Anticoagulant Factor Concentrates in Disseminated Intravascular Coagulation: Rationale for Use and Clinical Experience

Transcription

1 Anticoagulant Factor Concentrates in Disseminated Intravascular Coagulation: Rationale for Use and Clinical Experience Evert de Jonge, M.D., 1 Tom van der Poll, M.D., 2 Jozef Kesecioglu, M.D., 1 and Marcel Levi, M.D. 3 ABSTRACT Natural inhibitors of coagulation, in other words, antithrombin (AT), the protein C system, and tissue factor pathway inhibitor (TFPI), play an important role in controlling the activation of coagulation during disseminated intravascular coagulation (DIC). Furthermore, they may not only influence coagulation but also attenuate inflammatory responses during sepsis. Low circulating levels of AT and protein C have been associated with poor outcome. Replacement therapy with AT, activated protein C (APC), and TFPI has been shown to attenuate thrombin generation and to reduce mortality in experimental sepsis models. Experience with AT and APC in patients is promising. Data from large phase III trials of AT and APC as treatment of patients with severe sepsis will soon be available. Recombinant TFPI is currently in phase II clinical trials for severe sepsis. KEYWORDS: Sepsis, antithrombin, protein C, tissue factor pathway inhibitor, TFPI, inflammation Objectives: Upon completion of this article, the reader should be able to (1) describe the natural inhibitor systems of clotting and their role in DIC and (2) summarize the rationale and experience gained for using inhibitor concentrates in managing patients with DIC. Accreditation: Tufts University School of Medicine is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians. TUSM takes full responsibility for the content, quality, and scientific integrity of this continuing education activity. Credit: Tufts University School of Medicine designates this education activity for a maximum of 1.0 hours credit toward the AMA Physicians Recognition Award in category one. Each physician should claim only those hours that he/she actually spent in the educational activity. DIC is a frequent complication of a wide variety of clinical conditions such as septicemia, trauma, cancer, obstetric complications and some intoxications. Massive activation of the coagulation system may result in generation and deposition of fibrin, leading to microvascular thrombi and contributing to the development of multiorgan failure. As platelets and coagulation factors are consumed, it also may lead to an increased risk of bleeding. In patients with sepsis, the severity of DIC has been shown to correlate with poor outcome. 1 Cornerstone for the treatment of DIC is the optimal management of the underlying disease. Traditionally, Seminars in Thrombosis and Hemostasis, volume 27, number 6, Address for correspondence and reprint requests: Evert de Jonge, M.D., Department of Intensive Care, Academic Medical Center, P.O. Box 22660, 1100 DD Amsterdam, The Netherlands. e.dejonge@amc.uva.nl. 1 Department of Intensive Care, 2 Laboratory of Experimental Internal Medicine, and 3 Department of Internal Medicine, Academic Medical Center, Amsterdam, The Netherlands. Copyright 2001 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) ,p;2001,27,06,667,674,ftx,en;sth00769x. 667

2 668 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 27, NUMBER replacement therapy with platelets and plasma is given to reduce the risk of bleeding and heparin to inhibit the activation of coagulation. However, these treatments are not based on firm evidence. 2 In this article, we will briefly discuss the pathogenesis of the activation of coagulation in DIC, and we will focus on the importance of the natural inhibitors of coagulation: AT, the protein C system, and TFPI in the pathogenesis of DIC. Furthermore, we will present current experience with treating DIC with natural anticoagulant factor concentrates. PATHOGENESIS OF DIC The systemic formation of fibrin during DIC results from increased activation of coagulation, impaired fibrinolysis, and simultaneous suppression of the physiological anticoagulant mechanisms. The derangements of coagulation and fibrinolysis appear to be mediated by cytokines, which are produced by the host in response to various pathogenetic insults. For example, in sepsis, microorganisms and their products, such as endotoxins, induce increased circulating cytokine levels, mainly produced by mononuclear cells in response to these products. The principal mediator of the activation of coagulation appears to be interleukin (IL)-6, whereas tumor necrosis factor (TNF) is the pivotal mediator in the dysregulation of fibrinolysis and the anticoagulant pathways. 3 It has been shown in animal models of sepsis that the activation of coagulation in DIC is initiated exclusively by the (extrinsic) tissue factor (TF)/factor VIIa dependent pathway. Under physiological conditions, TF cannot be detected on the luminal surface of the vascular endothelium 4 and only in very low quantities in circulating blood cells. 5,6 However, during infection and after stimulation with endotoxin or TNF, TF can be rapidly induced on blood mononuclear cells 5,7,8 and on vascular endothelium. 9 TF expression is increased not only during infection but also in other conditions associated with DIC, such as cancer 10 and possibly hypoxia. 