- Email: [email protected]
Editorial III
Editorial III
choice of the best transfusion strategy. Curr Med Res Opin 1996; 8: 465±78 4 Lown JA, Barr AL, Jackson JM. A reappraisal of pre-transfusion testing procedures in a hospital blood bank. Pathology 1985; 17: 489±92 5 Association of Anaesthesists of Great Britain and Ireland. Blood
Transfusion and the Anaesthesist. Red Cell Transfusion. Association of Anaesthesists of Great Britain and Ireland, 2001 6 Scottish Intercollegiate Guidelines Network. Perioperative Blood Transfusion for Elective Surgery. A National Clinical Guideline. SIGN 54, Edinburgh, 2001
Editorial III In¯ammation and the coagulation system The syndrome of multiple organ failure has been recognised for at least 30 yr and is one of the leading causes of mortality in intensive care. Patients develop a spectrum of different organs being affected, as well as degrees of severity of failure, and the term multi-organ dysfunction syndrome (MODS) is now more commonly used.1 The aetiology of MODS is still not clear although it is considered to result from severe and generalized in¯ammation. There are two current theories: abnormal cellular metabolism, and abnormal tissue blood ¯ow. Abnormalities of cellular metabolism are common, and lactic acidosis in severe sepsis is frequently observed. It is believed that the lactate accumulation results from widespread anaerobic respiration caused by either alterations in the microcirculation (oxygen delivery to tissues) or abnormalities in oxygen utilization by the tissues. The simple idea that anaerobic metabolism occurs as a result of tissue hypoxia and hypoperfusion is no longer tenable since it is known that tissue PO2 is often increased above normal in the septic patient. The mechanism for the defect in the microcirculation is also not entirely understood, although altered rheology or a decrease in ¯ow resulting from interstitial oedema as a result of increased microvascular permeability have both been implicated. Another possibility is that the microvasculature is simply blocked by microthrombi or microvascular ®brin deposition. Activation of platelet and coagulation pathways is a common feature of sepsis,2 and consumption of clotting factors and endogenous anticoagulants such as antithrombin III (AT III) and protein C is well recognized. However, true disseminated intravascular coagulation (DIC) in sepsis is rare except in some speci®c conditions such as meningococcal septicaemia. Hence the severity of coagulation abnormality ranges from small changes in platelet count and subclinical alterations in global clotting time to fullblown DIC. Histological studies in patients with DIC induced by sepsis show areas of ischaemia and necrosis associated with ®brin deposition in small and mid-sized vessels of a variety of organs. Also, experimental animal studies of sepsis show ®brin deposition in various organs and this is prevented by a variety of haematological
treatments designed to correct the coagulation system defects.3 4 Clinical studies involving patients with and without MODS who all had evidence of DIC have shown that activation of ®brinolysis may be an important protective mechanism preventing MODS in patients with DIC.5 The crucial role of the endothelium in the aetiology of multiorgan dysfunction is clear and, perhaps even more importantly, that of the microvascular endothelium. However, the link between coagulation and in¯ammation, and how the effects of therapeutically modifying one will affect the other, is not so well appreciated. Figure 1 is a simpli®ed diagram of the coagulation and ®brinolytic systems and illustrates possible links in the in¯ammatory process. The links between in¯ammation and coagulation are becoming increasingly understood, and are thought to be very important. For example, it is now known that Factor Xa induces the expression of a range of in¯ammatory cytokines such as interleukin 6 (IL-6) and IL-8 as well as adhesion molecules in endothelial cells in culture.6 Thus, activation of the clotting system can promote in¯ammation by enhancing leucocyte adhesion and activation. A number of new strategies for altering this system have been suggested. The in vivo initiation of the coagulation cascade is triggered by the binding of plasma Factor VIIa to the cell surface receptor, tissue factor (TF). TF is constitutively expressed in adventitial cells and pericytes surrounding blood vessels, but not in cells that come into contact with blood, such as endothelial cells. Tissue injury, which disrupts the endothelial cell barrier, is normally required for Factor VIIa to come into contact with TF. However, in pathological states, monocytes and endothelial cells can be stimulated to express TF.7±9 The in¯ammatory response following exposure to lipopolysaccharide (LPS) or tumour necrosis factor (TNF) is one example. It is now known that TF expression is regulated by activator protein 1 (AP1) and nuclear factor kappa B (NFkB) (two transcription factors known to regulate other mediators of in¯ammation).10 Human volunteers infused with TNF and animals given endotoxin show no change in markers of the activation of the contact system of the clotting cascade. Furthermore, animal experiments where inhibition of the TF/Factor VIIa
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Fig 1 The link between coagulation, ®brinolysis and in¯ammation, with the interactions between the various components shown as either a negative effect (dashed line) or positive or enhancing effect (solid line). APC, activated protein C; EPCR, endothelial protein C receptor; NFkB, nuclear factor kappa B; PAI, plasminogen activator inhibitor; TF, tissue factor; TFPI, tissue factor pathway inhibitor.
