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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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Endothelial Cell Perturbation and Disseminated Intravascular Coagulation

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Introduction

It has long been recognized that the vascular endothelium is a major target of bacterial endotoxin and of a variety of microorganisms, and that damage to endothelial cells (ECs) is a hallmark of Gram-negative sepsis and many other infections. Earlier evidence to suggest a role for ECs in endotoxin-induced pathophysiological changes, including disseminated intravascular coagulation (DIC), has derived almost exclusively from animal studies showing that endotoxin administration causes severe endothelial injury in different tissues, ranging from morphological abnormalities to detachment of the cells, eventually leading to their appearance in the circulation.1–3 Accordingly, it was originally thought that damage to ECs could expose vascular basement membrane and thus provide a site at which the contact system of blood coagulation could become activated.

During the last 20 years, there has been an increasing awareness that vascular endothelium, far from being merely a passive vessel wall lining, is actually a multifunctional organ involved in maintaining homeostasis. Strategically located at the interface between blood and the extravascular space, ECs express a vast array of properties through which they exert numerous functions including selective transport, maintenance of vascular tone, repair and remodeling of the vessel wall, new blood vessel formation, trafficking of blood cells, and control of blood fluidity and vascular patency (for a review see ref. 4). One of these properties is the capacity to dynamically regulate the processes of coagulation and fibrinolysis. While “resting” endothelium essentially express anticoagulant activities, following “perturbation” by a variety of pathogenetically relevant agents or conditions, it acquires clot-promoting properties whereby blood coagulation is initiated and propagated on the cell surface. In addition, ECs play a central role in the control of plasmin generation because of their ability to synthesize and secrete plasminogen activators and their inhibitors and to express cell surface binding sites for both enzymes and substrate. Again, the dynamic modulation of fibrinolytic properties by numerous agents and conditions eventually will influence fibrin deposition. Importantly, these (and other) functional changes of ECs may occur in the total absence of morphological alterations leading to the concept of “endothelial cell perturbation” as a fundamental pathogenic mechanism in a number of diseases. This has prompted a number of in vitro and in vivo studies aimed at understanding the role of ECs in blood clotting activation and fibrin deposition during DIC associated with sepsis and other conditions.

This Chapter first provides a concise description, based mainly on in vitro experiments, of the vast repertoire of procoagulant and fibrinolytic properties expressed by ECs on perturbation by DIC-related agents. Moreover, it summarizes available evidence on the in vivo expression of these properties and the role thereof in animal and human DIC. Particular emphasis is given to sepsis-associated DIC, in which the pathophysiological role of EC perturbation has been rather extensively investigated.

Endothelial Cell Perturbation and Coagulation

The basal state of EC surface is thought to be essentially anticoagulant or non-thrombogenic. Control of coagulation is exerted by ECs at different critical steps of the clotting cascade (for a review see ref. 5). ECs are the main source of the Kunitz-type tissue factor (TF) pathway inhibitor (TFPI), which blocks the initiation of blood coagulation by TF (Fig. 1).5,6 TFPI manifests its action with the generation of limited quantities of factor Xa. It first binds to factor Xa and then to TF-factor VIIa thereby forming an inactive quaternary complex. Cultured ECs constitutively synthesize and secrete TFPI, which, by immunocytochemical methods using electron microscopy, has been localized to the cell surface, the Golgi apparatus and the endocytic compartment;7 the cell surface-associated fraction is thought to act as a direct vessel wall anticoagulant. Synthesis and/or release of TFPI are positively regulated by heparin, fluid flow and thrombin.8–10 The mobilization of the inhibitor into the circulation by heparin also occurs in vivo and it is thought to contribute substantially to the anticoagulant action of the drug.5,6 Studies on the distribution in normal human tissues revealed that TFPI mRNA was expressed in large amounts in placenta and lung, followed by liver, kidney and heart, whereas low expression was found in the brain.11 ECs also express heparan sulfate and related glycosaminoglycans that catalyze the antithrombin action, thus contributing to neutralization of major clotting enzymes factor Xa and thrombin (Fig. 2).5 Interestingly, ECs derived from microvasculature produce about 5 times the amount of heparin-like activity than cells from macrovasculature. Finally, ECs play a pivotal role in the protein C (PC) anticoagulant pathway, a major control mechanism of blood coagulation, through the expression of at least three key elements of the PC system, namely thrombomodulin (TM), protein S and the EC PC receptor (EPCR) (Fig. 3).12,13 TM is an integral membrane protein whose main function is to bind thrombin thus altering the molecular specificity of this enzyme. The latter, indeed, is no longer able to exert procoagulant activities (fibrinogen cleavage, platelet activation, activation of factors V, VIII and XIII, activation of other cells including ECs themselves) but, instead, it converts PC to activated PC (APC). PC activation by the thrombin-TM complex is augmented by the binding of PC to EPCR. APC, in turn, inactivates factors Va and VIIIa and, in this way, shuts down thrombin generation. Binding of thrombin to TM, however, also leads to the activation of thrombin-activatable fibrinolysis inhibitor (TAFI), a carboxypeptidase that dampens fibrinolysis (see below). The anticoagulant activity of APC is greatly enhanced by its cofactor protein S. Although protein S is produced by other cell types (liver cells, megakaryocytes), its level in plasma likely is regulated primarily by the endothelium, since patients with severe liver diseases do not exhibit significant protein S deficiency.14 Vascular TM is located primarily in capillaries, but it is also present in larger arteries, veins and lymphatics.15,16 TM mRNA was easily detectable in numerous human tissues, particularly in the heart and pancreas, followed by lung, kidney and liver, whereas in the brain it was expressed at low levels.11 Studies with human and animal tissues have shown that, unlike TM, EPCR is located mainly in the endothelium of large blood vessels, with very low levels being expressed in most capillaries, except the liver sinusoidal endothelium.16 ECs have been also shown to promote the APC function.17 This is probably a peculiar property of ECs since monocytes as well as other cell types, including platelets, were unable to serve as a cofactor of APC and, in some cases, even inhibited its anticoagulant activity, possibly through the expression of a membrane component (not present in ECs) that protects factor Va from inactivation by APC.18–21 These findings further strengthen the pivotal role of ECs in the control of blood coagulation.

Figure 1. Anticoagulant properties of endothelium.

Figure 1

Anticoagulant properties of endothelium.1. Tissue factor pathway inhibitor (TFPI). Endothelial cells (ECs) are the main source of TFPI, an inhibitor that comes into action when limited quantities of factor Xa are generated. TFPI, indeed, first binds to (more...)

Figure 2. Anticoagulant properties of endothelium.

Figure 2

Anticoagulant properties of endothelium. 2. Heparan sulfate and related glycosaminoglycans. These heparin-like substances are expressed on the surface of endothelial cells (ECs), especially at microcirculation level, and accelerate the inhibition of factor (more...)

Figure 3. Anticoagulant properties of endothelium.

Figure 3

Anticoagulant properties of endothelium. 3. The protein C (PC) anticoagulant pathway. Endothelial cells (ECs) contribute to this pathway through the expression of thrombomodulin (TM), endothelial cell protein C receptor (EPCR) and protein S (PS). TM binds (more...)