11 Recent evidence suggests that TF may be present in an inactive, encrypted form and that the mere presence of TF on the cell surface is not sufficient for initiating blood coagulation. Some additional stimulus may be required to express this latent procoagulant activity. 12 TF binds and activates factor VIIa. The TF factor VIIa complexes formed can activate coagulation factors X and IX, ultimately leading to thrombin formation. It may be assumed that thrombin plays a pivotal role in further activation of systemic coagulation. Not only may thrombin affect several positive feedback loops (e.g., activation of factor IX, thereby resulting in even more factor Xa and thrombin generation), but it can also potently activate platelets. Activated platelets further facilitate coagulation activation. Evidence for the role of TF factor VIIa in activation of the coagulation system is derived from studies in primates, showing that the coagulant response to bacteremia or endotoxemia could be completely blocked by the simultaneous administration of monoclonal antibodies that are able to inhibit TF or factor VIIa activity, and infusion of active site inhibited factor VIIa 16 or TFPI. 17 Activation of coagulation by itself is not sufficient to lead to full-blown DIC with consumption of coagulation factors. The importance of the anticoagulant systems in the pathogenesis of DIC is illustrated in a series of baboon experiments by Taylor et al. 15,16 They have shown that activation of coagulation by intravenous infusion of factor Xa plus phospholipid particles produces high circulating levels of thrombin but without consumption of platelets or coagulation factors. However, when the anticoagulant protein C system was inhibited by pretreatment with monoclonal antibodies to protein C, thrombin generation after infusion of factor Xa with phospholipid particles was even higher and associated with a consumptive coagulopathy with depletion of platelets and fibrinogen. To protect against uncontrolled activation of coagulation, the human body possesses three major anticoagulant systems: AT, the protein C system, and TFPI. NATURAL ANTICOAGULANT PATHWAYS AND DIC Antithrombin AT is a serine protease inhibitor secreted by the liver with a molecular weight of 58 kd. AT is an important physiological regulator of blood coagulation that affects the intrinsic, extrinsic, and common pathways of coagulation. It has the capacity to inhibit thrombin; factors IXa, Xa, XIa, and XIIa; and the factor VIIa TF complex. 18,19 Heparin or other glycosaminoglycans, such as heparan sulfate, found on endothelial cell surfaces can bind to AT, thereby potentiating its enzymatic activity as a serine protease inhibitor by several orders of magnitude. 20 In contrast to other coagulation factors affected by AT, additional binding of heparin is required for AT to inhibit thrombin. Low-molecularweight heparin (LMWH) does not possess this additional binding potential, thereby explaining the lack of direct thrombin inhibitory activity with retained anticoagulant activity against other clotting factors. 21 In the absence of heparin, AT will bind to endothelial surfaces and function as an anticoagulant on the cell surface of the microcirculation. 20 In situations associated with DIC, such as sepsis, circulating AT levels are decreased due to increased consumption 22 and degradation by elastase released from activated neutrophils. 23 In sepsis, low AT levels are associated with increased mortality. 1 The first report of

3 ANTICOAGULANT FACTORS IN DIC/DE JONGE ET AL. 669 the efficacy of AT in experimental inflammation was published in Since that time, many studies have shown that administration of AT concentrate can result in an improvement of coagulation abnormalities, amelioration of organ failure, and, in some instances, reduction of mortality Because AT has shown promising results in animal models of DIC, the use of AT concentrates in patients with DIC has been studied relatively intensively. A number of controlled clinical trials, most of them concerning patients with sepsis, have been conducted Two meta-analyses of these trials were performed that suggested a decreased mortality in patients with sepsis when AT was administered. 34,37 One trial compared AT infusion to a synthetic protease inhibitor (Gabexate mesilate) in obstetric patients with DIC 38 and found that a single infusion of AT was more effective in controlling the symptoms of DIC but without effect on survival. It is not clear from the literature which patients will benefit most from AT treatment, and there are no clear-cut data defining the optimal plasma level to be obtained during supplementation. However, animal experiments suggest that high doses of AT, resulting in supranormal plasma levels, are required to reduce mortality. 28 In patients with sepsis the elimination half-life of AT is significantly reduced. Recently, it was shown that the mean AT response in patients with severe sepsis was 1.7% per IU/kg and that the mean half-life was 18.6 hours in these patients. 39 Administration of high doses of AT, aiming at plasma levels of more than 120%, appears to be safe and well-tolerated. 32,39 However, in one study, increased blood loss was found when the combination of AT and heparin was given as compared with either AT or heparin alone. 