pathway was achieved by administration of monoclonal antibodies showed no evidence of DIC following infusion of endotoxin or live Escherichia coli.11 12 An impairment of the various natural regulators of coagulation activation may contribute to ®brin formation. Plasma levels of the most important inhibitor of thrombin, ATIII, are usually markedly reduced in sepsis. This is caused by a combination of increased consumption, degradation by elastase released from activated neutrophils, and also impaired synthesis. In addition, there is a decrease in the protein C/protein S system which also enhances the procoagulant state.13 It has now been suggested that, as well as being an important factor in the activation of coagulation, activated protein C (APC) may also play a role as a regulator of in¯ammation in sepsis.14 TF is inhibited by tissue factor pathway inhibitor (TFPI). This can effectively block the generation of thrombin in human volunteers given endotoxin,15 and reduces mortality in a lethal baboon model of sepsis.3 Unstimulated endothelial cells grown in culture constitutively express TFPI mRNA, and its levels either did not change or increased slightly (up to 1.5-fold) on stimulation with either LPS or TNF.16 Moreover, in vivo, unlike other coagulation system inhibitors, TFPI concentrations are usually not reduced in sepsis.
Heparin Heparin can at least partly inhibit the activation of coagulation in sepsis-induced as well as other forms of DIC.17 However, there is no evidence of a bene®cial role in
MODS. A recent study has shown that heparin causes an increase in release of TFPI into the medium of endothelial cells in culture and a rapid increase in TFPI mRNA.18 The procoagulant activity of the cells was downregulated by 36% and the anticoagulant potency of the cells was moderately increased after 24 h heparin stimulation. In a randomized double-blind placebo-controlled trial, 30 healthy male volunteers were given endotoxin (LPS) 2 ng kg±1 i.v. followed by an infusion of either unfractionated or fractionated heparin, or placebo.19 In the placebo group, activation of coagulation was observed, with marked increases in plasma levels of prothrombin fragment F(1+2) (P<0.01) and polymerized soluble ®brin; the number of TF-positive monocytes doubled in response to LPS, whereas levels of activated Factor VII slightly decreased and levels of TFPI remained unchanged. Both forms of heparin markedly decreased activation of coagulation caused by LPS, and TFPI values increased after either heparin infusion (P<0.01). Thus, heparin has been shown to be useful in established DIC and can prevent the activation of coagulation seen in sepsis. It has not, however, been shown to be useful in preventing MODS in patients with sepsis.
Inhibitors of TF activation There have been trials which have examined the possibility of blocking TF activation in sepsis. The use of recombinant TFPI to block endotoxin-induced thrombin generation has produced some promising results,15 20 but a large multicentre study which enrolled approximately 2000 patients
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has not con®rmed a bene®cial effect on outcome from severe sepsis.21 In a study in which eight human volunteers were given a bolus injection of endotoxin 4 ng kg±1 i.v. followed by a 6 h continuous infusion of either TFPI 0.2 mg±1 kg h±1 after a bolus of 0.05 mg kg±1 or placebo, activation of coagulation was completely prevented by TFPI.22 In contrast, TFPI did not in¯uence leucocyte activation, chemokine release, endothelial cell activation or the acute phase response. The conclusion would appear to be that complete prevention of coagulation activation by TFPI does not in¯uence activation of in¯ammatory pathways during human endotoxaemia. The reason for this differential effect is unclear; however, it is interesting that the large clinical study failed to demonstrate bene®t. Perhaps this lack of effect on the in¯ammatory process in sepsis explains the lack of ef®cacy in the clinical setting. Nitric oxide has antithrombotic actions in the vasculature, and its role in the regulation of TF expression has recently been studied in human microvascular endothelial cells stimulated with LPS or IL-1b to form TF.23 When added to the media, L-arginine (the principal substrate for nitric oxide synthases) signi®cantly suppressed the induction of TF activity (by 66%) at 24 h. D-Arginine had no effect, and inhibition of endogenous nitric oxide production failed to increase TF expression. It could therefore be envisaged that enhanced production of nitric oxide would reduce expression of TF and, thereby, the prothrombotic characteristics of the endothelial cells exposed to LPS. An alternative approach may be the new potent and speci®c inhibitor of the complex formed between TF/Factor VIIa and Factor Xa±rNAPc2.24 Nematode anticoagulant proteins (NAP) were originally isolated from hookworm nematodes and are currently being investigated in patients with DIC. Another possibility is to competitively block the cell surface receptor of TF by administration of an inactivated form of Factor VIIa. In a preliminary study in a rabbit model, infusion of a low concentration of inactivated Factor VIIa inhibited the endotoxin-induced decrease in platelet count and ®brinogen levels and abolished the deposition of ®brin in kidneys.25 In a model of acute lung injury, in which adult baboons were primed with killed E. coli (13109 CFU kg±1) and bacteraemic sepsis was induced 12 h later by infusion of live E. coli at 131010 CFU kg±1, inactivated FVIIa dramatically protected gas exchange and lung compliance, prevented lung oedema and pulmonary hypertension, and preserved renal function compared with placebo (all P<0.05).26 Treatment also attenuated ®brinogen depletion (P<0.01) and decreased the concentrations of proin¯ammatory cytokines.