Under normal circumstances, ECs, unlike cells surrounding the blood vessels (especially adventitial fibroblasts) that constitutively express significant amounts of TF, are almost unable to activate the coagulation pathways, as shown by immunohistochemical studies and by in situ hybridization.22,23 However, a large body of evidence indicates that cultured human or animal ECs, upon exposure to a number of agonists, undergo profound functional alterations that effectively transform the cell membrane from an anticoagulant to a procoagulant surface. The pivotal step causing this shift in the phenotype of endothelial properties is the synthesis and expression of TF. Synthesis of TF can be easily induced in vitro by a wide variety of stimulating agents or conditions that are implicated in a number of human diseases (for a review see ref.24). Stimuli that are of particular relevance in DIC accompanying different pathological conditions are listed in Table 1.25–53 Endotoxin, which is universally considered a major player in the pathophysiology of Gram-negative sepsis, is perhaps the most powerful inducer of TF in ECs and, likely, it is an important determinant of the endothelial procoagulant response to some whole Gram-negative microorganisms such as Neisseria meningitidis.25 The interaction of endotoxin with ECs is thought to occur through soluble CD14,54 but recent studies have shown that human umbilical vein ECs (HUVEC) do express CD14 on their surface (Fig. 4).55 Although DIC is classically associated with Gram-negative sepsis, it occurs as frequently in Gram-positive sepsis and, occasionally, in rickettsial, viral or parasitic (malaria) infections.56 Therefore, it is not surprising that numerous microorganisms are able to trigger TF expression in ECs.25–32 The effect of certain bacteria may be species- and strain-dependent.26 Endotoxin-independent mechanisms involved in EC stimulation by infectious agents are not completely understood. There are, however, several possible candidates including cell wall components that may be present also in Gram-positive bacteria, such as peptidoglycan and lipotheicoic acid, and exotoxins, such as staphylococcal a-toxin, hemolysin and verotoxin-1. These substances are able to induce activation of ECs57–59 and to contribute to shock and multiorgan failure;60 some of them, e.g., pneumococcal cell wall and verotoxin-1, have been shown to be potent triggers of TF synthesis in HUVEC.27,34 In certain circumstances, especially in viral and rickettsial diseases, TF expression can result from the direct infection of ECs.29–32 Moreover, microorganisms and/or their products may act indirectly, as they are known to induce the production of pro-inflammatory cytokines by mononuclear cells and by ECs themselves61 and there is quite compelling evidence that several cytokines , especially tumor necrosis factor a (TNF-α) and interleukin-1 (IL-1), are strong inducers of TF synthesis in ECs (Fig. 4).35,36Since cytokines can be released in vivo in DIC-associated pathological processes other than infectious diseases, such as trauma and malignancy,62,63 they are likely to play a major role in the up-regulation of TF also in these conditions. Other inducers of TF expression in ECs that may play a pathogenetic role in DIC-associated conditions include immunological stimuli, such as activation products of the complement system (C5a39 and C5b–9 complex at sublytic concentrations40), immune complexes41 and anti-proteinase 3 antibodies,64 and cell-cell interactions, such as adhesion to ECs of leukocyte microparticles,42 allogeneic lymphocytes,43 natural killer cells,44 monocytes,45 CD4-positive T cells,46 activated platelets47and smooth muscle cells (SMCs).48 The mechanisms underlying TF induction during cell-cell interaction have been elucidated, at least in part (Fig. 4), and may involve either direct CD40 engagement on ECs by CD154 (CD40 ligand)-expressing cells46,47,65 or the production of soluble mediators, including cytokines43,45and unknown factors.48 Finally, the observation that thrombin,49 factor Xa50 and fibrin,51 by binding to their respective receptors on ECs, up-regulate TF expression is of particular interest since it provides evidence for important amplification mechanisms of blood clotting activation by vascular cells (Fig. 4) The recent finding that stimulation of proteinase-activated receptor-2 (PAR-2) induces TF synthesis in ECs66,67 suggests yet another mechanism promoting blood coagulation, also because PAR-2 on ECs is strongly up-regulated by inflammatory mediators.68

Table 1. Agents and conditions inducing tissue factor synthesis/expression in endothelial cells.

Table 1

Agents and conditions inducing tissue factor synthesis/expression in endothelial cells.

Figure 4. Main mechanisms of tissue factor (TF) induction in endothelial cells (ECs).

Figure 4

Main mechanisms of tissue factor (TF) induction in endothelial cells (ECs). Lipopolysaccharide (LPS) induces TF synthesis through direct and indirect mechanisms. Direct induction occurs via interaction with CD14, either soluble or expressed on the EC (more...)

It is worth mentioning that most of the agents and conditions listed in Table 1 are also able to induce TF synthesis/expression in cells of the monocyte-macrophage lineage and that the latter are thought to be major players in different forms of DIC.24,69–71

Regulation of TF expression by ECs is complex and occurs at multiple levels. Although detailed studies are not available for all known stimulating agents, in general TF is induced at the level of gene transcription and possibly also by inhibition of mRNA degradation.72 In many instances, particularly with inflammatory stimuli, transcriptional activation of TF gene has been shown to involve nuclear factor kappa B (NF-κB).72 Details on regulation of TF expression are presented in the Chapter by Wolfram Ruf in this book. Quiescent ECs do not express TF, possibly due to promoter elements in the TF gene that repress its transcription under basal conditions.73 From the pathophysiological standpoint, an important level in the regulation of TF is the cellular distribution of the protein. Expression of TF on the luminal (apical) surface of stimulated EC monolayers has been reported by many investigators.33,35,39,40,74,75 However, in some studies, TF was either detected on the basolateral surface of cultured cells76 or in vesicular structures in the subendothelial matrix (and not surface-expressed at all).77 This discrepancy has been attributed mainly to differences in cell culture conditions.75,76 TF may be also localized to specific regions in the plasma EC membrane, i.e., to glycosphingolipid- and cholesterol-rich microdomains near and within particular microinvaginations of the cell membrane named caveolae.78,79 Such a localization would result from an active transport (translocation) of TF upon binding to its ligand factor VIIa and the generation of the product factor Xa and appears to be associated with an impairment of the proteolytic function of TF-VIIa complex, owing to the co-localization of TFPI with glycolipid microdomains/caveolae (Fig. 5).78,80Interestingly, in other cell types, for instance smooth muscle cells, TF is localized to caveolae in the absence of factor VIIa.81 Therefore, depending on the cell type, the role of TF in caveolae, which are deficient in anionic phospholipids (see later), would be either a latent pool of TF activity79,81 or a mechanism down-regulating its function.78,80

Figure 5. Localization of tissue factor (TF) to glycolipid-rich microdomains-caveolae.

Figure 5

Localization of tissue factor (TF) to glycolipid-rich microdomains-caveolae. Upon binding to its ligand factor VIIa and the generation of the product factor Xa, TF translocates into glycolipid-rich microdomains-caveolae. In this location, the proteolytic (more...)

Another relevant question to understand the pathophysiological role of TF is whether the protein present on the EC surface is active and functional. A common observation in studies on endothelial and other cells is that TF activity increases dramatically following cell lysis, the activity of intact cells (cell surface activity) being about 20–30% that of lysed cells. This suggests that TF activity is normally suppressed or “encrypted” on the surface of TF-bearing cells, that is, although TF antigen is detectable on the surface of the cells, its full procoagulant activity is not.33,82–84 Based on studies in purified systems and with different cell types, it is thought that a major mechanism for encryption is the cell membrane phospholipid asymmetry.84–87 Therefore, exposure of anionic phospholipids, particularly phosphatidylserine, on perturbed cells appears to be responsible for expression (de-encryption) of TF activity, although phospholipid-independent mechanisms have been proposed using other cell types.86,88

The expression of functional T activity may also depend on the origin of ECs. For instance, when stimulated with IL-1 or TNF-α, HUVEC expressed more functional TF than adult saphenous vein ECs, whereas TF mRNA was not different.89 Moreover, Shiga toxin, a toxin identical to verotoxin-1) produced by Shigella dysenteriae and Escherichia coli O157:H7 that has been strongly implicated in the pathogenesis of the hemolytic-uremic syndrome (HUS), augmented TF activity of TNF-α-stimulated ECs of glomerular, but not umbilical, vein origin. Since neither TF mRNA or antigen nor TFPI production was affected, the observed increase in TF activity was likely due to de-encryption of preformed TF. This observation suggests an important role of the TF pathway in glomerular fibrin deposition occurring in HUS.90 Finally, Grau et al91 reported that the procoagulant activity induced by TNF-α and LPS was higher in lung than in brain microvascular ECs, an observation that may be related to the frequency of lung involvement in septic shock.

Under particular conditions, ECs can initiate blood coagulation via TF-independent pathways. Cultured or native endothelium exposed to a hypoxic environment has been reported to generate a membrane-associated procoagulant that directly activates factor X.92 This mechanism, coupled with the hypoxia-induced up-regulation of endothelial TF,52,53 provides a basis for understanding the initiation of vascular pathology in hypoxemic states, including DIC-associated conditions. ECs infected with herpesvirus provide a viral-encoded cell surface glycoprotein (glycoprotein C) that may promote thrombin generation through direct binding and activation of factor X.93 Finally, a direct membrane-associated prothrombin activator has been reported in cultured human ECs stimulated by LPS or IL-1β94 and in porcine endothelium.95

There is also evidence that the endothelium may provide a suitable surface for assembly and activation of the contact system of blood coagulation.96,97 Cultured ECs express binding sites for factor XII and for high molecular weight kininogen (HK), the multidomain protein that is the major receptor for prekallikrein (PK) and factor XI on the cell surface, and, at the same time, the cofactor for activation of these proenzymes. It has been hypothesized that, when HK binds to ECs, it initiates a series of events that allow an EC-associated protease (most probably a cysteine protease) to activate PK and factor XI bound to HK on the cell membrane. Although, in this model, PK and factor XI activation clearly occurs independently of factor XII, the formed kallikrein can activate surface-bound factor XII to factor XIIa, which will amplify the reactions. Factor XIa, in turn, can activate factor IX on the EC surface.96–98 HK is also the substrate for kallikrein, leading to bradykinin generation. While these studies describe a novel mechanism for activation of the contact system, it is unclear so far which role this mechanism plays in haemostasis and thrombosis and whether it is influenced by endothelial perturbation caused by relevant pathogenic stimuli involved in DIC. Interestingly, treatment of ECs with bradykinin results in increased HK binding.96

Besides the potential for triggering clotting pathways, ECs may exhibit other properties promoting coagulation events on their surface including the production of factor V99,100 and of a putative factor V activator94 and the expression of specific membrane receptors for several clotting factors (Fig. 6). Although the identity and location of most of these receptors remain to be definitively established, several possible candidates have been proposed. Specific binding sites for factor IX/IXa have been described, which, together with putative receptors for factor VIIIa, might be operative in localizing a functional factor VIIIa/IXa complex activating factor X on the EC surface.101,102 TNF has been shown to enhance the number of these binding sites, thus potentially enhancing factor Xa generation.101 Although type IV collagen was identified as a major factor IX/IXa binding site and claimed to be the endothelial receptor,103 its physiological relevance remains obscure. ECs also express high affinity receptors for factor Xa that are related104 or identical105 to effector cell protease receptor 1 (EPR-1). Cell-bound factor Xa then promotes prothrombin activation in the presence of factor V.104,106 While the putative factor V receptor on cells remains unknown, recognition of prothrombin appears to be controlled by the agonist-induced activation of the integrin αVβ3 on ECs.107 Of note, factor Xa, when added to ECs, was a potent mitogen, increased intracellular free calcium levels and phosphoinositide turnover and induced TF expression,50 suggesting that endothelial factor Xa receptor may be of importance also in mediating cellular signaling and responses of the vessel wall. These effects were all abolished by direct or indirect (antithrombin-heparin) inhibition of factor Xa, indicating that these mechanisms are dependent on its catalytic activity. Finally, a number of stimulating agents, including pathogenic microorganisms and complement membrane attack complex C5b-9, were shown to efficiently promote the assembly of the prothrombinase complex on the endothelial surface, thereby increasing the rate of thrombin formation.31,108 This effect is own mainly to the induction of the key changes in membrane asymmetry (exposure of anionic phospholipids, especially phosphatidylserine) required for activation of prothrombin (and factor X). Due to this complex array of procoagulant properties, perturbed ECs may represent a surface onto which clotting pathways are initiated and propagated, eventually leading to fibrin formation (Fig. 6).

Figure 6. Thrombin generation on “perturbed” endothelial cells (ECs).

Figure 6

Thrombin generation on “perturbed” endothelial cells (ECs). Expression of tissue factor (TF) leads to generation of factors IXa and Xa (the latter is not shown). Specific receptors for clotting factors expressed on the EC surface along (more...)

Of particular interest is the observation that activated ECs can shed into the extracellular space fragments of their plasma membrane named microparticles or microvesicles that retain their procoagulant properties, including TF.108,109 This phenomenon, also observed with other cells,110,111 may represent an important mechanism for disseminating coagulation activation.

It should be emphasized that the stimuli inducing the expression of procoagulant activities may also cause the down-regulation of the physiological anticoagulant potential of ECs (Fig. 7). In this respect, the most extensively studied EC property is the expression of TM, which was found consistently to be reduced in response to most agents listed in Table 1.46,47,74,92,112 Mechanisms of TM down-regulation include inhibition of synthesis, acceleration of internalization and degradation, and inactivation by products of leukocytes released following their interaction with ECs, such as oxidants and/or lysosomal proteases.101,113–116 Leukocyte proteases could also contribute to the release of TM from the cell membrane in vitro and in vivo (see below). Inflammatory cytokines may further impair the PC pathway by reducing protein S secretion117 and EPCR expression12,13 in cultured ECs. Moreover, various agents, including cytokines, H2O2 and thrombin dramatically increase the shedding of EPCR from cells in culture medium.13 The central role of the PC anticoagulant pathway in modulating the thrombogenic properties of ECs is supported by the elegant study of Cadroy et al,118 using an ex vivo perfusion model of thrombogenesis.

Figure 7. Endothelial perturbation creates a prothrombotic microenvironment.

Figure 7

Endothelial perturbation creates a prothrombotic microenvironment. Different stimuli induce or enhance the synthesis of the prothrombotic factors tissue factor (TF) and plasminogen activator inhibitor-1 (PAI-1), and down-regulate the production or expression (more...)

As to the other endothelial anticoagulant properties, there is some evidence that inflammatory stimuli can down-regulate the expression and function of heparan sulphate proteoglycan in cultured cells.119–121 Finally, conflicting data have been published about TFPI. Recently, Shimokawa et al122 reported that LPS and TNF-α strongly decreases the steady state level of TFPI mRNA in vascular ECs. This is at variance with other studies showing that inflammatory mediators had no effect or slightly increased TFPI synthesis.123 Nevertheless, the role of endothelium-associated TFPI as a direct vessel wall anticoagulant is supported by the findings of Grabowski et al124 showing a marked increase in TF-induced factor X activation in a flow model after neutralization of TFPI on the endothelial surface. It is interesting to note that TFPI blocks the cellular effects of endotoxin by binding to it and interfering with transfer to CD14.125

The progressive reduction or loss of physiological anticoagulant mechanisms, coupled with the appearance of clot-promoting properties, will eventually favor uncontrolled fibrin formation on the EC surface (Fig. 7).

In Vivo Changes and Role in the Pathophysiology of DIC

The in vitro findings summarized above clearly demonstrate that numerous factors of prominent pathogenetic relevance in pathological conditions associated with DIC are able to cause profound alterations in the coagulation-related properties of vascular ECs leading to a prothrombotic phenotype. Moreover, in perfusion models of thrombogenesis, TF-expressing ECs, when exposed to non-anticoagulated blood under flow conditions, could indeed elicit thrombus formation.126,127 On the basis of this evidence, it is conceivable that ECs might be implicated in blood clotting activation and fibrin deposition occurring during DIC. Therefore efforts have been made to establish the extent to which the in vitro findings reflect mechanisms and reactions operating in vivo. Our knowledge on the relationship between endothelial perturbation and microvascular thrombosis in DIC stems from numerous animal studies and a more limited number of human studies.

Animal Studies

The model in which ECs have been most extensively investigated is DIC induced either by genuine endotoxin or by classical Gram-negative bacteria. In this model, several lines of evidence indicate a pivotal role of TF in blood clotting activation. First, administration of low-dose endotoxin to primates elicited a clear activation of coagulation, as reflected by the increase in plasma levels of factor X activation peptide, prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complexes (TAT) and fibrinopeptide A (FpA), but did not cause any change in markers for activation of the contact system.128 Moreover, in different animal species, the impairment of the TF pathway by various means (antibodies against TF or factor VII/VIIa, TFPI) prevented blood clotting abnormalities (including fibrin deposition in target tissues) and, in many instances, also the lethal effects of endotoxemia.128–132 Conversely, inhibition of the contact system, which may be also activated during experimental endotoxemia, did not prevent clotting activation.133 This strong evidence notwithstanding, the identification of the actual cellular source of TF in vivo and, in particular, the contribution of ECs still remain an incompletely resolved issue. To our knowledge, only a few data are available concerning the in vivo/ex vivo expression of TF on ECs during experimental endotoxemia. In rabbits treated with endotoxin134 or IL-1β,101 the luminal surface of thoracic aorta exhibited significantly higher levels of TF activity than vessels from control animals. At variance with other findings,135 the observed increase in TF could be completely ascribed to ECs, since almost no TF activity could be detected when the aorta was denuded of its endothelium.134 Induced expression of TF in vascular endothelium was also definitely documented by immunohistochemical methods in baboons with lethal E. coli septic shock.136 However, despite the profound stimulation with endotoxin in this model, TF antigen was detected only on ECs confined to the marginal zone of the spleen, while it was absent in ECs of the other organs examined. In a study on the role of NF-κB in vivo, Bohrer et al137 reported a possible causal relationship between NF-κB activation, TF expression and the outcome of sepsis in a particular mouse model of endotoxemia, in which the animals were treated with a mixture of LPS and D-galactosamine in order to sensitize them to the lethal effects of LPS. In this model, intravenous somatic gene transfer of plasmids overexpressing IκB-a or antisense TF reduced both TF expression in renal tissues and activation of the coagulation system (as evidenced by a decrease in TAT and in renal fibrin deposition), and increased survival. Of note, ECs in kidney and peripheral blood mononuclear cells were the major targets for somatic gene transfer with IκBa. Therefore, in this model, NF-κB-dependent de novo synthesis of TF, likely occurring in ECs and mononuclear phagocytes, might contribute, at least in part, to DIC and lethality. In other studies in rabbits, mice and rats, in which TF expression in vivo was analyzed following endotoxin administration, the endothelium of several tissues was TF-negative.72,138–143 Rather, aberrant expression of TF was found in other cell types that are localized in organs in which fibrin deposition is often observed during DIC, particularly in kidney and lung. It appears, therefore, that local triggering of coagulation by different TF-expressing cells may contribute significantly to endotoxin-induced DIC. It should be emphasized that, in these as well as other studies,144,145 the in vivo expression of TF by monocytes/macrophages in response to endotoxin or gram-negative bacteria was a frequent finding, thus pointing to an important role of these cells in sepsis-associated blood clotting activation. Similarly, in a mouse model of hypoxia, in which TF-dependent fibrin deposition was observed in the lung, the endothelium did not display TF antigen, at least at levels detectable immonohistochemically,146 which is in contrast with the in vitro findings. Again mononuclear phagocytes in the pulmonary microvasculature were TF-positive and were likely responsible for local activation of coagulation. Why it is so difficult to detect TF expression on ECs in vivo despite intense stimulation by LPS and other mediators involved in endotoxemia is unclear. Possible explanations are sensitivity limitations of the assays because of the low TF level, heterogeneous expression of TF possibly linked to vascular bed-specific response or to species-specific differences and a more tightly controlled TF induction in ECs. In this respect, it is of interest that nitric oxide, a physiological vasodilator and antiplatelet agent produced by ECs, down-regulates LPS- and IL-1β-induced TF expression by microvascular ECs, thus exerting its antithrombotic activity not only by inhibiting platelet function but also by suppressing the coagulation.147