30 Recently, a large double-blind placebocontrolled multicenter phase III trial involving an estimated 2300 patients was performed to define conclusively the role of AT supplementation to 120% or more of normal in patients with severe sepsis. This study finished patient enrollment early in The complete results of this study should be reported soon. 40 In the meantime, physicians involved in the care of patients with sepsis and DIC may consider AT replacement an optional therapy that is safe and well-tolerated. Because it cannot be inferred from the literature which patients will benefit in terms of increased survival or reduced morbidity, it seems reasonable to reserve this expensive treatment for cases where mortality attributable to DIC is expected to be high and for patients with active DIC leading to substantial morbidity. THE PROTEIN C SYSTEM The protein C system is an important regulator of blood coagulation initiated by thrombin and serves as an ondemand mechanism for limiting the coagulation response to injury. The protein C pathway is initiated when thrombin binds to thrombomodulin (TM). When thrombin is bound to TM it loses its procoagulant properties, such as fibrinogen clotting, activation of factors V and VIII, and platelet activation, and turns into an anticoagulant by activating protein C. This activation is enhanced when protein C binds to the endothelial cell protein C receptor (EPCR). Once APC is formed, it proteolytically inactivates coagulation cofactors Va and VIIIa, thereby limiting further thrombin generation. APC activity is facilitated by its cofactor protein S. APC has a half-life, estimated at approximately 15 minutes. Inactivation is mediated by 1 -antitrypsin, protein C inhibitor, and 2 -macroglobulin. The importance of the protein C system is illustrated by the severe thrombotic complications seen in patients with deficiencies of members of this pathway. Infants with complete deficiency of protein C suffer from microvascular thrombosis of the skin (purpura fulminans). These lesions can be prevented by infusion of protein C concentrate. 41 Heterozygous deficiencies of protein C and protein S are associated with a moderately increased risk for thrombosis. Another risk factor for thrombosis, factor V Leiden, is caused by a mutation of factor V, rendering it resistant to proteolysis by APC. 42 Depression of the protein C system may contribute to a fatal outcome in sepsis. 43,44 The protein C system is impaired during sepsis because of different mechanisms. First, circulating cytokines can induce downregulation of TM and EPCR on endothelial cells, resulting in decreased activation of protein C Endothelial cell TM activity may also be reduced by proteolytic cleavage of TM by neutrophil elastase 48 or by eosinophil products. 49 Furthermore, circulating protein C and protein S levels are decreased during sepsis, probably because of increased consumption. 50 Also, because 1 -antitrypsin is an acute phase protein that increases in response to inflammation, inhibition of APC will increase. Finally, the acute phase protein C4bBP, which can bind protein S, is increased during severe illness, leading to lower levels of the biologically active free protein S. 50 The fact that TM binds thrombin, thereby blocking fibrinogen clotting and activating protein C, suggests that administration of soluble TM could improve the coagulation abnormalities in DIC. Soluble TM had a beneficial effect on the coagulation abnormalities in endotoxin and tissue factor induced DIC in rats 51,52 and crab-eating monkeys. 53 Furthermore, the endotoxin-induced increase in pulmonary vascular permeability in rats was also inhibited by recombinant human soluble TM (rhstm), and this effect appeared to depend on the protein C activating property of TM. 54 At this time, no reports have been published on studies investigating the effects of TM on DIC in patients. However, rhstm has been given to healthy volunteers without adverse effects being reported. 55,56 A large multicenter trial of the effects of rhstm on DIC is now being designed. 57

4 670 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 27, NUMBER Administration of APC has been shown to prevent the activation of coagulation as well as the lethal effects of E.coli infusion in a lethal baboon model of sepsis. In contrast, blocking the protein C pathway with monoclonal antibodies against protein C made the animals hyperresponsive to sublethal doses of E.coli. 58 Successful treatment with protein C or APC of patients with sepsis, usually due to meningococcemia, has been reported A recent phase II study of patients with severe sepsis suggested a trend toward improved survival, decreased time in the intensive care unit (ICU) and decreased circulating levels of IL-6 in the group treated with APC. 64 A recently published multicenter randomized and placebo-controlled phase III trial involving 1690 patients with systemic inflammation and organ failure, about half of whom were treated with APC, revealed an absolute risk reduction of 6.1% (p = 0.005) in favor of the treated group. 