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Originally published online as doi:10.1189/jlb.0607373 on November 21, 2007

Published online before print November 21, 2007
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(Journal of Leukocyte Biology. 2008;83:536-545.)
© 2008 by Society for Leukocyte Biology

Inflammation, endothelium, and coagulation in sepsis

Marcel Schouten, Willem Joost Wiersinga, Marcel Levi and Tom van der Poll1

Center for Infection and Immunity Amsterdam (CINIMA), Center for Experimental and Molecular Medicine, Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

1Correspondence: Academic Medical Center, Meibergdreef 9, Room G2-130, 1105AZ, Amsterdam, The Netherlands. E-mail: t.vanderpoll{at}amc.uva.nl

ABSTRACT

Sepsis is a systemic response to infection, and symptoms are produced by host defense systems rather than by the invading pathogens. Amongst the most prominent features of sepsis, contributing significantly to its outcome, is activation of coagulation with concurrent down-regulation of anticoagulant systems and fibrinolysis. Inflammation-induced coagulation on its turn contributes to inflammation. Another important feature of sepsis, associated with key symptoms such as hypovolemia and hypotension, is endothelial dysfunction. Under normal conditions, the endothelium provides for an anticoagulant surface, a property that is lost in sepsis. In this review, data about the interplay between inflammation and coagulation in sepsis are summarized with a special focus on the influence of the endothelium on inflammation-induced coagulation and vice versa. Possible procoagulant properties of the endothelium are described, such as expression of tissue factor (TF) and von Willebrand factor and interaction with platelets. Possible procoagulant roles of microparticles, circulating endothelial cells and endothelial apoptosis, are also discussed. Moreover, the important roles of the endothelium in down-regulating the anticoagulants TF pathway inhibitor, antithrombin, and the protein C (PC) system and inhibition of fibrinolysis are discussed. The influence of coagulation on its turn on inflammation and the endothelium is described with a special focus on protease-activated receptors (PARs). We conclude that the relationship between endothelium and coagulation in sepsis is tight and that further research is needed, for example, to better understand the role of activated PC signaling via PAR-1, the role of the endothelial PC receptor herein, and the role of the glycocalyx.

Key Words: infection • tissue factor • protein C • fibrinolysis • microparticles • protease-activated receptors

INTRODUCTION

Sepsis is widely recognized as a clinical syndrome, resulting from an overwhelming, systemic host response to infection [1 , 2 ]. The key clinical manifestations of sepsis are not caused directly by the invading pathogens; rather, the hypotension, coagulopathy, and multisystem organ dysfunction that characterize severe sepsis are predominantly a result of dysregulation of host-derived mediators of inflammation. Sepsis is the most common cause of death among hospitalized patients in noncardiac intensive care units and has instigated a lot of preclinical and clinical research [3 , 4 ]. In the last years, tremendous progress has been made in understanding the complex triad of infection, inflammation, and coagulation during sepsis.

It is now well established that in sepsis, systemic inflammation invariably leads to activation of the coagulation system and inhibition of anticoagulant mechanisms and fibrinolysis (see Fig. 1 ). Activation of coagulation and subsequent fibrin deposition are essential parts of the host defense against infectious agents in an attempt to contain the invading microorganisms and the subsequent inflammatory response [5 ]. An exaggerated response, however, can lead to a situation in which coagulation itself contributes to disease in its most severe form causing microvascular thrombosis and organ dysfunction, a syndrome known as disseminated intravascular coagulation (DIC) [6 ]. It is becoming increasingly clear, that vice versa, components of the coagulation system are able to markedly modulate the inflammatory response [7 , 8 ].


Figure 1
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  		Figure 1. Extensive crosstalk exists between coagulation and inflammation during sepsis, which is characterized by inflammation-induced activation of coagulation with concurrent impairment of anticoagulant systems, fibrinolyis, and endothelial function. Furthermore, during sepsis, inflammation-induced coagulation contributes to inflammation.

One of the important hallmarks of sepsis is microvascular dysfunction, in which endothelial activation and dysfunction seem to play a pivotal role [7 , 9 , 10 ]. In infection and subsequent sepsis, components of the bacterial cell wall, such as LPS, activate pattern recognition receptors on the endothelial surface [11 , 12 ]. Once an inflammatory response has been instigated, a large number of host-derived mediators, including cytokines, chemokines, and products of the complement system, are also able to activate endothelial cells (ECs). The endothelium responds to these mediators with structural changes, such as cytoplasmic swelling and detachment and importantly, also with functional changes, such as the expression of adhesion molecules, resulting in increased platelet adhesion and leukocyte trafficking. An important feature of endothelial dysfunction in sepsis is increased vascular permeability, resulting in redistribution of body fluid and edema. Fluid leakage from the intravascular space contributes to hypovolemia and hypotension, which are important signs of the sepsis syndrome [9 ].