Antithrombin III ATIII is one of the most important physiological inhibitors of coagulation, and ATIII treatment in animal models of sepsis has shown promise. However, a large multicentre
randomized controlled trial in humans with severe sepsis failed to show any survival bene®t.27 A total of 2314 adults were randomized into two equal groups to receive either i.v. ATIII (30 000 IU in total over 4 days) or a placebo (1% human albumin). The overall mortality at 28 days was 38.9% in the ATIII treatment group and 38.7% in the placebo group (P=0.94). Secondary end-points, including mortality at 56 and 90 days and survival time in the intensive care unit, did not differ between the ATIII and placebo groups. Therefore, despite a compelling series of clinical and laboratory animal trials, the use of ATIII in high doses in this large carefully conducted multicentre clinical trial failed to achieve ef®cacy in the primary study end-point of 28-day all-cause mortality. Various possibilities have been suggested, including the lower than expected frequency of reduced levels of circulating ATIII at study entry, and the lower than expected levels of ATIII achieved in the blood after 24 h in recipients of ATIII. Also, to promote local anticoagulant activity and anti-in¯ammatory activities, ATIII must bind to glycosaminoglycans on endothelial surfaces and to in¯ammatory cells such as polymorphonuclear leucocytes. It has been demonstrated that heparin competitively inhibits the binding of ATIII to other glycosaminoglycans, and it is interesting in this context that the subgroup in this phase III study who were not receiving heparin appeared to bene®t from the high-dose ATIII therapy. ATIII has been reported to have some in¯uence on the in¯ammatory process but it appears to have this effect indirectly via enhanced prostacyclin release from endothelial cells.28
Protein C Protein C is a circulating protein which is an inactive precursor of a protease and is converted to APC in the presence of the thrombin/thrombomodulin complex. There is a protein C receptor, called endothelial protein C receptor, which facilitates activation of protein C; however it is not an absolute requirement for activation. APC inactivates Factors Va and VIIIa and so limits thrombin generation29 and also promotes ®brinolysis by inhibiting activity of plasminogen activator inhibitor 1.30 It is also suggested that APC may reduce in¯ammation by inhibiting cytokine production and white cell activation.31 APC was shown to have a protective role against MODS and mortality in a baboon model of sepsis,32 and has subsequently been shown to decrease mortality in humans with severe sepsis.33 34 In a recent study of 1690 patients with severe sepsis, recombinant human activated protein C (Drotrecogin alfa [activated]), 24 mg±1 kg h±1 for a total duration of 96 h, was shown to decrease the 28-day mortality rate from 30.8% to 24.7%.34 Treatment with Drotrecogin alfa (activated) was associated with a reduction in the relative risk of death of 19.4% (95% con®dence interval, 6.6±30.5) and an absolute reduction in the risk of death of 6.1% (P=0.005). The incidence of serious bleeding was higher in the Drotrecogin
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alfa (activated) treated group than in the placebo group (3.5 vs 2.0%, P=0.06). Results regarding longer term survival are still awaited. The interesting question is why this approach has succeeded when others have apparently failed. The antiin¯ammatory properties are clearly signi®cant and it is interesting that patients given Drotrecogin alfa (activated) consistently had lower plasma concentrations of IL-6. It is suggested that, in addition to its antithrombotic and pro®brinolytic properties, APC also acts as a modulator of the in¯ammatory process, possibly through its effect on the transcription factor NFkB. APC thereby alters the cytokine pro®le in patients with severe sepsis and it is this effect, rather than its antithrombotic properties, that have resulted in bene®cial effect.
Conclusion LPS is a major trigger of sepsis-induced DIC via the TF/ Factor VIIa-dependent pathway of coagulation. Various therapeutic options are now available to modulate the coagulation system in patients with sepsis. However, it is interesting that so far the only agent that has antithrombotic and pro®brinolytic properties and has proven useful in humans with sepsis also appears to have signi®cant effects on in¯ammatory mediators.
7
8 9 10 11
12 13 14 15
²
Nigel R. Webster Academic Unit of Anaesthesia and Intensive Care Institute of Medical Sciences Foresterhill Aberdeen AB25 2ZD UK
16 17 18
References 1 Bone RC, Balk RA, Cerra FB, et al. De®nitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 1992; 201: 1644±55 2 Mavorommatis AC, Theodorisis T, Ofanidou A, et al. Coagulation system and platelets are fully activated in uncomplicated sepsis. Crit Care Med 2000; 28: 451±7 3 Creasey AA, Chang AC, Feigen L, et al. Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock. J Clin Invest 1993; 91: 2850±6 4 Kessler CM, Tang Z, Jacobs HM, et al. The suprapharmacologic dosing of antithrombin concentrate for Staphylococcus aureus induced disseminated intravascular coagulation in guinea pigs: Substantial reduction in mortality and morbidity. Blood 1997; 89: 4393±401 5 Asakura H, Ontachi Y, Mizutani T, et al. An enhanced ®brinolysis prevents the development of multiple organ failure in disseminated intravascular coagulation in spite of much activation of blood coagulation. Crit Care Med 2001; 29: 1164±8 6 Senden NH, Jeunhomme TM, Heemskerk JW, et al. Factor Xa induces cytokine production and expression of adhesion
19 20
21 22 23 24
²
Declaration of interest: Professor N. R. Webster is a member of the Drotrecogin alfa (activated) Advisory Board of Eli Lilly and Company Ltd.