65 Serious bleeding was higher in the APC-treated patients (p = 0.06). The current experience with APC treatment for sepsis patients is thus promising. Because protein S levels may also be low in patients with sepsis and DIC, replacement therapy with protein S might be useful. In a baboon model of E. coli induced septic shock, protein S deficiency was induced by infusion of the acute phase protein C4bBP, which can bind to protein S. This acquired protein S deficiency exacerbated the response to sublethal levels of E. coli, leading to death. Supplementation with protein S to maintain normal free protein S levels prevented the excess mortality induced by C4bBP. 66 Thus, administration of protein S to patients with DIC might be effective. However, no data with protein S in humans are available at this time. Tissue Factor Pathway Inhibitor The rationale for use of TFPI for the treatment of DIC stems from the following observations: (1) the TF pathway plays a pivotal role in the initiation of coagulation in DIC, (2) TFPI is the major physiological inhibitor of the TF pathway, and (3) endothelial cells produce or release TFPI in response to injury. 67 Elimination of TF activity in endotoxemic or bacteremic primates results in a complete inhibition of coagulation activation. 13,15,17,68 In accordance with these results, we recently found that infusion of TFPI caused a dose-dependent reduction of the coagulant response to endotoxin in healthy humans in vivo. 69 TFPI is an approximately 43-kD, trivalent, Kunitz-type inhibitor that directly inhibits factor Xa with its second Kunitz domain. After factor Xa is bound, it rapidly inhibits the TF/factor VIIa complex with the first Kunitz domain. 70 Most of total TFPI is located in association with endothelial cells and only 10 to 25% is found in circulating blood. Circulating TFPI is predominantly bound to lipoproteins. 71 Blood platelets also carry native TFPI (about 10% of the plasma pool) that is released after stimulation by thrombin. 72 In vitro studies suggest that there might be a slight increase in TFPI produced by endothelial cells and monocytes by stimulation with endotoxin. 73 Furthermore, (slightly) increased levels of TFPI have been observed in a number of illnesses, including malignancy and septicemia In previous studies, plasma concentrations of TFPI increased only after severe injury. Thus, a sublethal dose of E. coli only induced a minimal, approximately 1.2-fold, increase in plasma TFPI levels, whereas infusion of an LD 100 dose of E. coli resulted in a twofold rise in plasma TFPI concentrations. 77 Thus, it may be hypothesized that TFPI increases during severe injury but relatively insufficiently during DIC. Administration of exogenous TFPI in disease states associated with DIC has been studied in many animal models. High-dose TFPI protected rabbits from the consumptive coagulopathy after administration of endotoxin and rabbit brain thromboplastin. 78,79 In different studies in septic baboons, TFPI prevented the activation of coagulation after E. coli bacteremia, 17,68,80 and in some studies reduced mortality. 68,69 In addition to the studies in bacteremic animals, TFPI was also tested in a rabbit peritonitis model. This model is possibly more relevant to human disease because it contains a localized focus of infection with relatively low numbers of bacteria, and it includes treatment with hydration and antibiotics. Furthermore, similar to human sepsis, the cause of death in this model is multiorgan failure and, specifically, respiratory failure. Septic rabbits treated with TFPI had reduced signs of DIC, lower mortality, and less organ failure. 81 Experience with recombinant TFPI administration in humans is limited to studies with healthy volunteers that showed that TFPI was safe, tolerable, and effective in inhibiting the endotoxin-induced thrombin generation. 69,82 Phase II trials of TFPI in septic patients are currently being conducted. NATURAL ANTICOAGULANTS AS MODULATORS OF THE INFLAMMATORY RESPONSE As described previously, natural anticoagulants can attenuate the coagulant response, reduce the clinical signs of DIC, and, in some cases, improve survival in septicemia. Experimental evidence suggests that the reduction in mortality may not be caused by the inhibition of coagulation itself but may rather be the result of diminished inflammatory responses. The diminished inflammatory response could be a direct result of preventing coagulation, because it has been shown that different

5 ANTICOAGULANT FACTORS IN DIC/DE JONGE ET AL. 671 activated coagulation factors such as thrombin, factor VIIa, and factor Xa can activate cells to release cytokines. 83 However, although AT, protein C, and TFPI 17,25,58 protected against DIC, as well as mortality, alternative anticoagulant treatments with heparin 84 and active site inhibited factor Xa 85 also effectively prevented the activation of coagulation in lethal primate models of sepsis, but without effect on lethality. Thus, it appears that other, noncoagulant, functions of the natural coagulation inhibitors are involved in their modulation of the inflammatory response. Antithrombin In a lethal baboon model of sepsis, AT not only inhibited thrombin generation but also attenuated the IL-6 and IL-8 response and increased survival. 26 In accordance with this, AT supplementation decreased circulating IL-6 and adhesion molecule levels in patients with severe sepsis. 35 These anti-inflammatory properties of AT could be a consequence of inhibition of activated coagulation factors, such as factor Xa, thrombin, and factor VIIa. Factor Xa, by binding to effector cell protease receptor-1 (EPR-1) independent of thrombin, has been found to trigger acute inflammatory responses in animal experiments. 