In normal situations, the endothelium functions as an antithrombotic surface, preventing inappropriate activation of coagulation on the cell membrane [13 ]. However, once in sepsis the endothelium becomes activated, it transforms into a prothrombotic interface, which is critically involved in the detrimental cascade leading to multiple organ failure.

New insights into the vital role of the balance between procoagulant, anticoagulant, and fibrinolytic pathways during sepsis and the role of the endothelium herein continue to challenge our understanding of the sepsis syndrome. Tissue factor (TF), thrombin, the protein C (PC) pathway, activators and inhibitors of fibrinolysis, and protease-activated receptors (PARs) have all been shown to play vital roles in the crosstalk between inflammation and coagulation in sepsis. In the last years, research in this field has expanded its focus, now also covering microparticles (MPs), apoptosis, and platelets. In this review, the key players in inflammation-induced coagulation and coagulation-induced inflammation will be discussed, with a special focus on their relation with the endothelium and endothelial activation and dysfunction in sepsis. Figure 2 provides an overview of the pathways discussed in this article.


Figure 2
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  		Figure 2. The role of the endothelium in normal situations and in sepsis. (A) In normal situations, the endothelial layer provides for an anticoagulant surface to prevent the blood from clotting by expressing thrombomodulin (TM) and endothelial PC receptor (EPCR), which support thrombin in generating activated PC (APC), by having TF pathway inhibitor (TFPI) and antithrombin (AT) attached to their surface and by secreting tissue-type plasminogen activator (tPA), which promotes fibrinolysis. (B) When in infection bacteria invade the bloodstream, systemic activation of inflammation leads to cytokine release and endothelial activation and dysfunction, resulting in release of MPs, apoptosis, detachment of ECs, and loss of barrier function. Coagulation is activated by induction of TF on monocytes and MPs and possibly on endothelium and by release of von Willebrand factor (vWF), which adds to platelet adhesion to the subendothelial surface and platelet aggregation. Production of glycosaminoglycans (GAGs) is down-regulated, and the anticoagulant proteins TFPI, AT, EPCR, and TM are cleaved from the EC surface and are impaired in action. Moreover, APC and AT are consumed. Fibrinolysis is impaired as a result of a rise in the main inhibitor of the PA (PAI-1), which outweighs a rise in tPA, and complement activation is enhanced by loss of activation of thrombin-activatable fibrinolysis inhibitor (TAFI), which normally inhibits complement factor C3a and C5a and bradykinin activity. Anticoagulant proteins in turn modulate cytokine-release: tissue factor-factor VIIa (TF-FVIIa), factor (F) Xa, and thrombin exert proinflammatory activity by cleaving mainly PAR-1 and PAR-2. APC cleaves PAR-1 in an EPCR-dependent manner and hereby modulates inflammation and apoptosis.

ACTIVATION OF COAGULATION

TF
The pivotal initiator of inflammation-induced activation of coagulation is TF, which initiates coagulation by catalyzing, in a newly formed complex with FVII(a), the conversion of the zymogens FIX and X into active proteases, which in turn, enhance the activation of FX and prothrombin, respectively. Prothrombin is converted to thrombin, which converts fibrinogen into fibrin. The importance of inflammation-induced activation of coagulation by the TF-FVIIa complex is substantiated by experiments in nonhuman primates and healthy human volunteers, demonstrating that blocking of TF-FVIIa activity in endotoxemia or bacteremia after i.v. challenge with Escherichia coli completely abrogated coagulation activation and, in lethal models, prevented DIC and lethality [14 15 16 17 18 ].