So far a few studies have examined the regulation of TFPI by inflammatory agents in vivo. Hara et al142 reported a decreased expression of TFPI mRNA in lungs of endotoxin-treated rats. By immunohistochemistry, pulmonary ECs of control animals showed positive reaction for TFPI antigen, but TFPI-positive cells markedly decreased in number after endotoxin administration. In mice, treatment with endotoxin or TNF-α, but not with IL-1, transiently suppressed the expression of TFPI mRNA in several organs (lung, kidney, heart and adipose tissue); such a decrease was attributed mainly to ECs, since down-regulation of TFPI mRNA was also observed in cultured mouse ECs.122 Finally, an impaired renal expression of TFPI was found in a rabbit model of glomerulonephritis.148 If confirmed by further in vivo studies, TFPI underexpression might represent an additional mechanism, together with up-regulation of TF, to augment the local procoagulant potential and thus contribute to fibrin formation at tissue level. The importance of TFPI in modulating the procoagulant response to endotoxin in vivo is supported by the observation that, in human experimental low-dose endotoxemia149 and in a baboon E. coli septic shock model,132 the administration of TFPI inhibited thrombin generation and, in the latter model, also reduced the mortality. This effect probably results not only from impaired coagulation but also from the capacity of TFPI to block the cellular effects of endotoxin.125

Because the in vitro studies consistently showed a down-regulation of the EC properties involved in the PC anticoagulant pathway, i.e., TM and EPCR, in response to endotoxin and other inflammatory stimuli, attempts have been made to understand the regulation of these properties in vivo during experimental endotoxemia. Nawroth et al150 reported that infusion of interleukin-1 in rabbits suppressed TM activity of aortic endothelium. However, in the same animal species and in contrast with in vitro results, no changes in aortic TM activity were observed by others following endotoxin administration.134 Notably, in the latter study, endotoxin treatment also failed to influence the in vivo generation of APC induced by a continuous infusion of a low dose of thrombin, thus making unlikely a widespread effect of endotoxin at the microcirculatory level. In agreement with this study, TM down-regulation could not be observed in vascular beds of different organs by immunohistochemistry in two distinct sepsis models in baboons and rats.136,151 On the other hand, a rise in soluble plasma TM was consistently observed during experimental endotoxemia, suggesting that direct and/or indirect (leukocyte-mediated) endothelial activation-damage by inflammatory mediators does occur in vivo.152–154 In one study, the increase in TM was found to correlate with haemostatic markers (TAT, soluble FM, PC, APC/inhibitor complex).154 Failure to demonstrate TM down-regulation at tissue level might be due to sensitivity problems of the assay technique or, more likely, to insufficient changes of the tissue TM pool despite shedding.

As to the EPCR, studies in rats and mice have shown that EPCR mRNA, rather than falling, as predicted from in vitro experiments with cultured cells, rapidly increased after treatment with lethal doses of endotoxin.13 This was associated with a marked rise in soluble plasma EPCR. The increase of both EPCR mRNA and soluble EPCR was shown to be mediated by thrombin, not by TNF-α.

Although the data summarized above may be difficult to interpret, the importance of the PC anticoagulant pathway for the development of DIC in sepsis and other conditions has been suggested by numerous in vivo studies.155 Treatment with APC could prevent E. coli-induced coagulopathy in baboons and rats. Conversely, blocking the PC anticoagulant pathway at different levels, e.g., by interfering with PC activation (through a monoclonal antibody) or by reducing the plasma levels of free protein S (with the administration of C4b binding protein), resulted in a marked worsening of DIC and organ failure in baboons challenged with sublethal doses of E. coli. Moreover, in the same model, treatment with an antibody that blocked the binding of EPCR to PC/APC strongly exacerbated the response to sublethal bacterial challenge, suggesting that PC/APC-soluble EPCR interaction might also play a critical role in regulating the inflammatory and coagulation response to infection.13 Finally, mice homozygous for a point mutation of the TM gene that specifically deletes the anticoagulant activity of the protein (poor binding to thrombin and thus reduced capacity to generate APC), as compared with wild-type mice, exhibited 10- to 30-fold greater amounts of fibrin in arterioles, capillaries and venules of several, but not all, organs (lung, heart, spleen and liver).156 Most important in the context of DIC, under hypoxic conditions, in which there is up-regulation of TF in the lung (see above), the mutant mice exhibited more extensive pulmonary fibrin deposition than normal animals,156 indicating the importance of local TM in the control of coagulation.

Human Studies

The pathophysiological role of endothelial perturbation is much more difficult to assess in man, mainly because ECs are not accessible for analysis and quantitation of their multiple activities. Therefore experimental and clinical studies rely on circulating levels of activation markers as a readout of endothelial dysfunction/damage. As observed in primates, administration of endotoxin or TNF-α to healthy volunteers induced an early and sustained sub-clinical activation of the coagulation system, as assessed by highly sensitive and specific assays (increase of the activation peptide of factor X, F1+2 and TAT).149,157 Neither treatment caused activation of the contact blood clotting system, thus suggesting the involvement of the TF pathway. On the other hand, many clinical studies have shown that blood clotting activation, assessed by a variety of assays ranging from common hemostatic variables to highly specific and sensitive markers, is almost invariably observed in pathological conditions associated with DIC, including sepsis and trauma.149,158 Examination of plasma TF antigen revealed increased levels in patients with these conditions, particularly in those with overt DIC.159–163 In general TF elevation was associated with raised concentrations of FpA, F1+2, TAT and D-dimer and, in some studies, a significant correlation was found between TF levels and organ dysfunction.161,163 These findings clearly implicate TF in blood clotting activation, although they do not allow definitive conclusions about the contribution of ECs. It is worth mentioning that, in a model of human low-grade endotoxemia, factor XI activation was shown to occur independently of factor XII, probably mediated by thrombin generated via the TF pathway.164 In vivo activation of factor XI was also reported in mice following thrombin injection.165 The site of factor XI activation is presently unknown. Recently, on the basis of in vitro studies, activated platelets have been proposed as a preferential site for thrombin-mediated activation of functionally significant amounts of factor XI.166 Whether perturbed ECs represent a suitable surface for this phenomenon to occur is an open question.