86 Factor Xa was shown to stimulate cultured human endothelial cells to produce IL-6, IL-8, monocyte chemotactic protein-1 (MCP-1), the adhesion molecules se-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 by a mechanism independent of thrombin and EPR Thrombin is also implicated in cell activation and inflammation. Thrombin has been shown to stimulate IL-1, IL-6, IL-8, TNF, and MCP-1 release by monocytes and endothelial cells, probably mediated by thrombin catalytic activity, 88,95 and by cleavage of cell surface protease activated receptors (PARs). 96 Finally, factor VIIa could contribute to the inflammatory response, possibly by activation of PAR 2 or PAR An alternative explanation for the effects of AT on sepsis-induced cytokine production or release would be that AT has direct anti-inflammatory actions, independent of coagulation factors and by an unknown mechanism. The Protein C System APC inhibits monocyte activation by endotoxin, 98 mononuclear cell intracellular calcium signaling, 99 and it inhibits leukocyte adhesion to selectins. 100 These effects of APC occur independent of its influence on the coagulation system. As early as 1984, it was found that pretreatment with thrombin induced an anticoagulant response and prevented mortality in dogs challenged with lethal doses of endotoxin. 101 Thrombin infusion under those conditions leads to systemic protein C activation in vivo. 102 Indeed, administration of APC reduced mortality in a lethal bacteremia model in baboons. 58 Furthermore, inhibition of the protein C-protein S pathway by C4bBP increased the activation of coagulation, signs of organ failure, cytokine release, and mortality after sublethal E. coli infusion in baboons. This increase in morbidity and mortality after C4bBP appeared to be mediated through its capacity to bind protein S because these toxic effects could be reversed by protein S addition. The augmented inflammatory response was not secondary to activation of coagulation, because anticoagulation with active site degraded factor Xa cannot prevent mortality in this model. 66 Consistent with these anti-inflammatory effects is the observation in phase II studies in human patients with severe sepsis that administration of APC resulted in decreased plasma IL-6 levels. 64 Tissue Factor Pathway Inhibitor The TFPI mechanism of prolonging survival in the lethal E. coli sepsis model in baboons may not necessarily be limited to inhibition of coagulation but may also be due to another mechanism that could be related to inhibition of TF factor VIIa or factor Xa. Several studies have shown that factor VIIa can induce proinflammatory changes in mononuclear cells, 103 possibly by activation of PAR 2 and to a lesser degree PAR 1. TF factor VIIa could elicit a variety of proinflammatory responses in macrophages, including reactive oxygen species and induction of major histocompatibility complex (MHC) class II and adhesion receptors. 103 The macrophage activation required both the active site of factor VIIa and the cytoplasmic tail of TF. Another inflammatory action of TF factor VIIa is the reverse migration of monocytes from the basal to apical surfaces of the endothelium. 104 We recently found that administration of recombinant factor VIIa induced an IL-6 and IL-8 response in healthy human volunteers (E. de Jonge et al, manuscript submitted). Accordingly, TFPI reduced mortality and attenuated the IL-6 response in baboon models of sepsis. 17 In contrast, endotoxin-induced cytokine levels in healthy human volunteers were not influenced by TFPI. 69 A possible explanation is that the amount of activated coagulation factors, such as thrombin and factor VIIa, is too low to contribute to the inflammatory response. Alternatively, it can be speculated that endothelial cells produce IL-6 and IL-8 during sepsis, whereas monocytes are the predominant source of cytokines during low-grade endotoxemia. If so, TFPI could attenuate the endothelial cell response with a much smaller effect on monocytes. 69 Clinical studies of recombinant TFPI as a therapeutic for severe sepsis have been initiated. However, results are not yet available.

6 672 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 27, NUMBER REFERENCES 1. Fourrier F, Chopin C, Goudemand J, et al. Septic shock, multiple organ failure, and disseminated intravascular coagulation. Compared patterns of antithrombin III, protein C, and protein S deficiencies. Chest 1992;101: de Jonge E, Levi M, Stoutenbeek CP, van Deventer SJ. Current drug treatment strategies for disseminated intravascular coagulation. Drugs 1998;55: Review 3. Levi M, ten Cate H. Disseminated intravascular coagulation. N Engl J Med 1999;341: Ryan J, Brett J, Tijburg P, et al. Tumor necrosis factorinduced endothelial tissue factor is associated with subendothelial matrix vesicles but is not expressed on the apical surface. Blood 1992;80: Key NS, Slungaard A, Dandelet L, et al. Whole blood tissue factor procoagulant activity is elevated in patients with sickle cell disease. Blood 1998;91: Wilcox JN, Smith KM, Schwartz SM, Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci USA 1989;86: Østerud B, Flaegstad T. Increased tissue thromboplastin activity in monocytes of patients with meningococcal infection: related to an unfavourable prognosis. Thromb Haemost 1983;49: Rivers RP, Hathaway WE, Weston WL. The endotoxininduced coagulant activity of