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25
molecules by human umbilical vein endothelial cells. J Immunol 1998; 161: 4318±24 Zhang Y, Deng Y, Wendt T, et al. Intravenous somatic gene transfer with antisense tissue factor restores blood ¯ow by reducing tumor necrosis factor-induced tissue factor expression and ®brin deposition in mouse meth-A sarcoma. J Clin Invest 1996; 97: 2213±24 Osterud B. Cellular interactions in tissue factor expression by blood monocytes. Blood Coagul Fibrinolysis 1995; 6 Suppl 1: S20±5 Osterud B. Tissue factor expression by monocytes: regulation and pathophysiological roles. Blood Coagul Fibrinolysis 1998; 9 Suppl 1: S9±14 Parry GC, Mackman N. Transcriptional regulation of tissue factor expression in human endothelial cells. Arterioscler Thromb Vasc Biol 1995; 15: 612±21 Levi M, ten Cate H, Bauer KA, et al. Inhibition of endotoxininduced activation of coagulation and ®brinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees. J Clin Invest 1994; 93: 114±20 Taylor FBJ, Chang A, Ruf W, et al. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock 1991; 33: 127±34 Conway EM, Rosenberg RD. Tumor necrosis factor suppresses transcription of the thrombomodulin gene in endothelial cells. Mol Cell Biol 1988; 8: 5588±92 Esmon CT. Introduction: are natural anticoagulants candidates for modulating the in¯ammatory response to endotoxin? Blood 2000; 95: 1113±6 De Jong E, Dekkers PE, Creasey AA, et al. Tissue factor pathway inhibitor (TFPI) dose-dependently inhibits coagulation activation without in¯uencing the ®brinolytic and cytokine response during human endotoxemia. Blood 2000; 95: 1124±9 Ameri A, Kuppuswamy MN, Basu S, Bajaj SP. Expression of tissue factor pathway inhibitor by cultured endothelial cells in response to in¯ammatory mediators. Blood 1992; 79: 3219±26 Du Toit H, Coetzee AR, Chalton DO. Heparin treatment in thrombin-induced disseminated intravascular coagulation in the baboon. Crit Care Med 1991; 19: 1195±200 Hansen JB, Svensson B, Olsen R, Ezban M, Osterud B, Paulssen RH. Heparin induces synthesis and secretion of tissue factor pathway inhibitor from endothelial cells in vitro. Thromb Haemost 2000; 83: 937±43 Pernerstorfer T, Hollenstein U, Hansen J, et al. Heparin blunts endotoxin-induced coagulation activation. Circulation 1999; 100: 2485±90 Abraham E, Reinhart K, Svoboda P, et al. Assessment of the safety of recombinant tissue factor pathway inhibitor in patients with severe sepsis: A multicenter, randomised, placebocontrolled, single blind, dose escalation study. Crit Care Med 2001; 29: 2081±9 Chiron press release http://www.prnewswire.com/cgi-bin/ micro_stories.pl?ACCT=131167&TICK=CHIR&STORY=/www/ story/11±21±2001/0001620218&EDATE=Nov+21,+2001/ de Jonge E, Dekkers PE, Creasey AA, et al. Tissue factor pathway inhibitor does not in¯uence in¯ammatory pathways during human endotoxemia. J Infect Dis 2001; 183: 1815±8 Yang Y, Loscalzo J. Regulation of tissue factor expression in human microvascular endothelial cells by nitric oxide. Circulation 2000; 101: 2144±8 Bergum PW, Cruikshank A, Maki S, et al. The potent Factor X(a)dependent inhibition by rNAPc2 of Factor VIIa/tissue factor involves the binding of its cofactor to an exosite on Factor VII, followed by occupation of the active site. Blood 1998; 92: 669a Holst J, Kristensen AT, Kristensen HI, Ezban M, Hedner U. Local
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26 27 28
29
application of recombinant active-site inhibited human clotting factor VIIa reduces thrombus weight and improves patency in a rabbit venous thrombosis model. Eur J Vasc Endovasc Surg 1998; 15: 515±20 Welty-Wolf KE, Carraway MS, Miller DL, et al. Coagulation blockade prevents sepsis-induced respiratory and renal failure in baboons. Am J Respir Crit Care Med 2001; 164: 1988±96 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 2001; 286: 1869±78 Hoffmann JN, Vollmar B, Inthorn D, Schildberg FW, Menger MD. Antithrombin reduces leukocyte adhesion during chronic endotoxemia by modulation of the cyclooxygenase pathway. Am J Physiol Cell Physiol 2000; 279: C98±107 Rosenberg RD, Aird WC. Vascular bed speci®c hemostasis and hypercoagulable states. N Engl J Med 1999; 340: 1555±64
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30 Sakata Y, Loskutoff DJ, Gladson CL, et al. Mechanism of protein C dependent clot lysis: Role of plasminogen activator inhibitor. Blood 1986; 68: 1218±23 31 Muakami K, Okajima K, Uchiba M, et al. Activated protein C attenuates endotoxin induced pulmonary vascular injury by inhibiting activated leukocytes in rats. Blood 1996; 87: 642±7 32 Taylor FB, Chank A, Esmon CT, et al. Protein C prevents the coagulopathies and lethal effects of E. coli infusion in the baboon. J Clin Invest 1987; 79: 918±25 33 Bernard GR, Ely E, Wright TJ, et al. Safety and dose relationship of recombinant human activated protein C for coagulopathy in severe sepsis. Crit Care Med 2001; 29: 2051±9 34 Bernard GR, Vincent JL, Laterre PF, et al. Ef®cacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699±709
choice of the best transfusion strategy. Curr Med Res Opin 1996; 8: 465±78 4 Lown JA, Barr AL, Jackson JM. A reappraisal of pre-transfusion testing procedures in a hospital blood bank. Pathology 1985; 17: 489±92 5 Association of Anaesthesists of Great Britain and Ireland. Blood