TF is normally not present in the vasculature; instead, it is found in considerable amounts in tissues that are not in direct contact with blood, such as the adventitial layer of larger blood vessels, cardiomyocytes, and the subcutis. Histologically, TF appears to be present in all blood tissue barriers, so that coagulation can be initiated quickly once the endothelial barrier has been disrupted [19 ]. In inflammatory conditions, however, TF is also induced in the circulatory system by the action of mediators such as cytokines, C-reactive protein, and advanced glycosylation end-products. This inducible TF is predominantly expressed by monocytes and macrophages, as was shown in patients with severe bacterial infection [20 ]. Inducible TF is stimulated by the presence of platelets and granulocytes in a P-selectin-dependent manner [21 ]. In in vitro conditions, different cytokines such as TNF-{alpha} and IL-1β induced TF on ECs, but whether this also occurs to a relevant extent in vivo is still unclear [19 , 22 ]. Studies in baboons infused i.v. with E. coli have suggested that TF expression is present in the vascular wall during sepsis, especially in areas exposed to disturbed blood flow [23 ]. There are, however, also arguments against a role for endothelial TF in sepsis in vivo, such as the finding that TF was not detectable in ECs from rabbits subjected to the generalized Shwartzman reaction by i.v. injection of two subsequent doses of endotoxin. Moreover, immunohistochemical staining for TF failed to detect endothelial TF in rabbits subjected to endotoxin [24 ].

TF has also been reported to be present in the circulation. Various forms of circulating TF have been described—as a component of blood cells or as MP-associated TF (see below). Recently, alternatively spliced TF (lacking exon 5) was discovered as a soluble form of TF, which circulates in blood, and may exert procoagulant activity, expanding the concept of circulating TF by a further element [25 , 26 ]. Alternatively spliced TF is released from ECs upon stimulation with proinflammatory cytokines [27 ]. The plasma concentration of alternatively spliced TF may be of use as a clinical marker for inflammation-induced coagulation activation.

MPs
MPs are circulating cell fragments derived from activated or apopototic cells. They contain significant amounts of surface-exposed, negatively charged phospholipids that are essential for thrombin generation. As such, they are anticipated to contribute to a procoagulant state. Many different cell types have been shown to shed MPs. For example, in plasma from fresh blood of healthy individuals, MPs originating from platelets, granulocutes, erythrocytes, and ECs were identified by using flow cytometry [28 , 29 ]. Also, in sepsis, circulating MPs derived from platelets, monocytes, and ECs have been identified.

MPs can express TF on their surface: In human endotoxemia, an increase in TF-containing MPs up to 800% has been observed [30 ]. Moreover, TF and associated procoagulant activity have been detected on MPs derived from platelets and granulocytes in patients with meningococcal sepsis [31 ]. MPs derived from platelets have been acknowledged to be thrombogenic [32 , 33 ]. In a model of thrombosis, low TF mice (1% expression) had small platelet thrombi, which lacked TF and fibrin. This lack of TF and fibrin was not observed in chimera with wild-type bone marrow, suggesting a role for hematopoietic TF in thrombus formation as well [34 ]. Monocyte-derived MPs have indeed been shown to express TF after stimulation with LPS [35 ]. More recently, it has been described that TF on monocyte-derived MPs is enhanced by platelets in a P-selectin-dependent manner [36 , 37 ]. P-selectin can be expressed by ECs when they are activated by thrombin [38 ].

MPs derived from TNF-{alpha}-stimulated HUVECs induced coagulation in vitro in a TF-FVIIa-dependent way [39 ]. Moreover, endothelial MPs were shown to express vWF-binding sites and to express ultralarge vWF multimers, which promote the formation of platelet aggregates and increase their stability [40 , 41 ]. Endothelium-derived MPs have been detected in normal human blood, and their levels were increased in patients with a coagulation abnormality characterized by the presence of lupus anticoagulant [42 ]. Whether endothelial MPs are also elevated in sepsis and indeed play a procoagulant role in this condition still needs to be determined.

Apoptosis and circulating ECs (CECs)
Normally, only a small portion of ECs is apoptotic (<0.1%). LPS has been shown to induce endothelial apoptosis in vitro and also, although not in all studies, in vivo [43 ]. Interaction with other cells may influence endothelial apoptosis; for example, LPS-activated monocytes have been shown to contribute to endothelial apoptosis in vitro [44 ]. Endothelial apoptosis has been shown to contribute to a procoagulant state in vitro [45 ]. Whether this also holds true in vivo has not been established to our knowledge.

As apoptotic ECs detach from the basement membrane and are then rapidly cleared from the circulation, they may be difficult to measure in vivo [43 ]. Recently, however, CECs have been identified as a cellular index of endothelial damage and apoptosis. Indeed, increased CECs have been described in various conditions, such as experimental endotoxemia and septic shock [46 , 47 ]. It is unknown whether these CECs have a relevant procoagulant function in sepsis.