Numerous investigators have measured circulating levels of markers that are more specific for endothelial perturbation, including TFPI and proteins of the PC pathway, in patients with DIC-associated conditions. Reports on plasma levels of TFPI (activity and/or antigen) in sepsis have shown conflicting results. Normal or increased levels were found in some studies, especially in patients with a fatal outcome, probably due to release from ECs.167–169 In contrast, in other studies, TFPI levels in septic patients were significantly reduced, likely as a result of consumption.170,171 In one of these studies low TFPI plasma levels were associated with high urinary levels, suggesting that, besides consumption, increased clearance from the circulation might contribute to the observed decrease in TFPI.171 Decreased plasma TFPI was also found in patients with thrombotic thrombocytopenic purpura (TTP),172 another condition in which the notion that ECs are dysfunctional is widely accepted. The plasma levels of soluble TM have been evaluated in various disease states associated with DIC, including sepsis and TTP, and, with a few exceptions,173 were found to be increased.160,172,174–177 TM levels, generally thought to reflect EC activation or damage, were often correlated with disease severity and poor outcome.160,172,176 Interestingly, Saito et al177 found significantly higher TM levels in septic patients with leukocytosis than in those with leukopenia induced by chemotherapy for hematological malignancies, and suggested an important role for leukocytes in EC perturbation. Finally, patients with sepsis or vascular injury also had increased plasma levels of EPCR,173 indicating that the latter can be used as an additional marker of EC activation. The lack of correlation between EPCR and TM levels in the same patients suggests that different mechanisms might be responsible for their shedding/release from ECs or may reflect the different distribution of these two surface proteins. While the preponderance of data summarized above clearly points to a significant role of endothelium in coagulation disturbances seen in patients with DIC-associated conditions, it should be considered that changes in circulating markers do not indicate whether endothelial perturbation is a cause or a consequence of the disorder and, more important, they do not provide information about the site of disease involvement.

In striking contrast with the extensive number of studies performed with human ECs in vitro, direct evidence that inflammatory stimuli cause local procoagulant changes in vessel wall endothelium in vivo is very limited and it is based on results of biopsy studies in various conditions. The presence of TF antigen, associated with fibrin deposition, was reported in vascular endothelium of severely inflamed and ulcerated mucosa from patients with inflammatory bowel disease178 and in ECs of the pulmonary artery and of small adventitial vessels in a specimen from a patient with tuberculosis.179 On the other hand, a marked depletion of surface TM was found immunohistochemically in vascular endothelium close to granulomatous and peri-phlebitis lesions in patients with cutaneous sarcoidosis180 and in vessels of inflamed bowel tissues from patients with active ulcerative colitis.181 Moreover, very recently, the expression of both TM and EPCR on morphologically intact ECs was found to be reduced in biopsy specimens of purpuric lesions from children with meningococcal sepsis.174 Plasma levels of APC were low in these patients even after treatment with unactivated PC concentrates, indicating a dysfunction of endothelial PC activation. In a recent immunohistochemical study performed in healthy volunteers, Speicer et al182 demonstrated that intradermally injected TNF-α caused, besides the typical inflammatory changes (up-regulation of adhesion molecules and accumulation of leukocytes), the expression of TF antigen in ECs of inflamed but not normal dermal vessels. They also found a much weaker staining for TM and TFPI in vascular endothelium of inflamed tissue as compared to endothelium in normal tissue. Finally, an early depletion of endothelial IκBa was observed at the site of TNF-α-induced inflammation, pointing to an activation of the NF-κB pathway. Thus, this interesting study strongly suggests that the characteristic procoagulant changes of the endothelial phenotype observed in cell culture experiments can be recapitulated in vivo in man. Whether and to which extent these findings are of relevance to human disease, particularly to sepsis-associated DIC, remains to be established. It should be noted that, in patients with severe infections (meningococcal sepsis, peritonitis), ARDS secondary to sepsis or other conditions associated with endotoxemia (severe obstructive jaundice, experimental endotoxemia), mononuclear phagocytes of different origin were found to express significantly increased levels of TF,183–187with the highest amounts seen in patients with lethal outcome.183,186 This would suggest an important role of these cells also in human DIC. Interestingly, in a clinical study in septic patients, NF-κB activation could be detected in peripheral blood mononuclear cells and was more intense in patients who died than in survivors,137clearly implicating this factor in human sepsis.

An alternative approach for a direct exploration of the endothelium in vivo might be the enumeration and phenotypic characterization of circulating ECs (CEC) released in peripheral blood as a consequence of vascular injury. These cells should be an acceptable surrogate for vessel wall endothelium because they would be subjected to the same blood-borne activating signals. Recently, the development of methods for identification and harvesting of CEC allowed the detection of high numbers of these cells in different disorders, including infectious, immunological and thrombotic diseases, while CEC were found to be very rare in normal subjects.188 Moreover, in patients with sickle cell anemia, a condition associated with increased CEC and ongoing activation of blood coagulation, Solovey et al189 found that, unlike CEC from normal donors, sickle CEC expressed functional TF. According to these investigators, the fact that ECs can express TF in vivo implies that the vast endothelial surface area does provide an important trigger for clotting activation. As it has long been known that endotoxin disrupts endothelial barrier function and induces the detachment of ECs from the underlying matrix in vitro and in vivo,1,2,190 the assessment of CEC properties might open new perspectives for investigation of the role of ECs in human sepsis as well as in other DIC-associated conditions.

As already mentioned, activated ECs can release into the extracellular space fragments of their plasma membrane (microparticles) that retain the procoagulant phenotype of the intact cell and thus might provide a mechanism for dissemination of clotting activation. Therefore studies on the presence and characterization of these microparticles could be useful to understand the role of EC properties in the development of DIC. Recently, Nieuwland et al191 reported that patients with meningococcal sepsis have increased numbers of circulating procoagulant microparticles derived from various cells including ECs and that the presence of strongly procoagulant microparticles was associated with elevated plasma levels of F1+2. Interestingly, the highest levels of microparticles, including those of endothelial origin, were detected in a patient with an extremely fulminant course of DIC. The procoagulant activity of microparticles was own to the expression of both TF and phosphatidylserine on their surface. Although the majority of TF-positive microparticles were of monocyte origin (CD14-positive), the possibility that endothelium-derived microparticles also express TF cannot be excluded. An increase in circulating microparticles of endothelial origin was shown in patients with lupus anticoagulant by Combes et al,192 who postulated a possible role of these microparticles in the known thrombotic tendency associated with this condition. Unfortunately, the expression of TF on patient microparticles was not investigated. In this context, an interesting observation was recently made by Ozcan et al,193 who demonstrated that substantial circulating TF activity, measured by a novel whole blood assay, remained detectable in samples obtained from patients undergoing bone marrow or peripheral blood stem cell transplantation during the period of profound aplasia. The observed increase of TF-positive CEC could not account for the measurable whole blood TF activity. Although the source(s) of this activity was not identified, it could well be represented by microparticles shed from ECs following cytokine-mediated activation or damage by the myelo-ablative therapy.

Endothelial Cell Perturbation and Fibrinolysis

Fibrinolysis provide an important mechanism contributing to the maintenance of blood fluidity. Plasmin generation is essential to remove fibrin that has been deposited intravascularly and might also prevent clotting if it occurs during fibrin formation. The role of fibrinolysis in DIC-associated conditions is rather well established. First, accumulation of fibrin deposits in the microcirculation may be greatly facilitated by an impairment of the fibrinolytic system. Infusion of des-A-fibrin or thrombin, at doses unable to induce fibrin accumulation in normal animals, caused diffuse renal microthrombosis in animals pretreated with antifibrinolytic agents.194,195 Interestingly, a single endotoxin injection was sufficient to render the animals sensitive to thrombogenic stimuli,194,196 most probably because of the inhibition of fibrinolysis. Moreover, administration of high doses of tissue-type plasminogen activator (t-PA) or low doses of plasminogen activator inhibitor-1 (PAI-1)-resistant t-PA prevented fibrin deposition in kidneys of endotoxin-treated rabbits.197 Likewise, in a rat model of endotoxemia, a decrease in fibrin deposition in lungs was induced by the treatment with an inhibitor of PAI-1.198 Finally, in DIC associated with sepsis or other inflammatory diseases, a reduction in blood fibrinolytic activity, mainly due to a very marked increase in PAI-1, has been well documented and there is ample evidence that this fibrinolytic shut-down contributes to microvascular thrombosis and organ dysfunction.62,199–206

Endothelium plays a pivotal role in the fibrinolytic process by regulating it at many levels. ECs synthesize and release the key components of the fibrinolytic system, namely t-PA, urokinase-type PA (u-PA) and PAI-1, and express surface receptors that serve to localize and facilitate plasminogen activation (Fig. 8).4 As regards t-PA, the endothelium is considered the main source of the circulating enzyme.207 In addition, in view of the complex interaction between coagulation and fibrinolysis, ECs may influence fibrin removal by altering thrombin generation and thrombin specificity.208 Virtually all these fibrinolytic properties may undergo profound changes in response to external signals so that the profibrinolytic surface of the resting endothelium is transformed to an antifibrinolytic surface. This phenotypic shift is an essential part of the “core changes” that characterize EC activation and greatly contributes to create a prothrombotic microenvironment.

Figure 8. Fibrinolytic properties of endothelial cells (ECs).