human monocytes. Br J Haematol 1975;30: Moldow CF, Bach RR, Staskus K, Rick PD. Induction of endothelial tissue factor by endotoxin and its precursors. Thromb Haemost 1993;70: Francis JL, Biggerstaff J, Amirkhosravi A. Hemostasis and malignancy. Semin Thromb Hemost 1998;24: Lawson CA, Yan SD, Yan SF, et al. Monocytes and tissue factor promote thrombosis in a murine model of oxygen deprivation. J Clin Invest 1997;99: Bach RR, Moldow CF. Mechanism of tissue factor activation on HL-60 cells. Blood 1997;89: Levi M, ten Cate H, Bauer KA, et al. Inhibition of endotoxin-induced activation of coagulation and fibrinolysis by pentoxifylline or by a monoclonal anti-tissue factor antibody in chimpanzees. J Clin Invest 1994;93: Nuijens JH, Huijbregts CC, Eerenberg-Belmer AJ, et al. Quantification of plasma factor XIIa-Cl(-)-inhibitor and kallikrein-cl(-)-inhibitor complexes in sepsis. Blood 1988; 72: Taylor FBJ, Chang A, Ruf W, et al. Lethal E. coli septic shock is prevented by blocking tissue factor with monoclonal antibody. Circulatory Shock 1991;33: Taylor FB, Chang AC, Peer G, et al. Active site inhibited factor VIIa (DEGR VIIa) attenuates the coagulant and interleukin-6 and -8, but not tumor necrosis factor, responses of the baboon to LD100 Escherichia coli. Blood 1998;91: Creasey AA, Chang AC, Feigen L, et al. Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock. J Clin Invest 1993;91: Rao LV, Nordfang O, Hoang AD, Pendurthi UR. Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity. Blood 1995;85: Mammen EF. Antithrombin: Its physiological importance and role in DIC. Semin Thromb Hemost 1998;24: Opal SM, Thijs LG. New potential therapeutic modalities: Antithrombin III. Sepsis 1999;3: Mammen EF. Clinical relevance of antithrombin deficiencies. Semin Hematol 1995;32: Büller HR, ten Cate JW. Acquired antithrombin III deficiency: Laboratory diagnosis, incidence, clinical implications, and treatment with antithrombin III concentrate. Am J Med 1989;87:44S 48S. Review 23. Seitz R, Wolf M, Egbring R, Havemann K. The disturbance of hemostasis in septic shock: Role of neutrophil elastase and thrombin, effects of antithrombin III and plasma substitution. Eur J Haematol 1989;43: Mann LTJ, Jensenius JC, Simonsen M, Abildgaard U. Antithrombin 3. Protection against death after injection of thromboplastin. Science 1969;166: Taylor FBJ, Emerson TEJ, Jordan R, Chang AK, Blick, KE. Antithrombin-III prevents the lethal effects of Escherichia coli infusion in baboons. Circulatory Shock 1988;26: Minnema MC, Chang AC, Jansen PM, et al. Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with Escherichia coli. Blood 2000;95: Kessler CM, Tang Z, Jacobs HM, Szymanski LM. 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: Dickneite G. Antithrombin III in animal models of sepsis and organ failure. Semin Thromb Hemost 1998;24: Giebler R, Schmidt U, Koch S, Peters J, Scherer R. Combined antithrombin III and C1-esterase inhibitor treatment decreases intravascular fibrin deposition and attenuates cardiorespiratory impairment in rabbits exposed to Escherichia coli endotoxin. Crit Care Med 1999;27: Blauhut B, Kramar H, Vinazzer H, Bergmann H. Substitution of antithrombin III in shock and DIC: A randomized study. Thromb Res 1985;39: Vinazzer H. Therapeutic use of antithrombin III in shock and disseminated intravascular coagulation. Semin Thromb Hemost 1989;15: Fourrier F, Chopin C, Huart JJ, et al. Double-blind, placebo-controlled trial of antithrombin III concentrates in septic shock with disseminated intravascular coagulation. Chest 1993;104: Baudo F, Caimi TM, de Cataldo F, et al. Antithrombin III (ATIII) replacement therapy in patients with sepsis and/or postsurgical complications: A controlled double-blind, randomized, multicenter study. Intensive Care Med 1998;24: Eisele B, Lamy M, Thijs LG, et al. Antithrombin III in patients with severe sepsis. A randomized, placebo-controlled, double-blind multicenter trial plus a meta-analysis on all randomized, placebo-controlled, double-blind trials with antithrombin III in severe sepsis. Intensive Care Med 1998; 24: Inthorn D, Hoffmann JN, Hartl WH, Muhlbayer D, Jochum M. Effect of antithrombin III supplementation on inflammatory response in patients with severe sepsis. Shock 1998;10: Inthorn D, Hoffmann JN, Hartl WH, Muhlbayer D, Jochum M. Antithrombin III supplementation in severe

7 ANTICOAGULANT FACTORS IN DIC/DE JONGE ET AL. 673 sepsis: Beneficial effects on organ dysfunction. Shock 1997; 8: Levi M, de Jonge E, van der Poll T, ten Cate H. Disseminated intravascular coagulation. Thromb Haemost 1999;82: Maki M, Terao T, Ikenoue T, et al. Clinical evaluation of antithrombin III concentrate (BI 6.013) for disseminated intravascular coagulation in obstetrics. Well-controlled multicenter trial. Gynecol Obstet Invest 1987;23: Ilias W, List W, Decruyenaere J, et al. Antithrombin III in patients with severe sepsis: A pharmacokinetic study. Intensive Care Med 2000;26: Riess H. Antithrombin in severe sepsis new indication of an old drug? Intensive Care Med 2000;26: (Editorial) 41. Dreyfus M, Magny JF, Bridey F, et al. Treatment of homozygous protein C deficiency and neonatal purpura fulminans with a purified protein C concentrate. N Engl J Med 1991;325: Esmon CT, Xu J, Gu JM, et al. Endothelial protein C receptor. Thromb Haemost 1999;82: Fijnvandraat K, Derkx B, Peters M, et al. Coagulation