Platelets and vWF
Platelets are involved in the pathophysiology of sepsis as marked by the frequent occurrence of thrombocytopenia in sepsis [48 ]. Platelets can be activated in sepsis, either directly by endotoxin or by proinflammatory cytokines [49 , 50 ]. The negatively charged outer membrane surface of the activated platelet provides an ideal surface for coagulation to take place. Hence, activated platelets can play an important role in inflammation-induced coagulation. Platelets on their turn can be activated by coagulation proteases such as thrombin, and when activated, they secrete proinflammatory proteins and growth factors and hence contribute to inflammation as well. As described above, platelets also interact with monocyte-derived MPs in a P-selectin-dependent manner and by this means enhance TF expression on MPs [36 , 37 ].

Platelets have the ability to form platelet clots on a damaged endothelial layer. A first step in this process is adhesion by interaction with vWF bound to collagen in the subendothelial layer. vWF is produced predominantly by ECs and to a lesser extent by platelets and also plays a role in subsequent platelet aggregation. When endothelium is activated or injured, vWF is released from preformed stores into the circulation [51 , 52 ]. Indeed, vWF levels are generally accepted as a marker of endothelial injury [53 ]. In HUVECs, various proinflammatory cytokines induce the release of ultralarge vWF multimers, which are potent platelet aggregators and moreover, inhibit vWF cleavage by a disintegrin-like and metalloprotease with thrombospondin type 1 domain 13 (ADAMTS-13) [54 ]. Indeed, levels of vWF antigen are increased in sepsis, and decreased levels of ADAMTS-13 have been linked to a poor prognosis in sepsis [55 ].

The endothelium thus can contribute to platelet adhesion and aggregation by releasing vWF, also in its more potent, ultralarge form. Moreover, when the endothelium is damaged, it plays an indirect role by exposing subendothelial, collagenous surfaces on which platelets can adhere in a vWF-dependent manner.

IMPAIRMENT OF ANTICOAGULANT MECHANISMS

Under physiological conditions, anticoagulant systems are continuously active to prevent blood from clotting on the EC surface. The endothelium plays a key function in maintaining this anticoagulant condition. Blood clotting is controlled by three major anticoagulant proteins: TFPI, AT, and APC [7 , 56 ].

TFPI
TFPI is a serine protease inhibitor that is secreted by ECs. TFPI inhibits the activation of FX to FXa by the TF-FVIIa complex. The role of endogenous TFPI in anticoagulation in sepsis is illustrated by the fact that immunodepletion of TFPI sensitized rabbits to LPS-induced DIC and the generalized Shwartzman reaction [57 , 58 ]. Conversely, administration of TFPI attenuated consumptive coagulopathy and improved survival in septic primates [59 ] and prevented coagulation activation during human endotoxemia [15 ]. In addition, recombinant human TFPI was able to attenuate coagulation activation in patients with severe sepsis, although this intervention did not result in a reduced mortality in this population [60 ].

TFPI is normally attached to the endothelium via proteoglycans (PGs), which are GAGs bound to a core protein, so that it can optimally exert its TF-FVIIa-FX-inhibiting properties on the endothelial surface [61 ]. In sepsis, proinflammatory cytokines reduce the synthesis of GAGs on the endothelial surface, which could therefore impact on TFPI function. Although reports on TFPI activity in situations of TF-induced coagulation have shown contradictory results, an increase in plasma TFPI activity in meningococcal sepsis has been associated with more severe coagulation and mortality, supporting the hypothesis that TFPI works less efficiently when it is not attached to the endothelium [62 , 63 ].

AT and heparin
AT predominantly inhibits FXa and thrombin and also has inhibitory properties toward TF-FVIIa and FIXa. The anticoagulant properties of AT have been shown extensively in vivo [64 ]. For example, treatment with AT improved survival, concurrently inhibiting the procoagulant response and hyperinflammation during severe sepsis in the baboon [65 , 66 ]. Most notably, infusion of AT dose-dependently reduced TF-triggered coagulation and moreover, ameliorated IL-6 production in a human model of endotoxemia [67 ].

Apart from its anticoagulant activities, AT has been described to possess direct anti-inflammatory activity. For example, on HUVECs, AT increased prostacyclin formation, and administration of heparin abolished this effect [68 ]. Indeed, AT decreased ischemia-reperfusion injury in the rat liver by increasing the hepatic level of prostacyclin [69 ], and in another model, it prevented gastric musocal injury in a prostacyclin-dependent manner [70 ]. Moreover, AT reduces leukocyte rolling on the endothelium [71 ].