Transfusion and the Anaesthesist. Red Cell Transfusion. Association of Anaesthesists of Great Britain and Ireland, 2001 6 Scottish Intercollegiate Guidelines Network. Perioperative Blood Transfusion for Elective Surgery. A National Clinical Guideline. SIGN 54, Edinburgh, 2001
Editorial III In¯ammation and the coagulation system The syndrome of multiple organ failure has been recognised for at least 30 yr and is one of the leading causes of mortality in intensive care. Patients develop a spectrum of different organs being affected, as well as degrees of severity of failure, and the term multi-organ dysfunction syndrome (MODS) is now more commonly used.1 The aetiology of MODS is still not clear although it is considered to result from severe and generalized in¯ammation. There are two current theories: abnormal cellular metabolism, and abnormal tissue blood ¯ow. Abnormalities of cellular metabolism are common, and lactic acidosis in severe sepsis is frequently observed. It is believed that the lactate accumulation results from widespread anaerobic respiration caused by either alterations in the microcirculation (oxygen delivery to tissues) or abnormalities in oxygen utilization by the tissues. The simple idea that anaerobic metabolism occurs as a result of tissue hypoxia and hypoperfusion is no longer tenable since it is known that tissue PO2 is often increased above normal in the septic patient. The mechanism for the defect in the microcirculation is also not entirely understood, although altered rheology or a decrease in ¯ow resulting from interstitial oedema as a result of increased microvascular permeability have both been implicated. Another possibility is that the microvasculature is simply blocked by microthrombi or microvascular ®brin deposition. Activation of platelet and coagulation pathways is a common feature of sepsis,2 and consumption of clotting factors and endogenous anticoagulants such as antithrombin III (AT III) and protein C is well recognized. However, true disseminated intravascular coagulation (DIC) in sepsis is rare except in some speci®c conditions such as meningococcal septicaemia. Hence the severity of coagulation abnormality ranges from small changes in platelet count and subclinical alterations in global clotting time to fullblown DIC. Histological studies in patients with DIC induced by sepsis show areas of ischaemia and necrosis associated with ®brin deposition in small and mid-sized vessels of a variety of organs. Also, experimental animal studies of sepsis show ®brin deposition in various organs and this is prevented by a variety of haematological
treatments designed to correct the coagulation system defects.3 4 Clinical studies involving patients with and without MODS who all had evidence of DIC have shown that activation of ®brinolysis may be an important protective mechanism preventing MODS in patients with DIC.5 The crucial role of the endothelium in the aetiology of multiorgan dysfunction is clear and, perhaps even more importantly, that of the microvascular endothelium. However, the link between coagulation and in¯ammation, and how the effects of therapeutically modifying one will affect the other, is not so well appreciated. Figure 1 is a simpli®ed diagram of the coagulation and ®brinolytic systems and illustrates possible links in the in¯ammatory process. The links between in¯ammation and coagulation are becoming increasingly understood, and are thought to be very important. For example, it is now known that Factor Xa induces the expression of a range of in¯ammatory cytokines such as interleukin 6 (IL-6) and IL-8 as well as adhesion molecules in endothelial cells in culture.6 Thus, activation of the clotting system can promote in¯ammation by enhancing leucocyte adhesion and activation. A number of new strategies for altering this system have been suggested. The in vivo initiation of the coagulation cascade is triggered by the binding of plasma Factor VIIa to the cell surface receptor, tissue factor (TF). TF is constitutively expressed in adventitial cells and pericytes surrounding blood vessels, but not in cells that come into contact with blood, such as endothelial cells. Tissue injury, which disrupts the endothelial cell barrier, is normally required for Factor VIIa to come into contact with TF. However, in pathological states, monocytes and endothelial cells can be stimulated to express TF.7±9 The in¯ammatory response following exposure to lipopolysaccharide (LPS) or tumour necrosis factor (TNF) is one example. It is now known that TF expression is regulated by activator protein 1 (AP1) and nuclear factor kappa B (NFkB) (two transcription factors known to regulate other mediators of in¯ammation).10 Human volunteers infused with TNF and animals given endotoxin show no change in markers of the activation of the contact system of the clotting cascade. Furthermore, animal experiments where inhibition of the TF/Factor VIIa
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Fig 1 The link between coagulation, ®brinolysis and in¯ammation, with the interactions between the various components shown as either a negative effect (dashed line) or positive or enhancing effect (solid line). APC, activated protein C; EPCR, endothelial protein C receptor; NFkB, nuclear factor kappa B; PAI, plasminogen activator inhibitor; TF, tissue factor; TFPI, tissue factor pathway inhibitor.
pathway was achieved by administration of monoclonal antibodies showed no evidence of DIC following infusion of endotoxin or live Escherichia coli.11 12 An impairment of the various natural regulators of coagulation activation may contribute to ®brin formation. Plasma levels of the most important inhibitor of thrombin, ATIII, are usually markedly reduced in sepsis. This is caused by a combination of increased consumption, degradation by elastase released from activated neutrophils, and also impaired synthesis. In addition, there is a decrease in the protein C/protein S system which also enhances the procoagulant state.13 It has now been suggested that, as well as being an important factor in the activation of coagulation, activated protein C (APC) may also play a role as a regulator of in¯ammation in sepsis.14 TF is inhibited by tissue factor pathway inhibitor (TFPI). This can effectively block the generation of thrombin in human volunteers given endotoxin,15 and reduces mortality in a lethal baboon model of sepsis.3 Unstimulated endothelial cells grown in culture constitutively express TFPI mRNA, and its levels either did not change or increased slightly (up to 1.5-fold) on stimulation with either LPS or TNF.16 Moreover, in vivo, unlike other coagulation system inhibitors, TFPI concentrations are usually not reduced in sepsis.
Heparin Heparin can at least partly inhibit the activation of coagulation in sepsis-induced as well as other forms of DIC.17 However, there is no evidence of a bene®cial role in
MODS. A recent study has shown that heparin causes an increase in release of TFPI into the medium of endothelial cells in culture and a rapid increase in TFPI mRNA.18 The procoagulant activity of the cells was downregulated by 36% and the anticoagulant potency of the cells was moderately increased after 24 h heparin stimulation. In a randomized double-blind placebo-controlled trial, 30 healthy male volunteers were given endotoxin (LPS) 2 ng kg±1 i.v. followed by an infusion of either unfractionated or fractionated heparin, or placebo.19 In the placebo group, activation of coagulation was observed, with marked increases in plasma levels of prothrombin fragment F(1+2) (P<0.01) and polymerized soluble ®brin; the number of TF-positive monocytes doubled in response to LPS, whereas levels of activated Factor VII slightly decreased and levels of TFPI remained unchanged. Both forms of heparin markedly decreased activation of coagulation caused by LPS, and TFPI values increased after either heparin infusion (P<0.01). Thus, heparin has been shown to be useful in established DIC and can prevent the activation of coagulation seen in sepsis. It has not, however, been shown to be useful in preventing MODS in patients with sepsis.
Inhibitors of TF activation There have been trials which have examined the possibility of blocking TF activation in sepsis. The use of recombinant TFPI to block endotoxin-induced thrombin generation has produced some promising results,15 20 but a large multicentre study which enrolled approximately 2000 patients
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has not con®rmed a bene®cial effect on outcome from severe sepsis.21 In a study in which eight human volunteers were given a bolus injection of endotoxin 4 ng kg±1 i.v. followed by a 6 h continuous infusion of either TFPI 0.2 mg±1 kg h±1 after a bolus of 0.05 mg kg±1 or placebo, activation of coagulation was completely prevented by TFPI.22 In contrast, TFPI did not in¯uence leucocyte activation, chemokine release, endothelial cell activation or the acute phase response. The conclusion would appear to be that complete prevention of coagulation activation by TFPI does not in¯uence activation of in¯ammatory pathways during human endotoxaemia. The reason for this differential effect is unclear; however, it is interesting that the large clinical study failed to demonstrate bene®t. Perhaps this lack of effect on the in¯ammatory process in sepsis explains the lack of ef®cacy in the clinical setting. Nitric oxide has antithrombotic actions in the vasculature, and its role in the regulation of TF expression has recently been studied in human microvascular endothelial cells stimulated with LPS or IL-1b to form TF.23 When added to the media, L-arginine (the principal substrate for nitric oxide synthases) signi®cantly suppressed the induction of TF activity (by 66%) at 24 h. D-Arginine had no effect, and inhibition of endogenous nitric oxide production failed to increase TF expression. It could therefore be envisaged that enhanced production of nitric oxide would reduce expression of TF and, thereby, the prothrombotic characteristics of the endothelial cells exposed to LPS. An alternative approach may be the new potent and speci®c inhibitor of the complex formed between TF/Factor VIIa and Factor Xa±rNAPc2.24 Nematode anticoagulant proteins (NAP) were originally isolated from hookworm nematodes and are currently being investigated in patients with DIC. Another possibility is to competitively block the cell surface receptor of TF by administration of an inactivated form of Factor VIIa. In a preliminary study in a rabbit model, infusion of a low concentration of inactivated Factor VIIa inhibited the endotoxin-induced decrease in platelet count and ®brinogen levels and abolished the deposition of ®brin in kidneys.25 In a model of acute lung injury, in which adult baboons were primed with killed E. coli (13109 CFU kg±1) and bacteraemic sepsis was induced 12 h later by infusion of live E. coli at 131010 CFU kg±1, inactivated FVIIa dramatically protected gas exchange and lung compliance, prevented lung oedema and pulmonary hypertension, and preserved renal function compared with placebo (all P<0.05).26 Treatment also attenuated ®brinogen depletion (P<0.01) and decreased the concentrations of proin¯ammatory cytokines.