activation and tissue necrosis in meningococcal septic shock: Severely reduced protein C levels predict a high mortality. Thromb Haemost 1995;73: Taylor FB Jr. Protein S, C4b binding protein, and the hypercoagulable state. J Lab Clin Med 1992;119: Editorial and comment 45. Nawroth PP, Handley DA, Esmon CT, Stern DM. Interleukin 1 induces endothelial cell procoagulant while suppressing cell-surface anticoagulant activity. Proc Natl Acad Sci USA 1986;83: Nawroth PP, Stern DM. Modulation of endothelial cell hemostatic properties by tumor necrosis factor. J Exp Med 1986;163: Fukudome K, Esmon CT. Identification, cloning, and regulation of a novel endothelial cell protein C/activated protein C receptor. J Biol Chem 1994;269: Boehme MW, Deng Y, Raeth U, et al. Release of thrombomodulin from endothelial cells by concerted action of TNFalpha and neutrophils: In vivo and in vitro studies. Immunology 1996;87: Slungaard A, Vercellotti GM, Tran T, Gleich GJ, Key NS. Eosinophil cationic granule proteins impair thrombomodulin function. A potential mechanism for thromboembolism in hypereosinophilic heart disease. J Clin Invest 1993;91: Hesselvik JF, Malm J, Dahlbäck B, Blombäck M. Protein C, protein S and C4b-binding protein in severe infection and septic shock. Thromb Haemost 1991;65: Takahashi Y, Hosaka Y, Imada K, et al. Human urinary soluble thrombomodulin (MR-33) improves disseminated intravascular coagulation without affecting bleeding time in rats: Comparison with low molecular weight heparin. Thromb Haemost 1997;77: Uchiba M, Okajima K, Murakami K, et al. Effect of human urinary thrombomodulin on endotoxin-induced intravascular coagulation and pulmonary vascular injury in rats. Am J Hematol 1997;54: Mohri M, Gonda Y, Oka M, et al. The antithrombotic effects of recombinant human soluble thrombomodulin (rhstm) on tissue factor-induced disseminated intravascular coagulation in crab-eating monkeys (Macaca fascicularis). Blood Coagul Fibrinolysis 1997;8: Uchiba M, Okajima K, Murakami K, et al. rhs-tm prevents ET-induced increase in pulmonary vascular permeability through protein C activation. Am J Physiol 1997;273: L889 L Nakashima M, Uematsu T, Umemura K, Maruyama I, Tsuruta K. A novel recombinant soluble human thrombomodulin, ART-123, activates the protein C pathway in healthy male volunteers. J Clin Pharmacol 1998;38: Nakashima M, Kanamaru M, Umemura K, Tsuruta K. Pharmacokinetics and safety of a novel recombinant soluble human thrombomodulin, ART-123, in healthy male volunteers. J Clin Pharmacol 1998;38: Maruyama I. Recombinant thrombomodulin and activated protein C in the treatment of disseminated intravascular coagulation. Thromb Haemost 1999;82: Taylor FBJ, Chang A, Esmon CT, et al. Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987;79: Ettingshausen CE, Veldmann A, Beeg T, et al. Replacement therapy with protein C concentrate in infants and adolescents with meningococcal sepsis and purpura fulminans. Semin Thromb Hemost 1999;25: Rintala E, Kauppila M, Seppala OP, et al. Protein C substitution in sepsis-associated purpura fulminans. Crit Care Med 2000;28: Rivard GE, David M, Farrell C, Schwarz HP. Treatment of purpura fulminans in meningococcemia with protein C concentrate. J Pediatr 1995;126: Smith OP, White B, Vaughan D, et al. Use of protein-c concentrate, heparin, and haemodiafiltration in meningococcus-induced purpura fulminans. Lancet 1997;350: Gerson WT, Dickerman JD, Bovill EG, Golden E. Severe acquired protein C deficiency in purpura fulminans associated with disseminated intravascular coagulation: Treatment with protein C concentrate. Pediatrics 1993;91: Bernard GR, Hartman DL, Helterbrand JD, Fisher CJ. Recombinant human activated protein C (rhapc) produces a trend toward improvement in morbidity and 28 day survival in patients with severe sepsis. Crit Care Med 1998;27:S4 65. Bernhard GR, Vincent J-L, Laterre P-F, et al. Efficacy and safety of recombinanat human activated protein C for severe sepsis. N Engl J Med 2001;344: Taylor F, Chang A, Ferrell G, et al. C4b-binding protein exacerbates the host response to Escherichia coli. Blood 1991; 78: Creasey AA. New potential therapeutic modalities: Tissue factor pathway inhibitor. Sepsis 1999;3: Carr C, Bild GS, Chang AC, et al. Recombinant E. coliderived tissue factor pathway inhibitor reduces coagulopathic and lethal effects in the baboon gram-negative model of septic shock. Circulatory Shock 1994;44: de Jonge E, Dekkers PE, Creasey AA, et al. Tissue factor pathway inhibitor dose-dependently inhibits coagulation activation without influencing the fibrinolytic and cytokine response during human endotoxemia. Blood 2000;95: Girard TJ, Warren LA, Novotny WF, et al. Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor. Nature 1989; 338: Novotny WF, Girard TJ, Miletich JP, Broze GJ Jr. Purification and characterization of the lipoprotein-associated coag-

