticoagulant as well as an anti-inflammatory agent. People with low circulating levels of TFPI have been shown to be at increased risk for venous thromboembolism.27 A study determined that TFPI was increased after oral supplementation with a fatty acid in people with chronic atherosclerotic disease, a disease characterized by high levels of TF. This increase in TFPI could promote its use as a mild antithrombotic agent to help to dampen the TF pathway.28 Recombinant TFPI (rTFPI) has been examined for its role as an antithrombotic agent, and it was determined that local administration (injections of rTFPI near a created wound) prevented thrombosis and was equivocal to heparin and dextran.29 Several studies have evaluated recombinant TFPI supplementation in animal models.30,31 Human studies focus importance on the levels of TFPI secondary to heparin therapy and use those levels as a marker for postoperative bleeding.32,33 The use of rTFPI showed great promise in animal models of sepsis and DIC. A study evaluating intravenous rTFPI determined that the rTFPI prevented thrombosis and progression into DIC in a rabbit model.34 Administration of rTFPI reduced mortality in baboons and rabbits with Escherichia coli–induced septic shock.35,36 Unlike animal studies using rTFPI, the use of TFPI in humans currently shows no benefit as a therapeutic agent in sepsis. Initially phase I and II studies were promising37 and rTFPI was safe in humans, but when taken to a phase III level, there was no survival benefit to its use in sepsis.38 Antithrombin Background AT, previously termed antithrombin III, is a plasma-derived glycoprotein. AT is a serpin (serine protease inhibitor) and shares approximately 30% homology in amino acid sequence with other serine pro-
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Figure 2. Antithrombin’s actions with unfractionated heparins and low-molecular-weight heparins. When antithrombin binds to UFH at a specific pentasaccharide sequence, it causes a conformation change in AT at its reactive site. This change accelerates AT interaction with FXa. Both heparin forms are able to interact with and inactivate FXa, but only the long chains (at least 18 saccharide units) of UFH are capable of forming a heparin-AT-thrombin complex, making UFH a stronger inhibitor of thrombin than LMWH. UFH, Unfractionated heparins; LMWH, low-molecular-weight heparins; AT, antithrombin; FXa, activated factor X. (Adapted from Weitz JI. Low-molecular-weight heparins. N Engl J Med 337:688-698, 1997.)
tease inhibitors.39 AT’s activity is 2-fold in that it is known to act as a potent endogenous anticoagulant, but newer studies have also shown that AT also has powerful anti-inflammatory properties.40-42 Structure AT is produced by the liver and consists of 432 amino acids and contains 3 disulfide bonds and 4 potential glycosylation sites.43 Plasma AT exists as 2 isoforms, ␣ and . The -isoform lacks 1 of 4 carbohydrate side-chains that are present on the ␣-isoform. The -isoform constitutes about 5% to 10% of AT in plasma and has a higher affinity for heparin and heparan sulfate glycosaminoglycans (HSG) than does the ␣-isoform. The -isoform of AT appears to be concentrated in the vessel wall, whereas the ␣-isoform exists predominately in plasma.44 Mechanism of Action AT influences the activity of many coagulation factors. In addition to the inhibition of thrombin (FIIa), AT also inhibits FIXa, FXa, FXIa, and FXIIa. In the presence of heparin, FVIIa is also inhibited.40,45-47 Endogenous heparins are termed heparans. Heparans are glycosaminoglycans expressed on endothelial cell surfaces. Heparans bind to and activate AT via allosteric activation. Synthetic heparins and heparan sulfates possess repeating sequences of a specific pentasaccharide sequence that binds to AT with high affinity.48 AT binds to its target and irreversibly inhibits the protease, and the reticuloendothelial system of the liver then removes the complex.46,49 Heparin is an important but not essential cofactor for AT. Without heparin or HSG, AT inhibits coagulation very slowly and methodically.40 In the presence of heparin/HSG, AT’s activity is increased 1000-fold.49 Heparin/HSG binds to AT in a distinct manner. A conformational change in AT is induced when heparin binds to AT by a
specific pentasaccharide sequence. Heparin must specifically attach to thrombin while simultaneously bind to AT for the inactivation of thrombin. This is possible with unfractionated heparins (UFH), which have complex, long-chain structures with multiple pentasaccharide sequences. This expanded binding capacity is not needed for AT to inactivate other serine proteases such as FXa. This explains why lowmolecular-weight heparins (LMWHs) fail to directly inhibit thrombin yet retain their intrinsic anticoagulant activity against other coagulation factors50,51 (Fig. 2). LMWHs are small, with an average weight of 5000 Da, whereas UFH weigh up to 30,000 Da. LMWHs and UFH both exert their anticoagulant activity by activating AT. The activation of AT is mediated by binding through a unique pentasaccharide sequence that is present on one third of the chains of all UFH, but only 15% to 25% of LMWH chains.52 LMWHs produce a more predictable anticoagulant response than UFH.53 The plasma half-life of LMWH is 2 to 4 times longer than UFH.52 Some of this behavior is attributable to a decreased likelihood of LMWHs to bind to plasma proteins, endothelial cells, and macrophages like UFH do.54 The inhibition of FXa by LMWH persists longer than their inhibitory activity against thrombin.55 LMWHs cause less bleeding than UFH and with its more predictable behavior does not require laboratory monitoring.56 In vivo and in vitro studies have demonstrated that AT has potent anti-inflammatory effects.42,57-59 It is well known that thrombin is a central procoagulant factor with many inflammatory side effects.60 By inhibiting thrombin, the pro-inflammatory response is attenuated. AT is an inhibitor of the protease FXa, which has several pro-inflammatory effects including stimulation and production of pro-inflammatory cytokines (IL-8, IL-6), monocyte chemotactic protein-1, E-selectin, and vascular cell adhesion molecule.61 By successfully inhibiting leukocyte adhesion, this results in reduced or diminished neutrophil rolling and adhesion to the endothelium. The exact mechanism of this
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action is under investigation, but it is suspected to be secondary to downregulation of P-selectin by AT in the lipopolysaccharide-stimulated endothelium.42,62 More information regarding AT’s role in inflammation can be found in the inflammation and coagulation article in this issue.
Measurement AT can be measured with an ELISA or immunologic (quantitative) or functional (qualitative) chromogenic assays and is typically done to evaluate congenital or acquired deficits.63 The ELISA detects AT antigen. With the chromogenic assays, plasma is incubated with an excess of the relevant substrate in the presence of heparin. The heparin binds to and causes a conformational change in the structure of AT, significantly increasing its inhibitory activity. A chromogenic substrate specific for the protease is then added and any residual protease causes the cleavage of the substrate and a color change. The absorbance is inversely proportional to the AT activity concentration in the plasma sample.64 AT measurement can be performed in veterinary species but is used in a research or an academic setting and is not considered a routine test in practice.65-68
AT in Disease Congenital AT deficiency occurs in humans and is classified into either type I or type II.69 Type I AT deficiency is characterized by a decrease in both AT activity and concentration, and type II deficiency is characterized by normal levels but reduced AT activity.70 Acquired AT deficiency has been documented in humans and animals and can result from diverse disorders including liver dysfunction, sepsis, kidney disease, and secondary to major surgery or cardiopulmonary bypass.71 In veterinary medicine, AT deficits have been documented with disease processes that may contribute to a hypercoagulable state. The best-characterized diseases affecting canine patients with AT deficiency include protein-losing nephropathy (PLN) and protein-losing enteropathy (PLE). Dogs with PLN and renal failure had significantly lower AT levels compared with normal dogs. In addition to reduced AT levels, other coagulation abnormalities were discovered that could also contribute to a hypercoagulable state.66 AT concentrations were borderline low in dogs with PLE compared with healthy dogs and were thought to contribute to thromboembolic complications.67 Dogs with naturally occurring sepsis were also noted to have significantly lower AT levels.72
Therapeutic Applications of AT AT administration has been evaluated as a therapeutic option in patients with sepsis to reduce the severity of DIC or to decrease the inflammatory response. Multiple studies have been evaluated with mixed results; some studies indicated AT could reduce the mortality associated with sepsis,80,81 whereas more recent studies revealed there was no effect of AT treatment versus placebo.82,83 Evaluation of the KyberSept study84 revealed that those patients who received high-dose AT without concomitant heparin had decreased mortality compared with those that received both.85 According to Surviving Sepsis Campaign guidelines, high-dose AT therapy is not recommended at this time.86 Protein C Background PC is a vitamin K– dependent protein synthesized predominantly in the liver as a single polypeptide chain that circulates as a zymogen.87 It undergoes modifications, both co-translational and posttranslational, before it expresses anticoagulant activity. Once activated, PC, termed activated PC (APC), plays a fundamental role in balancing coagulation through the inactivation of FVa and FVIIIa.88 PC has a short half-life, approximately 6 hours, which is shorter than other vitamin K– dependent factors. Because of the short halflife, it is rapidly depleted with warfarin administration, resulting in an initial and transient hypercoagulable state.89 Heparin and APC display anticoagulant synergy in plasma. The mechanisms include heparin enhancement of AT-dependent inhibition of FV, inactivation of FVa by APC, and proteolytic inactivation of factor V by APC.90 Structure PC in plasma exists primarily as a 2-chain molecule containing both a light and heavy chain linked by a disulfide bond. Initially, PC is secreted as a single chain molecule but is cleaved by a protease into the 2-chain form.91 Conversion of PC to APC is catalyzed by the thrombin-thrombomodulin (TM) complex that resides on the endothelium.92 Alternatively, thrombin by itself can function as a catalyst to convert PC to APC, although much less efficiently than when combined with TM.93 APC has a specific receptor, endothelial protein C receptor (EPCR), which is located on the endothelial cells of large blood vessels. EPCR enhances the activation of PC by binding the protein on cell surfaces and presenting it to the thrombin-TM complex.94 Mechanism of Action
AT as a Prognostic Indicator During severe inflammatory diseases like sepsis, AT levels are markedly decreased secondary to impaired synthesis, degradation by elastase from activated neutrophils, and consumption secondary to ongoing thrombin generation.73 Pro-inflammatory cytokines also cause reduced synthesis of HSG on endothelial cells contributing to reduced AT function.74 Lower AT levels in patients with sepsis worsen survival prediction indexes.75 Lower AT levels have also had prognostic significance in neoplastic diseases and DIC.76,77 AT levels have been used as a prognostic indicator in veterinary medicine. A study by Kuzi et al revealed that when evaluating dogs with immune-mediated hemolytic anemia, pancreatitis, hepatopathy, or neoplasia, AT levels ⬍ 60% normal were associated with an increased mortality.65 AT has been evaluated in horses with colic, and it was found that low AT levels are a negative prognostic indicator.78,79 Dogs with naturally occurring sepsis and low AT levels that did not increase over time had a worse prognosis than those with increases in AT levels.72
APC catalyzes the inactivation of FVa and FVIIIa in the presence of calcium, phospholipids, and its cofactor, PS.95 This significantly dampens the production of thrombin.87 Binding of APC to EPCR speeds up the activation of PC 20-fold, thus inhibiting coagulation at a more rapid pace. EPCR are located on endothelial cells of large blood vessels, thus minimizing clot formation in these larger blood vessels.94 PC is also known for inactivation of the fibrinolytic inhibitor, plasminogen activator inhibitor-1.96 Measurement Clotting-based and colorimetric (chromogenic substrate-based) assays have been developed to evaluate PC in plasma. The majority of these assays involve an activation step in which the zymogen is converted into a functional enzyme, followed by a step that measures PC activity.97 PC tests have been validated for use in veterinary species, but they are modifications of human assays and used in research or academic settings.98,99
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PC in Disease There are 2 main types of PC deficiency, type I and type II. With type I PC deficiency, there is a quantitative defect of PC. Type II deficiency is a qualitative defect in the function of PC.100 In patients with sepsis, the levels of APC are found to be low and shift the system into a hypercoagulable state.101 In dogs, low PC levels are found with hepatobiliary diseases,99,102 sepsis,72 congestive heart failure,103 and aflatoxicosis.104 The PC pathway has been found to be defective in human patients with chronic inflammatory conditions like inflammatory bowel disease,105 asthma,106 vascular inflammation and stroke,107 glomerulonephritis,108 and rheumatoid arthritis.109 Therapeutic Applications of APC APC has been extensively evaluated for its effectiveness as a treatment in sepsis because of the anticoagulant and anti-inflammatory properties in both animal models and human trials. One of the first studies in human medicine using recombinant human-activated PC (rhAPC), drotrecogin alfa, revealed that treatment in patients with sepsis decreased the risk of death by 19.4%.110 After these promising results, the use of rhAPC as an adjunctive treatment for humans with sepsis was promoted through the Surviving Sepsis Campaign.86 The Surviving Sepsis Campaign was sponsored by the manufacturer of drotrecogin alfa (Xigris) and this connection plagued the product with controversy from the start.111 