Figure 8

Fibrinolytic properties of endothelial cells (ECs). ECs synthesize both tissue-type plasminogen activator (t-PA) and prourokinase (pro-uPA) as well as the main PA inhibitor PAI-1, most of which is localized in the extracellular matrix. ECs also express (more...)

t-PA is costitutively synthesized by cultured ECs as an active single-chain enzyme.4 It is partly released and partly stored in intracellular granules, identified as Weibel Palade bodies by some investigators209 and as new small-dense granules by others.210 The continuous release of t-PA would serve to maintain a basal level of fibrinolytic activity in blood (which will be the result of a dynamic equilibrium with PAI-1, normally present at higher concentration) whereas the intracellular pool is released upon demand to rapidly enhance the local fibrinolytic capacity. One main feature of t-PA is its ability to bind to fibrin, which is essential for efficient plasminogen activation.211 If fibrin is formed in the presence of free t-PA the latter will be incorporated within the clot and cause a rapid lysis even in the presence of inhibitors outside the clot.212,213 There is evidence that following the injection of a high dose of thrombin, fibrin deposition within lung vessels, which occurs within minutes, is enhanced if the animal is treated with antifibrinolytic agents such as EACA, a lysine analogue that competitively inhibits the binding of t-PA and plasminogen to fibrin.165 This suggests that physiologic levels of t-PA may limit fibrin accumulation even after massive and widespread stimulation of coagulation. Studies in knock-out mice indicate that the lack of t-PA does not cause spontaneous thrombosis but makes the animal more susceptible to thrombogenic stimuli.214 This is at variance with plasminogen-deficient mice which develop spontaneous thrombosis and suggests that, in the absence of t-PA, plasminogen activation may be catalyzed by other PAs such as u-PA.214

The production of t-PA can be induced in cultured ECs by a number of stimuli or conditions207and is usually regulated at the level of gene transcription and cellular release.207,215Among the agents involved in sepsis and other DIC-associated conditions, some, such as TNFa, IL-1, endotoxin and herpes simplex virus, have no effect or decrease t-PA synthesis in cultured ECs,216–219 while others, such as thrombin219,220 and factor Xa,50 increase t-PA production. It should be noted, however, that most of the above stimuli, including those that augment t-PA release, also stimulate PAI-1 synthesis, the net effect being almost invariably antifibrinolytic. The assumption that the production/release of t-PA is a property of all ECs, as suggested by in vitro findings, has not been supported by in vivo studies. In situ hybridization and immunohistochemical techniques have demonstrated that t-PA expression may be very heterogeneous depending on tissue type and vessel size. For instance, in mouse and primate lung tissue, t-PA antigen and mRNA expression was limited to ECs of bronchial arteries and pulmonary vessels of discrete size.221 Moreover, the exposure of mice to hyperoxic conditions promoting lung injury and inflammation caused an increase in the number of t-PA producing ECs, which, however, remained confined to the same vessels as in control animals.221 It thus appears that endothelial t-PA expression, either constitutive or induced, is a function of a limited group of ECs, defined by vessel size and anatomic location. In human specimens of normal saphenous vein and internal mammary artery, ECs expressed both t-PA mRNA and antigen, but at relatively low level.222

ECs express cell surface binding sites for plasminogen and plasminogen activators that allow enzyme and substrate to assemble on cell surface and facilitate the generation of the active protease plasmin.223 Moreover, binding of the activator and plasmin to their cell receptors appear to relatively protect these molecules from inhibition by PAI-1 and a2-plasmin inhibitor, respectively, thus conferring to the cell an efficient mechanism for pericellular proteolysis. Different plasminogen receptors on ECs have been reported.223 One such receptor is annexin II, a phospholipid binding protein that has the peculiar property of binding also t-PA, but not u-PA. Since plasminogen and t-PA bind to annexin II noncompetitively, the receptor is able to colocalize enzyme and substrate in a manner that enhances the catalytic efficiency of t-PA-dependent plasmin generation by 60-fold (Fig. 8).224,225 The relevance of cell surface plasmin generation is suggested by recent evidence showing that disregulated fibrinolytic assembly may be associated with either atherothrombotic disease or bleeding diathesis.226

Cultured ECs also synthesize single-chain urokinase (scu-PA, prou-PA) and express a urokinase receptor (uPAR) with the same characteristics of that expressed by other cell types.4,227,228 Scu-PA is a zymogen and is activated by proteolytic digestion by plasmin or kallikrein.229 Upon binding to u-PAR, it is more easily converted to the two-chain form which, in turn, is more active in converting plasminogen to plasmin (Fig. 8).230,231 Receptor bound scu-PA, moreover, is partially protected from inhibition by PAI-1 and PAI-2.232,233 u-PA-dependent plasminogen activation by ECs is thought to be involved mainly in wound healing and angiogenesis,234 consistent with the hypothesized role of u-PA in pericellular proteolysis rather than in fibrinolysis. This concept, however, has been recently challenged on the basis of several observations that underscore the importance of u-PA also in vascular homeostasis.235 For example, mice genetically deficient in u-PA not only manifest an increased susceptibility to thrombogenic stimuli but, at variance with t-PA deficient animals, they also develop spontaneous intravascular fibrin deposits.236u-PA appears not to be produced by most quiescent ECs in culture. A major variable in u-PA synthesis by ECs is their vascular origin, renal, cerebral and lung microvascular cells being particularly rich sources, in contrast to HUVECs and other ECs.237–239 Up-regulation of u-PA production in cultured ECs has been observed with a variety of stimulating agents, among which TNF-α,240 IL-1, endotoxin,241 thrombin,238 fibrinogen degradation product fragment D242 and vascular endothelial growth factor (VEGF).243 It is not yet clear whether resting ECs in vivo express u-PA. Indeed, while in some studies u-PA could not be detected in human vascular endothelium of different origin by immunohistochemistry,244–246 Salame et al222 were able to show the expression, though at low level, of the activator (antigen and mRNA) and of u-PAR protein in ECs of normal saphenous vein and internal mammary artery. Moreover, a highly variable expression of u-PA (activity, antigen and mRNA) by human venous endothelium was reported by Camoin et al,247 using ECs dislodged by venipuncture.

Cultured ECs of arterial, venous and microvascular origin produce PAI-1 in large quantities.248 The majority of the inhibitor released by ECs binds to vitronectin associated with the extracellular matrix. In this location it is protected from the spontaneous inactivation that occurs in solution, and is accessible to soluble activators.249 PAI-1 appears to be one of the most important regulators of fibrinolysis. Transgenic mice overexpressing human PAI-1 develop spontaneous thrombosis,250 while PAI-1 deficient mice show an enhanced capability of dissolving pulmonary emboli.214

PAI-1 synthesis by cultured ECs is stimulated by a wide variety of agents and conditions associated with numerous pathological conditions.251 Of particular interest in the context of DIC are endotoxin, some microorganisms, cytokines IL-1 and TNF-α, hypoxia, thrombin, factor Xa and others,50,199,216,217,252–256 all of which not only greatly reduce the cellular fibrinolytic potential by increasing PAI-1 but also make the endothelial surface procoagulant (see above) (Fig. 7). The induction of PAI-1 synthesis is due mostly to enhanced transcription of the PAI-1 gene. Analysis of normal human tissues revealed relatively high levels of PAI-1 protein in different organs, especially in liver and spleen,257 and of PAI-1 mRNA in a number of highly vascularized tissues including placenta, uterus, myocardium and liver.258 A variable PAI-1 expression could be detected in both ECs and smooth muscle cells (SMCs) of human arteries and veins.222,259–262 However, it is unclear whether these findings actually reflect the basal expression of PAI-1 in vivo, since most human tissues were obtained under stressed conditions and PAI-1 is an acute phase protein in humans.263 In more controlled animal models, PAI-1 mRNA was easily detectable in most of the normal tissues examined.264–266 However, in situ hybridization or immunohistochemical analysis showed that quiescent endothelium express little or no PAI-1 under normal conditions.251,267 Rather, in the mouse, the inhibitor was consistently expressed in SMCs in the aorta and in the vasculature of other tissues (kidney, heart, liver, adipose tissue).251 Therefore, both in animals and humans, SMCs might be an important source of PAI-1 under normal conditions and contribute, together with other cells, to circulating PAI-1. ECs, instead, may become a main actor in the dynamic regulation of PAI-1 under stress conditions.

In Vivo Changes and Role in the Pathophysiology of DIC

Since t-PA represents perhaps the best marker of endothelial function207 and PAI-1 is the major biosynthetic product of ECs in response to DIC-associated stimuli (see above), the plasma levels of these proteins have been extensively examined. In non-human primates268 and in healthy volunteers receiving low-dose endotoxin or TNF-α,157,269,270 a sudden increase in plasma t-PA levels, indicative of EC activation, was observed which coincided with the activation of the fibrinolytic system, as measured by plasmin-α2-plasmin inhibitor complexes. However, the fibrinolytic activity was rapidly offset by the subsequent and long-lasting increase in the plasma levels of PAI-1.157,268–270 A dramatic increase in plasma PAI-1 during endotoxemia has been consistently found also in every animal species studied (rat, rabbit, mouse).199,217,251 In view of these observations, studies at tissue level have attempted to elucidate the nature of the cells involved in the synthesis of fibrinolytic proteins and, in particular, the contribution of ECs. Aortic vascular segments freshly removed from endotoxin-treated rabbits, when incubated in culture medium, released much more PAI-1 activity than vessels from control (saline-treated) animals.271 Studies using in situ hybridization revealed that endotoxin administration induced a rapid elevation of PAI-1 mRNA in rat and murine tissues, including those most commonly affected by microthrombi during sepsis (kidney, adrenals, lung and liver), whereas much lower levels were observed in tissues from control animals.140,264–266,272–274 In mice, PAI-1 mRNA was also positively regulated by TNF-α, with a tissue-specific response pattern similar to that elicited by endotoxin, and by TGF-β, though with a distinct pattern.264 PAI-1 (mRNA and protein) expression was detected primarily in ECs at all levels of the vasculature (arteries, veins and capillaries) in various organs, including kidneys (glomerular and peritubular ECs), adrenals and liver.140,265,266,272,274 In liver, PAI-1 mRNA was also induced in hepatocytes,266,272 albeit with a different mechanism.272 The dramatic induction of PAI-1 gene expression in cells on the luminal surface of the vascular wall is likely to contribute to endotoxin-induced microvascular thrombosis in involved tissues. This concept is strongly supported by the observation that in mice with targeted disruption of the PAI-1 gene (PAI-1−/− mice) fibrin deposition in kidneys and adrenals could not be detected following treatment with endotoxin.140 Moreover, the endotoxin-induced up-regulation of PAI-1 synthesis in ECs of multiple tissues clearly suggests that plasma PAI-1 may originate from these cells during endotoxemia. In this respect, a possible contribution of platelets seems unlikely since, in a rat model of endotoxemia, neither induced thrombocytopenia nor antiplatelet or anticoagulant agents affected the increase in plasma PAI-1.275

Endotoxin administration also caused changes in t-PA and u-PA expression in rats and mice. In both animal species, an increase in t-PA mRNA was observed in different tissues,140,266,273 although the precise cellular localization was not reported. However, this increase did not appear to be associated with increased t-PA activity, in view of the very large amounts of PAI-1 produced in the same tissues. u-PA mRNA behaved differently in rats and mice, being increased in rat kidney266 but markedly decreased in murine tissues, particularly in adrenals and kidneys (tubular epithelial cells).273 Interestingly, this decrease in u-PA expression was shown to be of pathogenetic importance in fibrin deposition occurring in kidneys and adrenals of endotoxin-treated mice.140 It is worth mentioning that, in rabbits, a marked reduction in fibrinolytic activity was observed in glomeruli harvested after a continuous infusion of endotoxin or TNF and that this decrease was mainly own to a reduced production of PAs.276Altogether these data indicate that suppression of the fibrinolytic system through increased PAI-1, mostly mediated by ECs, and other tissue- and species-specific alterations, such as decreased u-PA in the mouse model, are essential for fibrin deposition in tissue-specific vasculature, at least in experimental models of sepsis. That local changes in fibrinolysis may indeed contribute to fibrin deposition in a target organ is also illustrated by other experiments. Administration of low-dose endotoxin to chimpanzees caused, simultaneously with blood clotting activation, a marked depression of fibrinolytic activity (mediated by u-PA, not t-PA) in the bronchoalveolar fluid, due to increased levels of PAIs, especially PAI-2.277 In this case, however, macrophages, rather than ECs, were probably responsible for down-regulation of fibrinolysis. Similarly, in a mouse model of hypoxia, a clear increase in PAI-1 mRNA in the lung tissue was observed, which was associated with a decrease in both t-PA and u-PA mRNAs.278,279 These local fibrinolytic changes were shown to play an important role in hypoxia-induced vascular fibrin deposition in the lung. Again, immunolocalization studies identified macrophages, rather than ECs, as the predominant source of increased PAI-1 within the hypoxic lung. Therefore, the cellular origin of fibrinolytic components may be quite different, depending on the tissue, on the animal species, and on the pathological condition.

In human DIC-associated diseases, studies of endothelial fibrinolysis-related properties at tissue levels are lacking, with the notable exception of malignancy (see below). It is worth mentioning that, in patients with adult respiratory distress syndrome (ARDS) secondary to sepsis, the fibrinolytic activity of bronchoalveolar fluid (mediated by u-PA) was markedly lower than that of patients with interstitial lung disease or normal subjects, due to the appearance of PAI activity.185,280 However, in situ hybridization studies of acute-phase ARDS lung biopsies showed that, as in animal models of lung injury (see above), macrophages, rather than ECs, were the main source of PAI-1.

The majority of studies in DIC patients has been restricted to analysis of circulating fibrinolytic markers. In patients with sepsis or other conditions associated with activation of intravascular coagulation, such as trauma and thrombotic microangiopathy, a sustained increase in plasma PAI-1 has been consistently reported by numerous investigators62,199–206 and, in some studies, PAI-1 has been proposed as a prognostic marker in patients with septic shock.62,203–206 Plasma t-PA antigen was also found to be elevated in these patients.175,281–283 This increase, however, might not reflect exclusively an enhanced release since it might partly be due to a reduced rate of clearance of t-PA in complex with PAI-1.284 Whatever the mechanism leading to elevated plasma t-PA, the net effect of the changes in t-PA and PAI-1 in conditions associated with DIC is definitely antifibrinolytic. Assuming that, at least under pathological states, circulating PAI-1 is mostly of vascular origin, as clearly suggested by animal studies, its increase could represent an additional marker of endothelial stimulation in vivo. That impaired fibrinolysis, mainly mediated by PAI-1 increase, is an important factor in the pathophysiology of sepsis is supported by the finding that a 4G/5G polymorphism in the PAI-1 promoter leading to high PAI-1 expression is associated with poor outcome of meningococcal septic shock.285,286

There is also some evidence that plasma levels of u-PA are increased in sepsis but this activator seems to originate from leukocytes rather than ECs.282

Thrombin Generation, Thrombomudulin Expression and TAFI Activation

Thrombin has profound influence on fibrinolysis. Besides its capacity to stimulate the synthesis and release of fibrinolytic factors in different cells, including ECs, thrombin activates FXIII and TAFI, giving rise to two enzymes that down-regulate fibrinolysis by modifying the fibrin structure. FXIIIa is a transamidase that makes the fibrin clot more resistant to lysis by catalyzing the cross-linking between fibrin monomers and between α2-plasmin inhibitor and fibrin.287 Activated TAFI (TAFIa) is a type B carboxypeptidase and dampens the fibrinolytic process through the cleavage of C-terminal lysines from partially degraded fibrin thereby reducing plasminogen binding and activation.288 Enhanced thrombin generation induced by activated ECs, therefore, is another mechanism by which these cells inhibit fibrin removal by the fibrinolytic system. As regards TAFI activation, the contribution of ECs goes beyond their influence on thrombin generation since TM has been shown to enhance thrombin-induced TAFI activation by more than 1000-fold.289 TM is normally expressed by most quiescent ECs and since its discovery it has been considered one of the main antithrombotic factors of the endothelium thanks to its ability to change the substrate specificity of thrombin whereby the enzyme loses its procoagulant properties and become a potent activator of PC.12 This view, now, needs to be revisited since TM may serve both as antithrombotic, by dampening thrombin generation via PC, and as prothrombotic by retarding the removal of fibrin via TAFI (Fig. 9). Remarkably, TM increases the catalytic efficiency of thrombin-induced PC activation to an extent similar to that of thrombin-induced TAFI activation.288 Moreover, based on the kinetics of the reactions, PC and TAFI activation may occur simultaneously on the endothelial surface, without any significant competition, since the plasma concentration of the two substrates is below their Km.12,290 A critical point, thus, is to understand which is the net effect of the simultaneous generation of the anticoagulant APC and of the antifibrinolytic TAFIa and whether changes in TM expression may influence the balance between PC and TAFI activation. Available experimental data clearly indicate that the reduction in TM activity, as in mice with a targeted point mutation156 or following injection of anti-TM antibodies,291 makes the animal more susceptible to a thrombogenic challenge. Accordingly, the administration of exogenous TM has definitely an antithrombotic effect.291–293 In agreement with these in vivo findings, Mosnier et al,294 using an in vitro model of plasma clots, showed that TM, either soluble or EC-bound, inhibit fibrinolysis at low concentrations (in the range of 0.5 nM) because of increased TAFI activation, whereas it stimulates fibrinolysis at high concentrations (> 5 nM), through the enhancement of PC activation. This concentration-dependent behavior is not a new theme in haemostasis. Thrombin has also a dual function, acting essentially as anticoagulant at low concentrations and as procoagulant at high concentrations.295 It is conceivable, therefore, that the reduced expression of TM, associated with endotoxemia and other inflammatory conditions, along with an increase in the procoagulant potential of ECs, will favour TAFI activation, thus providing an additional mechanism for the inhibition of the fibrinolytic process.