In sepsis, AT levels are markedly decreased as a result of a combination of an impaired synthesis as a result of a negative acute-phase response, degradation by elastase from activated neutrophils, and, quantitatively the most important, consumption as a result of ongoing thrombin generation [56 ]. The anticoagulant activities of AT are normally accelerated, to a large extent by heparin-like GAGs, such as heparan sulfate (HS). As described above, proinflammatory cytokines reduce the synthesis of GAGs on the endothelial surface, which contributes to reduced AT function in sepsis. Of note, i.v. infusion of AT did not alter mortality in patients with sepsis in a large, multinational trial; in this population, the possible AT effect may have been obscured by concurrent heparin treatment, considering that heparin, which is a highly sulfated version of HS, has been found, like other soluble GAGs, to antagonize the anti-inflammatory and microcirculatory effects of AT [68 , 72 ].

HS polysaccharides are ubiquitously expressed as HSPGs on cell surfaces such as the endothelium. Direct binding of bacteria to HSPGs on alveolar epithelium has been described; however, this phenomenon has not been reported for HSPGs on endothelium, nor has this finding been substantiated in vivo [73 ]. GAGs have been shown to interfere with antibacterial properties of the antimicrobial cathelicidin LL-37. The same phenomenon could play a role with heparin [74 ].

GAGs have been described to be able to play an important, proinflammatory role by participating in almost every stage of leukocyte transmigration through the vessel wall. This holds true especially for HSPGs: Endothelial HSPGs facilitate adhesion of leukocytes to the inflamed endothelium by binding to L-selectin expressed by leukocytes. HSPGs also play a role in endothelial transcytosis and subsequent presentation of chemokines such as IL-8, which is important for leukocyte activation and subsequent production of integrins that tighten leukocyte binding to the endothelium [75 ]. Moreover, HSPGs facilitate leukocyte transmigration through the vessel wall, possibly by binding proteins that regulate vascular permeability, such as kininogen [76 ]. To cross the endothelial basement membrane, leukocytes secrete various proteases such as heparanase, which releases growth factors that are normally associated with basement membrane HSPGs. These growth factors play a role in the establishment of an acute and chronic inflammatory reaction by modulating angiogenesis and tissue remodeling [75 ].

HS polysaccharide structure can vary substantially by different positioning of sulfate groups and by epimerization of glucoronic acid residues to iduronic acid. Together with varying negative-charge densities, this provides for much structural heterogeneity. Binding of different HSPGs to different proteins can affect their biological properties, for example, in growth hormone and chemokine signaling [76 , 77 ].

Although the synthesis of GAGs has been described to be reduced by proinflammatory cytokines, HSPGs specifically can be up-regulated in inflammatory conditions [76 ]. That implies they could play an important proinflammatory role in sepsis. Exogenous heparin, however, could play an anti-inflammatory role by interfering in the interaction between leukocytes and (sub)endothelial HSPGs, for example, by binding to P- and L-selectin [78 ].

The PC system
The PC system provides important control of coagulation by virtue of the capacity of APC to proteolytically inactivate the cofactors Va and VIIIa. APC is generated by TM-bound thrombin; hence, thrombin also has anticoagulant besides procoagulant properties. TM is present on the vascular endothelium in high concentrations, mainly in the microcirculation [79 , 80 ]. The activation of PC to APC by TM-bound thrombin is augmented by the presence of the EPCR. TM-bound thrombin is inhibited efficiently by AT and PC inhibitor. As a consequence, TM inhibits coagulation by generating the anticoagulant APC, by accelerating the inhibition of thrombin, as well as by preventing thrombin to exert procoagulant properties on fibrinogen or platelets. The TM-thrombin complex also activates TAFI, an endogenous inhibitor of fibrinolysis, which removes C-terminal lysine residues from fibrin, thereby rendering fibrin less sensitive to the action of plasmin.

Several preclinical and clinical studies have supported the anticoagulant potency of the PC system in vivo. Infusion of APC into septic baboons prevented hypercoagulability and death, and inhibition of activation of endogenous PC with a mAb exacerbated the response to a lethal E. coli infusion and converted a sublethal model produced by an LD10 into a model of severe shock associated with DIC and death [81 ]. Treatment of baboons with an anti-EPCR mAb, reducing the efficiency by which PC can be activated, was also associated with an exacerbation of a sublethal E. coli infection to lethal sepsis with DIC [82 ]. Moreover, interference with the bioavailability of protein S (PS), an important cofactor for the anticoagulant functions of APC, by administration of C4b-binding protein, which causes a decrease in PS levels, resulted in similar changes [83 ]. Lastly, administration of recombinant human APC ameliorated coagulation in patients with severe sepsis and moreover, reduced absolute mortality by 6% [84 ].

During sepsis, the PC system is impaired [8 ]. This impairment is not only the result of increased consumption of PS and PC and decreased production of PC by the liver but also as a result of decreased activation of PC by less expression of TM on ECs. TM expression can be down-regulated by inflammatory mediators such as TNF-{alpha} [85 ]. Also, LPS has been shown to stimulate neutrophil activation on the endothelial surface, leading to the release of elastase, which subsequently cleaves TM from the endothelium. This results in a rise in soluble TM, which is a much less active protein than endothelium-bound TM, given the fact that it has no EPCR on its side as a cofactor [86 , 87 ]. Moreover, neutrophils release oxidants, which have been shown to oxidize TM, leaving behind a less-active protein [87 ]. In patients with severe meningococcal sepsis, down-regulation of TM and consequent impaired PC activation were confirmed in vivo by immunohistochemistry [88 ]. EPCR also has been shown to be cleaved from the EC membrane, resulting in higher levels of soluble EPCR in sepsis [86 , 89 , 90 ]. It should be noted, however, that in a study that investigated soluble TM and soluble EPCR on the one hand and coagulation and survival in sepsis on the other hand, plasma levels of soluble TM and soluble EPCR did not correlate well with F1+ 2/APC ratios, a marker for the procoagulant state. Moreover, the levels of soluble TM and soluble EPCR did not differ between survivors and nonsurvivors in this study, indicating that the precise relationship between these soluble proteins and coagulation and outcome in sepsis has not been completely elucidated [86 ].

IMPAIRMENT OF FIBRINOLYSIS

Hemostasis is further controlled by the fibrinolytic system, in which plasmin is the key enzyme, degrading fibrin clots. Plasmin is generated from plasminogen by different proteases, most notably, tPA and urokinase-type PA (uPA). The main inhibitor of the PAs is PAI-1, which is produced by the endothelium and the liver and binds to tPA and uPA. In inflammatory states, the first fibrinolytic response is a release of tPA and uPA that is stored inside ECs. This increase, however, is counteracted by a delayed but sustained increase in PAI-1 levels in response to stimulation with TNF-{alpha} and IL-1β [91 ]. The net effect is impairment of fibrinolysis. The importance of the fibrinolytic system for inflammation-induced coagulation in sepsis has been shown by experiments in genetically modified mice, showing that upon endotoxin administration, mice deficient for tPA or uPA had more extensive fibrin deposition in organs than wild-type mice, and the opposite held true for PAI-1-deficient mice [92 ]. The endothelium plays an obvious role in fibrinolysis in sepsis, being the main producer of profibrinolytic factors as well as one of the main producers of PAI-1.

COAGULATION-INDUCED INFLAMMATION

Inflammation not only leads to activation of coagulation, coagulation on its turn also influences inflammation [7 , 8 ]. For example, heterozygous PC-deficient mice and transgenic mice expressing low endogenous PC concentrations demonstrated higher levels of proinflammatory cytokines and increased neutrophil invasion in their lungs after i.p. injection with endotoxin [93 , 94 ]. AT has been found to diminish the expression of β2-integrins on leukocytes and by binding to neutrophils, can inhibit chemokine-induced neutrophil migration. In addition, AT can enhance prostacyclin formation, hereby inhibiting NF-{kappa}B signaling in ECs and decrease IL-6 production by monocytes and endothelium [7 ]. In vitro, APC attenuates inflammation by inhibiting monocyte expression of TF and TNF-{alpha}, NF-{kappa}B translocation, cytokine signaling, TNF-{alpha}-induced up-regulation of cell surface leukocyte adhesion molecules, and leukocyte-EC interactions [7 , 80 ]. TM also exerts anti-inflammatory effects at multiple levels. First, TM is essential for the activation of PC to APC; as such, TM plays a key role in the anti-inflammatory properties of APC. Second, TM binds thrombin, thereby preventing it from exerting proinflammatory properties (see below). Third, TM-bound thrombin activates TAFI, which has been demonstrated to suppress bradykinin activity and complement activation [95 ]. Furthermore, the lectin domain of TM likely plays a direct role in the orchestration of inflammatory reactions. Indeed, genetically modified mice that lack the N-terminal, lectin-like domain of TM displayed an increased neutrophil recruitment to the lungs and diminished survival after systemic endotoxin administration [96 ]. Importantly, deletion of the lectin-like domain of TM did not influence the capacity of TM to activate PC, indicating that the anti-inflammatory effects of this part of TM are not mediated by APC.

Multiple interactions also exist between inflammation and mediators of the fibrinolytic system [56 , 97 ]. Fibrinolytic activators and inhibitors may modulate the inflammatory response by their effect on inflammatory cell recruitment and migration. For instance, uPAR, the receptor for uPA, mediates leukocyte adhesion to the vascular wall and extracellular matrix components, and its expression on leukocytes is strongly associated with their migratory and tissue-invasive potential. This is illustrated in a mouse model of bacterial pneumonia, in which uPAR-deficient mice displayed a profoundly reduced neutrophil influx in the pulmonary compartment [98 ]. tPA-deficient mice demonstrated an impaired defense against abdominal sepsis caused by E. coli, as indicated by higher bacterial loads at the primary site of the infection, enhanced bacterial dissemination, and reduced survival; this protective effect of endogenous tPA was independent of its role in the generation of plasmin [99 ]. Plasma concentrations of the fibrinolysis inhibitor PAI-1 are strongly elevated in patients with sepsis, and the level of PAI-1 is highly predictive for an unfavorable outcome [100 ]. It remains to be established whether the elevated PAI-1 levels merely are indicative of a strong inflammatory response of the host or indeed have any pathophysiological significance. Recent findings that a sequence variation in the gene encoding PAI-1 influences the development of septic shock in patients and relatives of patients with meningococcal infection has provided circumstantial evidence that PAI-1 might play a functional role in the host response to bacterial infection [101 ]. However, recent studies using PAI-1-deficient mice and mice with transiently enhanced expression of PAI-1 have pointed to a protective rather than a detrimental role of this mediator in severe gram-negative pneumonia and sepsis [102 ].

PARs
In linking coagulation to inflammation, PARs seem to play a pivotal role [103 , 104 ]. The PAR family consists of four members, PAR-1 to PAR-4, which are localized in the vasculature on different cell types such as ECs, mononuclear cells, platelets, fibroblasts, and smooth muscle cells [103 ]. PARs serve as their own ligand: proteolytic cleavage by, for example, a coagulation factor, such as TF-FVIIa, FXa, or APC, leads to exposure of a neo-N-terminus, which serves as a ligand for the same receptor, hereby initiating transmembrane signaling (see Fig. 3 ). Low concentrations of thrombin have been shown to activate PAR-1, whereas high concentrations also activate PAR-3 and PAR-4. In humans, thrombin activates platelets by cleavage of PAR-1 and PAR-4; in mice, thrombin activates platelets by cleavage of a PAR-3–PAR-4 complex [105 ]. On ECs, all four PARs have been identified [106 ].


Figure 3
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  		Figure 3. Key role of PARs in the interplay between coagulation and inflammation during sepsis. (A) Proteases that are able to activate PARs. During sepsis, increased TF expression leads to activation of the coagulation proteases VIIa, Xa, Va, and thrombin. PAR-1 is activated by thrombin, FXa, APC, granzyme A, and trypsin. PAR-2 is activated by trypsin, tryptase, FVIIa, FXa, proteinase 3, Der P3, P9, and acrosien. PAR-3 is activated by thrombin, and PAR-4 is activated by thrombin, trypsin, and cathepsin G. (B) Activation of PARs. Proteases cleave and activate PARs in a common way: The receptor is cleaved at a specific site within its extracellular N-terminus; this cleavage exposes a new N-terminus that serves as a tethered ligand domain, which binds to conserved regions in the second extracellular loop of the cleaved receptor, resulting in the initiation of signal transduction. (C) Effects of PAR activation. Activated PARs cause activation of platelets (PAR-1, -2, and -4) and will result in proapoptotic (PAR-1) and proinflammatory (PAR-1 to -4) effects during sepsis. In contrast, APC cleavage of PAR-1 will lead to anti-inflammatory and antiapoptotic effects.