Antithrombin III ATIII is one of the most important physiological inhibitors of coagulation, and ATIII treatment in animal models of sepsis has shown promise. However, a large multicentre
randomized controlled trial in humans with severe sepsis failed to show any survival bene®t.27 A total of 2314 adults were randomized into two equal groups to receive either i.v. ATIII (30 000 IU in total over 4 days) or a placebo (1% human albumin). The overall mortality at 28 days was 38.9% in the ATIII treatment group and 38.7% in the placebo group (P=0.94). Secondary end-points, including mortality at 56 and 90 days and survival time in the intensive care unit, did not differ between the ATIII and placebo groups. Therefore, despite a compelling series of clinical and laboratory animal trials, the use of ATIII in high doses in this large carefully conducted multicentre clinical trial failed to achieve ef®cacy in the primary study end-point of 28-day all-cause mortality. Various possibilities have been suggested, including the lower than expected frequency of reduced levels of circulating ATIII at study entry, and the lower than expected levels of ATIII achieved in the blood after 24 h in recipients of ATIII. Also, to promote local anticoagulant activity and anti-in¯ammatory activities, ATIII must bind to glycosaminoglycans on endothelial surfaces and to in¯ammatory cells such as polymorphonuclear leucocytes. It has been demonstrated that heparin competitively inhibits the binding of ATIII to other glycosaminoglycans, and it is interesting in this context that the subgroup in this phase III study who were not receiving heparin appeared to bene®t from the high-dose ATIII therapy. ATIII has been reported to have some in¯uence on the in¯ammatory process but it appears to have this effect indirectly via enhanced prostacyclin release from endothelial cells.28
Protein C Protein C is a circulating protein which is an inactive precursor of a protease and is converted to APC in the presence of the thrombin/thrombomodulin complex. There is a protein C receptor, called endothelial protein C receptor, which facilitates activation of protein C; however it is not an absolute requirement for activation. APC inactivates Factors Va and VIIIa and so limits thrombin generation29 and also promotes ®brinolysis by inhibiting activity of plasminogen activator inhibitor 1.30 It is also suggested that APC may reduce in¯ammation by inhibiting cytokine production and white cell activation.31 APC was shown to have a protective role against MODS and mortality in a baboon model of sepsis,32 and has subsequently been shown to decrease mortality in humans with severe sepsis.33 34 In a recent study of 1690 patients with severe sepsis, recombinant human activated protein C (Drotrecogin alfa [activated]), 24 mg±1 kg h±1 for a total duration of 96 h, was shown to decrease the 28-day mortality rate from 30.8% to 24.7%.34 Treatment with Drotrecogin alfa (activated) was associated with a reduction in the relative risk of death of 19.4% (95% con®dence interval, 6.6±30.5) and an absolute reduction in the risk of death of 6.1% (P=0.005). The incidence of serious bleeding was higher in the Drotrecogin
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alfa (activated) treated group than in the placebo group (3.5 vs 2.0%, P=0.06). Results regarding longer term survival are still awaited. The interesting question is why this approach has succeeded when others have apparently failed. The antiin¯ammatory properties are clearly signi®cant and it is interesting that patients given Drotrecogin alfa (activated) consistently had lower plasma concentrations of IL-6. It is suggested that, in addition to its antithrombotic and pro®brinolytic properties, APC also acts as a modulator of the in¯ammatory process, possibly through its effect on the transcription factor NFkB. APC thereby alters the cytokine pro®le in patients with severe sepsis and it is this effect, rather than its antithrombotic properties, that have resulted in bene®cial effect.
Conclusion LPS is a major trigger of sepsis-induced DIC via the TF/ Factor VIIa-dependent pathway of coagulation. Various therapeutic options are now available to modulate the coagulation system in patients with sepsis. However, it is interesting that so far the only agent that has antithrombotic and pro®brinolytic properties and has proven useful in humans with sepsis also appears to have signi®cant effects on in¯ammatory mediators.
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Nigel R. Webster Academic Unit of Anaesthesia and Intensive Care Institute of Medical Sciences Foresterhill Aberdeen AB25 2ZD UK
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References 1 Bone RC, Balk RA, Cerra FB, et al. De®nitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Chest 1992; 201: 1644±55 2 Mavorommatis AC, Theodorisis T, Ofanidou A, et al. Coagulation system and platelets are fully activated in uncomplicated sepsis. Crit Care Med 2000; 28: 451±7 3 Creasey AA, Chang AC, Feigen L, et al. Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock. J Clin Invest 1993; 91: 2850±6 4 Kessler CM, Tang Z, Jacobs HM, et al. The suprapharmacologic dosing of antithrombin concentrate for Staphylococcus aureus induced disseminated intravascular coagulation in guinea pigs: Substantial reduction in mortality and morbidity. Blood 1997; 89: 4393±401 5 Asakura H, Ontachi Y, Mizutani T, et al. An enhanced ®brinolysis prevents the development of multiple organ failure in disseminated intravascular coagulation in spite of much activation of blood coagulation. Crit Care Med 2001; 29: 1164±8 6 Senden NH, Jeunhomme TM, Heemskerk JW, et al. Factor Xa induces cytokine production and expression of adhesion
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Declaration of interest: Professor N. R. Webster is a member of the Drotrecogin alfa (activated) Advisory Board of Eli Lilly and Company Ltd.
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molecules by human umbilical vein endothelial cells. J Immunol 1998; 161: 4318±24 Zhang Y, Deng Y, Wendt T, et al. Intravenous somatic gene transfer with antisense tissue factor restores blood ¯ow by reducing tumor necrosis factor-induced tissue factor expression and ®brin deposition in mouse meth-A sarcoma. J Clin Invest 1996; 97: 2213±24 Osterud B. Cellular interactions in tissue factor expression by blood monocytes. Blood Coagul Fibrinolysis 1995; 6 Suppl 1: S20±5 Osterud B. Tissue factor expression by monocytes: regulation and pathophysiological roles. Blood Coagul Fibrinolysis 1998; 9 Suppl 1: S9±14 Parry GC, Mackman N. Transcriptional regulation of tissue factor expression in human endothelial cells. Arterioscler Thromb Vasc Biol 1995; 15: 612±21 Levi M, ten Cate H, Bauer KA, et al. Inhibition of endotoxininduced activation of coagulation and ®brinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees. J Clin Invest 1994; 93: 114±20 Taylor FBJ, Chang A, Ruf W, et al. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circ Shock 1991; 33: 127±34 Conway EM, Rosenberg RD. Tumor necrosis factor suppresses transcription of the thrombomodulin gene in endothelial cells. Mol Cell Biol 1988; 8: 5588±92 Esmon CT. Introduction: are natural anticoagulants candidates for modulating the in¯ammatory response to endotoxin? Blood 2000; 95: 1113±6 De Jong E, Dekkers PE, Creasey AA, et al. Tissue factor pathway inhibitor (TFPI) dose-dependently inhibits coagulation activation without in¯uencing the ®brinolytic and cytokine response during human endotoxemia. Blood 2000; 95: 1124±9 Ameri A, Kuppuswamy MN, Basu S, Bajaj SP. Expression of tissue factor pathway inhibitor by cultured endothelial cells in response to in¯ammatory mediators. Blood 1992; 79: 3219±26 Du Toit H, Coetzee AR, Chalton DO. Heparin treatment in thrombin-induced disseminated intravascular coagulation in the baboon. Crit Care Med 1991; 19: 1195±200 Hansen JB, Svensson B, Olsen R, Ezban M, Osterud B, Paulssen RH. Heparin induces synthesis and secretion of tissue factor pathway inhibitor from endothelial cells in vitro. Thromb Haemost 2000; 83: 937±43 Pernerstorfer T, Hollenstein U, Hansen J, et al. Heparin blunts endotoxin-induced coagulation activation. Circulation 1999; 100: 2485±90 Abraham E, Reinhart K, Svoboda P, et al. Assessment of the safety of recombinant tissue factor pathway inhibitor in patients with severe sepsis: A multicenter, randomised, placebocontrolled, single blind, dose escalation study. Crit Care Med 2001; 29: 2081±9 Chiron press release http://www.prnewswire.com/cgi-bin/ micro_stories.pl?ACCT=131167&TICK=CHIR&STORY=/www/ story/11±21±2001/0001620218&EDATE=Nov+21,+2001/ de Jonge E, Dekkers PE, Creasey AA, et al. Tissue factor pathway inhibitor does not in¯uence in¯ammatory pathways during human endotoxemia. J Infect Dis 2001; 183: 1815±8 Yang Y, Loscalzo J. Regulation of tissue factor expression in human microvascular endothelial cells by nitric oxide. Circulation 2000; 101: 2144±8 Bergum PW, Cruikshank A, Maki S, et al. The potent Factor X(a)dependent inhibition by rNAPc2 of Factor VIIa/tissue factor involves the binding of its cofactor to an exosite on Factor VII, followed by occupation of the active site. Blood 1998; 92: 669a Holst J, Kristensen AT, Kristensen HI, Ezban M, Hedner U. Local
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application of recombinant active-site inhibited human clotting factor VIIa reduces thrombus weight and improves patency in a rabbit venous thrombosis model. Eur J Vasc Endovasc Surg 1998; 15: 515±20 Welty-Wolf KE, Carraway MS, Miller DL, et al. Coagulation blockade prevents sepsis-induced respiratory and renal failure in baboons. Am J Respir Crit Care Med 2001; 164: 1988±96 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 2001; 286: 1869±78 Hoffmann JN, Vollmar B, Inthorn D, Schildberg FW, Menger MD. Antithrombin reduces leukocyte adhesion during chronic endotoxemia by modulation of the cyclooxygenase pathway. Am J Physiol Cell Physiol 2000; 279: C98±107 Rosenberg RD, Aird WC. Vascular bed speci®c hemostasis and hypercoagulable states. N Engl J Med 1999; 340: 1555±64
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30 Sakata Y, Loskutoff DJ, Gladson CL, et al. Mechanism of protein C dependent clot lysis: Role of plasminogen activator inhibitor. Blood 1986; 68: 1218±23 31 Muakami K, Okajima K, Uchiba M, et al. Activated protein C attenuates endotoxin induced pulmonary vascular injury by inhibiting activated leukocytes in rats. Blood 1996; 87: 642±7 32 Taylor FB, Chank A, Esmon CT, et al. Protein C prevents the coagulopathies and lethal effects of E. coli infusion in the baboon. J Clin Invest 1987; 79: 918±25 33 Bernard GR, Ely E, Wright TJ, et al. Safety and dose relationship of recombinant human activated protein C for coagulopathy in severe sepsis. Crit Care Med 2001; 29: 2051±9 34 Bernard GR, Vincent JL, Laterre PF, et al. Ef®cacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699±709