Figure 9. The opposing roles of thrombomodulin (TM).

Figure 9

The opposing roles of thrombomodulin (TM). TM greatly enhances the capacity of thrombin to activate protein C (PC) and TAFI (thrombin-activatable fibrinolysis inhibitor). In this way TM may serve both as antithrombotic, by inhibiting fibrin formation (more...)

Endothelial Cell Perturbation in Malignancy

Coagulation-Related Properties

There is strong evidence that malignant disease is associated with a high incidence of macro or microvascular thrombotic manifestations and that activation of coagulation and fibrin deposition frequently occur locally at sites of tumor growth.296 Although the pathogenesis of tumor-related blood clotting activation is extremely complex and may vary depending on the tumor type and on the characteristics of the individual patient, it is generally recognized that TF plays a major role.71,297 Many studies on the in vivo expression of TF in malignancy have been focused on malignant cells themselves both in hematological diseases (mainly in acute leukemias, in which malignant cell TF appears to contribute significantly to hemostatic abnormalities) and in solid tumors.71,297 More recently, however, evidence has accumulated that TF can be expressed in vivo also by cells in tumor stroma.298,299 In particular, although the in vivo expression of TF in the endothelium has been difficult to demonstrate in disease states, several reports have shown that this may happen in cancer. In mice bearing a methyl-cholanthrene A-induced fibrosarcoma, TNF-α infusion resulted in TF expression and fibrin formation on the EC surface of the tumor capillary bed.300 The subsequent microthrombosis and reduced blood flow within the tumor likely contributed to the antineoplastic activity of TNF-α in this model. The observation that TNF-α induced clotting activation in the tumor capillaries even if tumor cells expressed virtually no TF indicates that the endothelium was an important target of TNF-α-mediated TF expression. Up-regulation of TF synthesis by ECs most probably resulted from the combined action of TNF-α and tumor-derived VEGF and was dependent, at least in part, on NF-κB activation.300,301 Interestingly, TNF-α infusion also caused a marked loss of TM immunoreactivity in the tumor vascular endothelium that was associated with an increase in circulating soluble TM.116 In man, with the use of immunohistochemistry and, in some studies, in situ hybridization or a functional in situ assay, TF was detected in vascular ECs of different tumor types, including Hodgkin's disease,302 Kaposi's sarcoma,303 invasive intraductal breast cancer (not benign breast tumors),304 lung carcinoma,305 pancreatic cancer,306 and gliomas (especially glioblastoma and anaplastic astrocytoma, not low-grade astrocytoma).307 TF production by ECs, probably elicited by local secretion of cytokines, appears to be instrumental in blood clotting activation since the distribution of intratumoral fibrin was virtually identical to that of TF.302,304,305 This suggests that EC TF might contribute to fibrin formation within the tumor and to the pathogenesis of the thrombotic complications in cancer patients.

During the last years evidence has been provided that TF expression in tumor tissue is linked with angiogenesis and the malignant phenotype.304–310 In view of the already mentioned resistance of vascular endothelium to induction of TF in vivo, the rather intense expression of EC TF in invasive malignant tumors, for instance breast cancer304 and gliomas,307 would suggest that the neoangiogenic vascular ECs in tumors differ from normal ECs. Thus, TF production by ECs, probably “activated” by tumor cell products such as VEGF, might represent a marker for the neoangiogenic response.

The role of EC TF in systemic activation of coagulation in malignancy remains to be addressed. As many tumor cells express TF, systemic hemostatic abnormalities could result from the access to the circulation of some cells (during the course of metastasis) or of tumor-derived microvesicles.296,297 However, the release into the circulation of mediators (either tumor-derived or manufactured by host cells as a result of their interaction with cancer cells) that are capable of activating distant ECs (and mononuclear phagocytes) for TF synthesis might also account, at least in part, for intravascular clotting activation in malignancy.296–299 Although high plasma and urinary levels of TF have been frequently observed in cancer patients,159,311–314 the source of this material and its clinical significance are as yet unknown.

Elevated plasma levels of TFPI315–317 and TM318–320 have been also reported in patients with different neoplastic diseases and, in some studies, they were found to correlate with the progression of malignancy,316,320 suggesting the involvement of ECs in this condition.

Fibrinolysis-Related Properties

During the last 20 years, there has been an explosion in research on the involvement of the fibrinolytic system in malignancy and its thromboembolic complications and it now appears firmly established that the u-PA-mediated pathway of plasminogen activation plays a central role in tumor growth and metastasis formation. In addition, a number of reports indicate that, in patients with different types of malignant disease, high levels of u-PA, PAI-1 and u-PAR in tumors are associated with a poor prognosis.321 Studies on the in vivo expression of the fibrinolytic components in tumors by means of immunohistochemistry and in situ hybridization revealed a very complex picture, with u-PA, u-PAR and PAI-1 being present not only in cancer cells but also in host cells. Although there are discrepancies in the results obtained by different investigators, the expression pattern of fibrinolytic proteins by different cell types in tumors appears to vary, depending on several factors, including the tumor type and stage, and the tissue involved.321

Few data are presently available on the localization of the fibrinolytic proteins within vessels in tumors. u-PA-expressing ECs were found in colon adenocarcinoma,322 breast carcinoma323 and astrocytoma,324 and u-PAR-expressing ECs in breast carcinoma325,326 and glioma.327 PAI-1 was expressed by ECs in breast carcinoma,323,328–330 colon adenocarcinoma,331,332 astrocytoma,333neuroblastoma334 and high-grade gliomas.335 Interestingly, in neuroblastoma specimens, PAI-1 was expressed almost exclusively by ECs and was correlated with metastasis and tumor recurrence.334 Although the relative importance of the expression of fibrinolytic components in cancer cells, ECs and other stroma cells (fibroblasts, macrophages) remains to be definitively established, the presence of the proteins in ECs would suggest a role in angiogenesis.

Increased plasma levels of t-PA, u-PA and, more frequently, PAI-1 have been reported in several (but not all) studies.336–339 However, the relationship between these changes and those at tumor level as well as their precise clinical significance are still a matter of debate. Increased PAI-1 and subsequent inhibition of fibrinolysis might contribute to fibrin deposition and to thrombotic complications, including DIC, in cancer patients.

Concluding Remarks

During the last years, quite a lot of studies have attempted to establish the role of EC coagulation- and fibrinolysis-related properties in DIC associated with different pathological conditions. There are, however, some limitations in these studies. An important one is that most of them have been performed in vitro with cultured ECs, i.e., under conditions far from the in vivo situation. Moreover, in vivo studies have been carried out mainly in animals and thus their relevance to human diseases is not easy to establish. Finally, the majority of clinical studies rely on measurements of circulating levels of activation markers that do not necessarily reflect EC changes. These caveats notwithstanding, a significant role of endothelium in mediating DIC-associated microvascular thrombosis can be postulated on the basis of available information. In sepsis, the condition most extensively investigated so far, inappropriate expression of TF and anticoagulant proteins by ECs has been documented, at least in certain experimental settings. However, one should consider that even subtle changes (not detectable by available techniques) might be pathogenetically relevant, in view of the very high endothelial surface/blood volume ratio in the microcirculation. Moreover, the dynamic regulation of EC properties and their heterogeneous expression in different vascular beds are other factors that must be considered in interpreting the “negative” results of studies on selected human vascular specimens. In contrast with coagulation-related properties, in vivo changes in the fibrinolytic potential of endothelium, particularly the in vivo expression of PAI-1, and the role thereof in sepsis-associated DIC appear much better documented. In malignancy, the in vivo expression of both TF and fibrinolytic proteins has been clearly demonstrated. Although EC-driven coagulation-fibrinolysis pathways may contribute to intratumoral fibrin turnover and to enhanced thrombogenesis that complicates cancer, recently it has become apparent that some of the proteins in these pathways, particularly TF, fulfill other roles in the regulation of tumor growth, among which angiogenesis.

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