Thrombin can induce the expression of proinflammatory cytokines and chemokines by endothelium and by cleaving PAR-1 in vitro [38 , 107 ]. Endotoxin and TNF-{alpha}-induced IL-6 production by cultured ECs was enhanced by the activation of PAR-1 and PAR-2. Moreover, endotoxin and proinflammatory cytokines also induced PAR-2 and PAR-4 expression in cultured ECs. In vivo data pointing to a role of coagulation protease-induced induction of inflammation come from recent experiments, showing that the administration of recombinant FVIIa to healthy human subjects causes a small but significant, three- to fourfold rise in plasma levels of IL-6 and IL-8 [108 ]. Most probably, the activation of multiple PARs enhances inflammation during sepsis. This is underscored by a recent study showing that genetically modified mice expressing low levels of TF exhibited reduced IL-6 expression and increased survival in a mouse model of endotoxemia compared with control mice [109 ]. Although hirudin inhibition of thrombin or a deficiency in PAR-1 or PAR-2 did not affect IL-6 expression or mortality in this model, combining hirudin treatment to inhibit thrombin signaling through PAR-1 and PAR-4 with PAR-2 deficiency in contrast, did reduce IL-6 expression and moreover, increased survival. Taken together, these studies suggest that activation of multiple PARs by coagulation proteases may contribute to inflammation in endotoxemia and sepsis [109 ].

Much effort has been made to elucidate the mechanisms by which APC exerts its anti-inflammatory properties as reported. APC inhibits inflammation indirectly through reducing thrombin generation and, thereby, thrombin-induced inflammation via PARs. In primary ECs, APC itself also signals through PAR-1, whereas TF-FVIIa-FXa has been indicated to signal through PAR-2. PAR-1-dependent APC signaling induces a number of genes that are known to down-regulate proinflammatory signaling pathways and inhibit apoptosis in an EPCR-dependent manner [110 111 112 113 ]. APC also promotes endothelial barrier enhancement in vitro in an EPCR and PAR-1-dependent manner, a property that could be of special interest, given the central role of loss of endothelial barrier function in sepsis [114 115 116 117 118 ]. Whether the anti-inflammatory, antiapoptotic, and barrier-stabilizing properties of APC play an important role during sepsis in vivo and whether these indeed are mediated by PAR-1 are, however, still a matter of debate [116 117 118 ].

A NEW PLAYER IN THE FIELD: THE GLYCOCALYX

It was already mentioned that in sepsis, GAGs are down-regulated as a result of proinflammatory cytokines, which can thereby impact on the function of AT and TFPI and also on leukocyte adhesion and transmigration. HSPGs and GAGs are part of a broad, negatively charged network on the luminal side of the endothelium called glycocalyx [119 ]. Besides GAGs and HSPGs, this glycocalyx consists of glycoproteins, hyaluronic acid, and other membrane-associated proteins. The glycocalyx has been described to play a role, not only in coagulation but also in other endothelial functions including maintaining vascular barrier function, NO-mediated vasodilation, and antioxidant functions, all processes that are known to be involved in sepsis [120 , 121 ]. It was shown recently that specific disruption of the glycocalyx results in thrombin generation and platelet adhesion within a few minutes [122 ]. Moreover, loss of glycocalyx in vivo has been associated with subendothelial edema formation [123 ]. It seems conceivable that the glycocalyx is disturbed in sepsis also, although evidence for this is still preliminary. The role of the glycocalyx in modulating endothelial function, including anticoagulation, and the role of the endothelium in modulating the glycocalyx in sepsis surely need further research.

CONCLUSIONS

There is ample evidence that activation of coagulation and down-regulation of anticoagulation and fibrinolysis are prominent features of the proinflammatory condition in sepsis that contribute to the outcome of the disease. The endothelium plays an important part in these sequelae, possibly by expression of TF, secretion of MPs and vWF, and interactions with platelets, by becoming apoptotic and by detaching from the basement membrane. Moreover, the endothelium undergoes several other alterations, most notably, down-regulation of anticoagulant proteins such as TM and EPCR and loss of surface-associated GAGs and different expression of endothelial HSPGs that impact on TFPI and AT function.

The procoagulant state in sepsis on its turn enhances inflammation, presumably especially through activation of PARs, although most data supporting an important role for PARs in sepsis come from in vitro experiments. Whether, for example, APC exerts its beneficial functions in sepsis through PAR-1 and EPCR in vivo needs further study, as does the possible role in sepsis for the glycocalyx, which provides an interesting new research subject, that could possibly help us in integrating the different pathophysiological sequelae that have been described regarding endothelial function and activation of coagulation in sepsis.

Received June 9, 2007; revised September 28, 2007; accepted October 4, 2007.

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