The initial study that showed promise could not be successfully replicated, and in October 2011, Xigris was withdrawn from the market and is no longer recommended as a therapeutic option for sepsis.112,113 Besides a lack of outcome improvement, the major complication associated with the use of rhAPC was bleeding. Ecchymoses was the most frequently reported form of bleeding, but in 2.4% of all enrolled participants, serious internal hemorrhage occurred that required blood transfusions.114 Protein S Background and Structure PS is a vitamin K– dependent plasma glycoprotein synthesized by the endothelium and liver and stored in endothelial cells and platelet alpha granules.115 Unlike other vitamin K– dependent clotting factors, PS is non-enzymatic and acts as an antithrombotic cofactor in an APCdependent and APC-independent manner.116 The APC-independent function involves its action with TFPI.117,118 PS is a 635-amino acid protein that circulates in plasma; approximately 40% of PS is free, whereas 60% exists in a high-affinity complex with C4b-binding protein (C4BP), which is a complement regulatory factor.119
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cofactor for APC-FVa inactivation. PS-C4BP can still mediate inactivation of FVIIIa but does so with less intensity than free PS. Therefore, the interaction of PS with C4BP may alter the activity of PS rather than inhibit it.119 Protein S and TFPI Early experiments involving PS showed that it possessed anticoagulant activity independent of PC. These studies went on to show that in fact PS acted as a cofactor to TFPI. PS decreases the rate-limiting step of FXa-TFPI complex formation 10-fold, hastening FXa inhibition.92 The Kunitz-3 domain on TFPI is suspected to be the binding site for PS. The PS-C4BP complex has incomplete activity as a TFPI cofactor (approximately 60% as effective as PS alone).119 Measurement PS can be measured in plasma, but the measurement is complicated by the fact that it exists in 2 forms: free and as a complex with PS-C4BP.92 Clot-based activity assay tests are based on the ability of PS to act as a cofactor for APC. Other tests include a free PS antigen assay (ELISA and latex particle agglutination) and a total PS antigen assay.115 As stated above, PS-C4BP has incomplete activity as a cofactor, and the free PS is considered to have more biological activity. Therefore, it is important to measure the allocation of these 2 forms, and this can be achieved by crossed immunoelectrophoresis.123 PS in Disease Natural PS deficiency is uncommon.124 There are 3 types of congenital PS deficiencies in humans. In type I, there is a low amount of activity, total, and free PS. Type II deficiency is associated with a functional abnormality with a normal amount of PS, and in type III, there is low free PS but normal total PS.125 PS deficiency is an autosomal-dominant disorder. Homozygous deficiency is potentially fatal after birth if untreated,126 whereas heterozygous deficiency of PS is associated with increased risk of venous thrombosis and stroke.127 In severe sepsis, a deficit of PS levels occurs secondary to a decrease in production, an increase in consumption, or utilization and inhibition.128 Therapeutic Applications of PS PS is currently not used as a therapeutic intervention in either human or veterinary medicine. Individuals with PS deficiency are treated supportively for thromboembolic disease or ischemic stroke if this occurs.129
Mechanism of Action
Conclusions
PS’s ultimate role as an antithrombotic is to downregulate thrombin formation.115 Smaller roles involve complement regulation, fibrinolysis, and other defense mechanisms.116,120
The endogenous anticoagulants are a varied group of proteins that act to limit or minimize coagulation either alone or in concert with each other. Endogenous anticoagulants can act as markers for mortality and have been investigated as therapeutic interventions for sepsis and other disease states. The scientific exploration into the role and use of endogenous anticoagulants will hopefully increase in the future in veterinary medicine.
Protein S and APC PS increases the reaction rate of proteolysis by APC of FVa by approximately 20-fold and inhibits FXa-dependent protection of FVa to the actions of APC.92 PS also augments the APC-mediated inactivation of FVIIIa.119 In order for PS to exert activity as a cofactor for APC, PS must bind to phospholipid membranes via its glargine (Gla) domain as is required by other vitamin K– dependent factors.121 Previous literature has reported that adding C4BP to plasma will counteract the PS-dependent prolongation of clotting time in the presence of APC, and secondary to this, it was thought that the cofactor activity for APC was lost upon binding of PS to C4BP.122 More recent literature has shown that the PS-C4BP complex is still an active
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