8 674 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 27, NUMBER ulation inhibitor from human plasma. J Biol Chem 1989; 264: Novotny WF, Girard TJ, Miletich JP, Broze GJ Jr. Platelets secrete a coagulation inhibitor functionally and antigenically similar to the lipoprotein associated coagulation inhibitor. Blood 1988;72: Ameri A, Kuppuswamy MN, Basu S, Bajaj SP. Expression of tissue factor pathway inhibitor by cultured endothelial cells in response to inflammatory mediators. Blood 1992;79: Lindahl AK. Tissue factor pathway inhibitor: From unknown coagulation inhibitor to major antithrombotic principle [see comments]. Cardiovascular Research 1997;33: Review 75. Takahashi H, Sato N, Shibata A. Plasma tissue factor pathway inhibitor in disseminated intravascular coagulation: Comparison of its behavior with plasma tissue factor. Thromb Res 1995;80: Novotny WF, Brown SG, Miletich JP, Rader DJ, Broze GJ Jr. Plasma antigen levels of the lipoprotein-associated coagulation inhibitor in patient samples. Blood 1991;78: Sabharwal AK, Bajaj SP, Ameri A, et al. Tissue factor pathway inhibitor and von Willebrand factor antigen levels in adult respiratory distress syndrome and in a primate model of sepsis [published erratum appears in Am J Respir Crit Care Med 1995;151:following 2118]. Am J Respir Crit Care Med 1995;151: Day KC, Hoffman LC, Palmier MO, et al. Recombinant lipoprotein-associated coagulation inhibitor inhibits tissue thromboplastin-induced intravascular coagulation in the rabbit. Blood 1990;76: Bregengard C, Nordfang O, Wildgoose P, et al. The effect of two-domain tissue factor pathway inhibitor on endotoxininduced disseminated intravascular coagulation in rabbits. Blood Coagul Fibrinolysis 1993;4: Randolph MM, White GL, Kosanke SD, et al. Attenuation of tissue thrombosis and hemorrhage by ala-tfpi does not account for its protection against E. coli a comparative study of treated and untreated non-surviving baboons challenged with LD100 E. coli. Thromb Haemost 1998;79: Camerota AJ, Creasey AA, Patla V, Larkin VA, Fink MP. Delayed treatment with recombinant human tissue factor pathway inhibitor improves survival in rabbits with gramnegative peritonitis. J Infect Dis 1998;177: Kemme MJ, Burggraaf J, Schoemaker RC, et al. The influence of reduced liver blood flow on the pharmacokinetics and pharmacodynamics of recombinant tissue factor pathway inhibitor. Clin Pharmacol Ther 2000;67: Esmon CT. Introduction: Are natural anticoagulants candidates for modulating the inflammatory response to endotoxin? Blood 2000;95: Coalson JJ, Benjamin B, Archer LT, et al. Prolonged shock in the baboon subjected to infusion of E. coli endotoxin. Circulatory Shock 1978;5: Taylor FBJ, Chang AC, Peer GT, et al. DEGR-factor Xa blocks disseminated intravascular coagulation initiated by Escherichia coli without preventing shock or organ damage. Blood 1991;78: Cirino G, Cicala C, Bucci M, et al. Factor Xa as an interface between coagulation and inflammation. Molecular mimicry of factor Xa association with effector cell protease receptor-1 induces acute inflammation in vivo. J Clin Invest 1997;99: 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 1998;161: Johnson K, Choi Y, DeGroot E, et al. Potential mechanisms for a proinflammatory vascular cytokine response to coagulation activation. J Immunol 1998;160: Jones A, Geczy CL. Thrombin and factor Xa enhance the production of interleukin-1. Immunology 1990;71: Hoffman M, Cooper ST. Thrombin enhances monocyte secretion of tumor necrosis factor and interleukin-1 beta by two distinct mechanisms. Blood Cells Mol Dis 1995;21: Kranzhofer R, Clinton SK, Ishii K, et al. Thrombin potently stimulates cytokine production in human vascular smooth muscle cells but not in mononuclear phagocytes. Circ Res 1996;79: Ueno A, Murakami K, Yamanouchi K, Watanabe M, Kondo T. Thrombin stimulates production of interleukin-8 in human umbilical vein endothelial cells. Immunology 1996; 88: Grandaliano G, Valente AJ, Abboud HE. A novel biologic activity of thrombin: Stimulation of monocyte chemotactic protein production. J Exp Med 1994;179: Colotta F, Sciacca FL, Sironi M, et al. Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin. Am J Pathol 1994;144: Anrather D, Millan MT, Palmetshofer A, et al. Thrombin activates nuclear factor-kappab and potentiates endothelial cell activation by TNF. J Immunol 1997;159: Kahn ML, Nakanishi-Matsui M, Shapiro MJ, Ishihara H, Coughlin SR. Protease-activated receptors 1 and 4 mediate activation of human platelets by thrombin. J Clin Invest 1999;103: Camerer E, Huang W, Coughlin SR. Tissue factor- and factor X-dependent activation of protease-activated receptor 2 by factor VIIa. Proc Natl Acad Sci USA 2000;97: Grey ST, Tsuchida A, Hau H, et al. Selective inhibitory effects of the anticoagulant activated protein C on the responses of human mononuclear phagocytes to LPS, IFNgamma, or phorbol ester. J Immunol 1994;153: Hancock WW, Grey ST, Hau L, et al. Binding of activated protein C to a specific receptor on human mononuclear phagocytes inhibits intracellular calcium signaling and monocyte-dependent proliferative responses. Transplantation 1995; 60: Grinnell BW, Hermann RB, Yan SB. Human protein C inhibits selectin-mediated cell adhesion: Role of unique fucosylated oligosaccharide. Glycobiology 1994;4: Taylor FBJ, Chang A, Hinshaw LB, et al. A model for thrombin protection against endotoxin. Thromb Res 1984; 36: Comp PC, Jacocks RM, Ferrell GL, Esmon CT. Activation of protein C in vivo. J Clin Invest 1982;70: Cunningham MA, Romas P, Hutchinson P, Holdsworth SR, Tipping PG. Tissue factor and factor VIIa receptor/ligand interactions induce proinflammatory effects in macrophages. Blood 1999;94: Randolph GJ, Luther T, Albrecht S, Magdolen V, Muller WA. Role of tissue factor in adhesion of mononuclear phagocytes to and trafficking through endothelium in vitro. Blood 1998;92: