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REVIEW ARTICLE
From the Department of Cell and Molecular Biology,
Section for Molecular Pathogenesis, Lund University, Lund,
Sweden.
Life-threatening complications from bacterial
infections are a major and growing clinical problem, aggravated by the
emergence and spread of antibiotic resistance in bacterial pathogens
and by an increase in the number of immunocompromised
patients.1-3 During bacterial infection, the host responds
to invading microbes with a number of different defense mechanisms.
Some bacteria or bacterial products, however, can modulate the host
response and cause serious complications from the disease. In severe
infections, such as sepsis and septic shock, the coagulation and
fibrinolytic systems are targets for such modulation. These cascades
are normally activated upon tissue injury and/or blood vessel damage.
During infection, inflammatory mediators of the parasite and/or host can manipulate the procoagulant/anticoagulant equilibrium and cause
severe bleeding disorders.4,5 Although such effects induced by pathogenic bacteria often lead to similar clinical pictures,
different microbes seem to interfere at different stages of the
coagulation and fibrinolytic systems. This review aims to provide an
overview of how pathogenic bacteria can manipulate the coagulation and
fibrinolytic cascades in infectious diseases.
The coagulation cascade can be divided into an extrinsic and an
intrinsic pathway (Figure 1). The
extrinsic pathway is triggered upon complex formation of exposed tissue
factor (TF) with coagulation factor VII (F VII). This is followed by an
activation of factor IX (F IX) and/or factor X (F X) by the TF-F VII
complex and subsequent conversion of prothrombin to thrombin, which
finally induces the formation of fibrin from fibrinogen.6
The intrinsic pathway is initiated after activation of the contact
system, a process involving F XI, factor XII (F XII), plasma
kallikrein (PK), and high molecular weight kininogen
(HK).7,8 In addition to its procoagulative functions,
activation of the contact system also leads to the release of
bradykinin, which is generated by cleavage of HK by PK. Kinins are
considered to be important pro-inflammatory mediators.9
At present, it is generally accepted that coagulation is driven mainly
by the extrinsic pathway.6 This notion is based on the
observation that patients with deficiencies in the contact factors F
XII, HK, and PK do not suffer from any bleeding
disorders.8 It therefore seems likely that these proteins
play only a secondary role in hemostasis. However, F XI deficiency
leads to minor bleeding abnormalities, a dysfunction explained by the
ability of thrombin to activate F XI by a mechanism that is independent
of the contact system.10 Thrombin-activated F XI triggers
an augmentation stage of coagulation and allows continuation of
clotting after down-regulation of TF activity by TF-pathway inhibitor
(TFPI).11,12 Work published to date indicates that
activation of F XI by thrombin also attenuates fibrinolysis inside a
fibrin clot.13,14
Coagulation is controlled at 3 levels. First, thrombin changes to an
anticoagulant factor upon binding to thrombomodulin (TM), a
glycoprotein bound to the endothelium.15 The formation of the thrombin/TM complex allows a rapid cleavage and activation of the
anticoagulant protein C. Activated protein C then down-regulates further formation of thrombin by proteolytically inactivating the
coagulation cofactors factor V (F V) and factor VIII (F VIII). Additionally, thrombin, in complex with TM, loses its ability to clot
fibrinogen. Second, in order to prevent an expansion of clotting in the
periphery of the damaged tissue, coagulation factors are inactivated by
circulating proteinase inhibitors such as TFPI, C1 inhibitor (C1 INH),
and antithrombin III (AT III).16-18 Finally, fibrinolysis
is hindered inside the clot by a thrombin-dependent activation of the
plasma protein thrombin-activatable fibrinolysis inhibitor (TAFI). TAFI
exhibits carboxypeptidase-B-like activity and suppresses fibrinolysis,
most likely by removing carboxy-terminal lysine residues. Since plasmin
binds to carboxy-terminal lysines of progressively degraded fibrin,
this mechanism might protect the clot from degradation.19
At the site of tissue injury, fibrinolysis is initiated by the
conversion of plasminogen to plasmin.20 Plasmin has
multiple functions, such as degradation of fibrin, inactivation of the cofactors F V and F VIII, and activation of metalloproteinases, which
play an important role in wound healing and tissue-remodeling processes.21-23 Plasminogen is activated by tissue
plasminogen activator (tPA) and urokinase-type plasminogen activator
(uPA),24 proteins released from endothelial cells in
response to thrombin and upon cell damage.25 Plasminogen
activity is regulated by plasmin inhibitors such as
Lately, the connection between hemostasis and inflammation has
attracted much interest.28 This link was established by
recent work showing that the coagulation factors thrombin, F X, and F VII trigger intracellular signal cascades by binding to specific cell-membrane-spanning receptors, which have no known role in hemostasis.29-32 The receptors for thrombin
(protease-activated receptors [PARs]) are the best characterized.
PARs belong to the family of G-protein-coupled receptors, which are
activated upon binding to a protease that cleaves the exodomain of the
receptor. This manipulation unmasks a tethered peptide ligand that then stimulates its own receptor.33 So far, 4 receptors that
are triggered by this mechanism have been identified. Thrombin
activates PAR-1, PAR-3, and PAR-4, whereas PAR-2 is activated by
trypsin, mast cell tryptase, and a cysteine proteinase from
Porphyromonas gingivalis.34-37 The receptors for
F X (effector cell protease receptor-1 [EPR-1]) and F VII (TF) do
not belong to the family of G-protein-coupled receptors and are
activated by different mechanisms.38,39 Additionally, a
receptor for protein C (endothelial cell protein C [EPCR]) has been
described.40 Whether this receptor triggers intracellular
signaling is currently under investigation.41-43 Patients
with sepsis have significantly increased levels of a soluble form of
EPCR in plasma, indicating that EPCR can be used as a marker protein in
infectious diseases.44 The binding of coagulation factors
to these receptors and their subsequent activation trigger inflammatory
reactions such as leukocyte recruitment, release of cytokines and
nitric oxide, and the production of reactive oxygen
species.30,39,45-47
As mentioned above, the coagulation cascade is initiated mainly by
binding of F VII to cell-surface-bound TF. The physiological importance of TF was demonstrated by experiments in transgenic mice,
which show that interruption of the gene for TF is associated with
lethal embryonic bleeding and impaired vascular
development.48,49 TF is expressed in many tissues,
including brain, lung, placenta, and kidney.50 Cells that
constitutively produce TF are usually not in contact with blood, but
are found in perivascular tissues and stroma.11 Peripheral
blood cells and endothelium do not normally produce TF. However, TF
activity in these cells is increased after stimulation with such
substances as endotoxin, tumor necrosis factor Recently, Gando et al55 reported that TF values from
sepsis patients are significantly higher than from trauma patients, indicating an important role for TF-triggered coagulation complications during septic conditions. The observation that TF activity is enhanced
during infection is in accordance with the finding that several
bacterial species, such as Mycobacterium leprae,
Neisseria meningitidis, Rickettsia conorii,
Rickettsia rickettsii, Staphylococcus aureus, and
Streptococcus sanguis, are able to trigger TF expression in
monocytes and endothelial cells.56-64 It seems likely,
therefore, that the onset of coagulation in sepsis results partially
from induction of TF expression in endothelial cells and monocytes by
bacterial products. The finding that Gram-positive microorganisms also
trigger TF activity indicates that bacterial products other than
endotoxin could be involved in the up-regulation of procoagulant activity. Gram-positive bacteria may induce this indirectly, as various
exotoxins and peptidoglycans have been shown to trigger the induction
of pro-inflammatory cytokines.6,65-69 Notably, cytokines
such as interferon Numerous studies provide accumulating evidence that the contact
system is activated during sepsis.71 It was demonstrated as early as 1970 that patients with hypotensive septicemia have significantly decreased levels of contact factors.72 More
recently, it has been reported that activation of the contact system
occurs in children with meningococcal septic shock and that low levels of F XII and HK in systemic inflammatory response syndrome (SIRS) patients correlate with a fatal outcome of the
disease.73-75 In one of the more recent studies,
it was also shown that levels of PK- In 1996, Ben Nasr et al79 reported that contact factors
bind to the surface of E coli and Salmonella spp
bacteria that express curli organelles or thin aggregative fimbriae.
Subsequent studies demonstrated that the contact system is activated as
a consequence of the assembly of contact factors on the bacterial
surface. This is followed by the release of bradykinin, a potent
inducer of fever, pain, and hypotension.80 Moreover, the
absorption of contact system proteins and fibrinogen by these surface
organelles led to a depletion of relevant coagulation factors and
caused a hypocoagulatory state in mice.80 Apart from
Gram-negative bacteria, cell-wall peptidoglycan products from
Streptococcus pyogenes have been shown to trigger the
contact system.81 In fact, most streptococcal serotypes
bind kininogens by their M proteins,82 which are
considered to be important virulence factors on the outer membrane of
the microorganism.83 Once HK is absorbed on the bacterial
surface, it is susceptible to proteolytic cleavage by PK, leading to
the formation of bradykinin.84
In addition to bacterial-surface molecules, secreted microbial products
have been demonstrated to activate the contact system. Early
observations identified bacterial endotoxin as a potent activator of F
XII.85 Recently, with the use of a low-grade endotoxemia
model in humans, it has become apparent that F XI is also activated by
a contact-system-independent mechanism.86 Apart from
endotoxin, many bacterial proteinases that are able to trigger the
release of kinins have been isolated (Table
1).87-93 In order to
generate bioactive kinins from HK, bacterial proteinases act on
kininogens directly92,93 or activate the contact factors F
XII and PK, which in turn cleave HK, causing the release of bradykinin.87,91 As kinins increase vascular permeability, the generation of these pro-inflammatory peptides in infectious foci
might be a bacterial strategy that facilitates penetration and
spreading of the pathogen into the tissue.
The systemic activation of coagulation during infection results in the cleavage of fibrinogen by thrombin. The fibrin monomers generated can then become deposited in various organs, leading to microvascular and macrovascular thrombosis. The concomitant stimulation and aggregation of platelets, either by thrombin or by bacterial substances, may also cause thrombocytopenia.94 As a result of widespread thrombus formation, tissue ischemia and organ failure may occur. At the same time, fibrinogen degradation products can trigger the release of monocyte/macrophage-derived IL-1, IL-6, and PAI-1. Whereas IL-1 and IL-6 induce additional vascular endothelial damage, PAI-1 inhibits fibrinolysis, which accelerates further thrombus formation.95 Apart from these functions, fibrinogen is an important factor that regulates cellular interactions in the vasculature, such as the binding of leukocytes to the endothelial cells.96 As shown for intra-abdominal infections, fibrinous exudates can incorporate large numbers of bacteria, viz E coli and Bacteroides fragilis. Once bacteria are sequestered within the fibrin deposit, they are protected from phagocytosis, which might be a microbial strategy for avoiding the host defense machinery. Inside the clot, the microorganism can continue to proliferate, leading to the formation of abscesses. However, the host can also benefit from the encapsulation of the microorganism, since the entrapment of bacteria in a fibrin deposit might act to diminish the magnitude of bacterial dissemination, thus preventing host mortality.97 Fibrinogen-binding proteins (FgBPs) have been studied mostly in
staphylococci and streptococci. To date, 5 different FgBPs have been
characterized in S aureus.98-102 Three of them
are secreted: (1) an 87-kd coagulase, which binds to
prothrombin and forms a complex that has thrombinlike activity and
converts fibrinogen to fibrin,103 (2) a 60-kd FgBP, which
binds to fibrinogen and prothrombin,98 and (3) a 16-kd
FgBP, which binds to the Most group A streptococcal serotypes (S pyogenes) express FgBPs on their surface.82 Apart from fibrinogen-mediated adherence of these bacteria to endothelial and epithelial cells, streptococcal FgBPs seem to have an important antiphagocytic function owing to their ability to impair deposition of complement.110-113
Plasmin(ogen) plays a central role in fibrinolysis as it dissolves insoluble fibrin matrices.114 In addition to its fibrinolytic properties, plasmin degrades extracellular matrix proteins such as laminin and fibronectin and activates metalloproteinases.23,115 Studies with plasminogen-deficient mice have shown an effect of the enzyme on cell migration and tissue remodeling.116 In addition to plasmin(ogen), plasminogen activators have been demonstrated to be crucial factors in fibrinolysis. Transgenic mice deficient in tPA and uPA showed impaired clot lysis and suffered extensive spontaneous fibrin deposition.117 Also, these mice developed venous thrombosis upon endotoxin administration to a higher extent than wild-type animals. In sepsis, progressive failure of multiple organs is often accompanied
by fibrin deposition and formation of microthrombi. Clinical studies
have provided evidence that plasminogen concentrations in septic
patients are significantly decreased.118 Moreover, plasminogen levels or the ratio of plasminogen to
As shown in Table 2, many bacterial
species have been demonstrated to interact with human
plasmin(ogen).122 Interestingly, the mode of interaction
varies for different species.123 For example, most group A
streptococci secrete the nonenzymatic plasminogen activator
streptokinase. Binding of streptokinase to plasminogen converts the
zymogen into an active enzyme by inducing a conformational change
without hydrolyzing a peptide bond, which is normally required to
activate plasminogen.124 Also, staphylococci secrete a
nonenzymatic plasminogen activator, staphylokinase. The mechanism of
plasminogen activation by staphylokinase differs from its activation by
streptokinase, however, in that small amounts of active plasmin are
required for an efficient activation.125 The crystal
structures of the catalytic domain of human plasmin in complex with
streptokinase or staphylokinase have been solved
recently.126,127 In contrast to nonenzymatic activators, a
surface-bound plasminogen-binding protein (Pla) that exhibits protease
activity has been found in Yersinia pestis.128
Bacterially bound plasmin is thought to trigger dissemination of
Y pestis from a peripheral site and/or to facilitate the
escape of the microorganism from fibrin-mediated
entrapment.129 In addition to microbial plasminogen
activators, many pathogenic bacteria express nonactivating
plasminogen-binding molecules on their surface (Table 2). Once
plasminogen is bound to the bacterial surface, the zymogen can be
activated by different mechanisms. Whereas streptococci and
staphylococci produce their own activators, Borrelia
burgdorferi, Salmonella enteritidis, and E
coli can activate bound plasminogen by recruited tPA or
uPA.130,131 Studies with B burgdorferi, S
aureus, and S pyogenes have demonstrated that plasmin,
mobilized to the bacterial surface, is protected from inhibition by
host proteinase inhibitors, in particular
Blood coagulation is normally limited to a site of vascular injury, indicating that clotting is regulated in a highly controlled manner. The mechanism requires, apart from procoagulative factors, a number of specific proteinase inhibitors or enzymes that down-regulate clotting activity.137 It is important to stress that the correct ratio of procoagulative enzymes and their inhibitors is necessary to ensure that clotting is restricted to a damaged site. Once this delicate balance is disturbed, an efficient wound-healing process is hindered either by the inability to form a fibrin network, by uncontrolled coagulation, or by impaired fibrinolysis. In patients with sepsis, severe sepsis, or septic shock, the levels of many natural inhibitors are markedly lowered, and this may result in an unfavorable outcome for the disease.138-140 In several animal models and clinical trials, therapeutic substitution of coagulation inhibitors has been demonstrated to improve survival in cases of severe infection. In what follows, some of these treatments are described.
Expression of TFPI is, under normal conditions, restricted to
megakaryocytes, to endothelium of the small capillaries, and to
macrophages. TFPI levels in plasma are increased under inflammatory conditions.50 However, despite the elevated levels of
plasma TFPI in sepsis patients, efficacy studies indicate that in
addition, administration of a 10-fold higher concentration of
recombinant TFPI might be needed to compensate for an uncontrolled
activation of the extrinsic pathway of
coagulation.141 Treatments with recombinantly expressed variants of TFPI showed improved survival rates in several rabbit and baboon sepsis models,142-145 whereas, in pigs,
TFPI treatment attenuated the response of the inflammatory mediators
TNF-
AT III is the main inhibitor of thrombin and F X, but other serine
proteinases, including F IX, F XI, F XII, PK, uPA, tPA, and plasmin,
are also inactivated by AT III.149 In septic shock, AT III
levels drop drastically in plasma, and low AT III levels are
predictive of a fatal outcome for the disease.139 Several mechanisms can cause low AT III levels, such as consumption of AT III,
degradation by elastase released from neutrophils, and extravascular
leakage due to increased vascular permeability.150 Experiments with baboons infected with a lethal dose of E
coli showed that treatment with AT III increased the formation of
thrombin-AT III complexes and diminished fibrinogen consumption as
compared with a nontreated control group. The analysis of the cytokine response to the AT III administration revealed that levels of IL-6,
IL-8, and IL-10 in plasma were significantly reduced in the AT
III-treated group, whereas TNF-
C1 INH, the major plasma inhibitor of activated complement
C1-esterase, PK, and F XII, is an acute-phase protein, and its levels
can increase up to 2-fold during uncomplicated sepsis.154 However, the ratio between functional and total levels of C1 INH is
lower in patients with sepsis than in healthy
volunteers.155 It has been assumed that degradation of the
active inhibitor, probably by neutrophil elastase, is the cause of this
phenomenon.154 Administration of C1 INH to baboons
suffering from lethal E coli sepsis results in a reduction
of circulating cytokine levels, such as TNF-
TM is a cofactor expressed on endothelial cell surfaces, which modifies the activity of thrombin, leading to an activation of protein C and subsequent initiation of the anticoagulative pathway. The expression of TM is down-regulated by endotoxin and cytokines such as IL-1.160 Several animal studies provide evidence that application of a recombinantly produced soluble variant of TM prevents fatal acute thromboembolism and diminishes glomerular fibrin deposition in endotoxin-induced disseminated intravascular coagulation (DIC).161 A double-blind study to evaluate the effect of recombinant soluble TM in DIC is in progress.161 Protein C and its cofactor protein S function as important regulatory factors of the blood coagulation cascade.162 Protein S is found in 2 forms, as free protein or in a noncovalent bimolecular complex with C4b-binding protein (C4BP).163 The latter protein is a regulatory protein in the complement system that down-regulates the classical pathway of complement activation. C4BP binds approximately 60% of the circulating protein S and inactivates its anticoagulant properties.162 During sepsis, protein C levels are lowered, and especially in meningococcal septic shock, severely reduced protein C levels contribute to mortality and morbidity.94,164,165 Levels of C4BP and protein S, however, are within normal range, indicating that it is mainly the deficiency of protein C that determines the severity of meningococcal septic shock.165 In animal models, the administration of protein C had favorable effects on the hemodynamic parameters of endotoxin-treated animals and led to an improved survival.166,167 Smith and White168 recently published a clinical study involving 30 patients with meningococcaemia and severe protein C deficiency. Treatment of the patients with protein C resulted in a significant improvement of the host response to meniningococcal infection. Protein C and activated protein C are currently being investigated in clinical trials for septic shock.169 The receptor for protein C (EPCR) seems to play an important role in regulating the activity of protein C in infectious diseases. As shown in baboons that were challenged with a sublethal dose of E coli, the administration of a monoclonal antibody that blocks protein-C binding to EPCR converts the response to sublethal concentrations of bacteria into a lethal challenge.169 Interestingly, C4BP has been shown to bind to most strains of the species S pyogenes and Bordetella pertussis.170,171 Functional studies indicate that the bacteria could interact with C4BP in order to protect themselves from a complement-mediated attack. However, any possible effect on the coagulation cascade was not studied.
Despite an improved intensive care system, morbidity and mortality associated with severe bacterial infections constitute an increasing problem. This dilemma is caused by, among other factors, the ability of bacteria to develop antibiotic resistance and by an increasing number of immunocompromised patients. In order to develop novel therapeutic strategies that can successfully fight severe diseases caused by bacteria, it is necessary to have a thorough understanding of the molecular mechanisms involved in their interactions with host effector systems. Much knowledge about sepsis and septic shock has been achieved by studying the effect of endotoxin in different in vitro and in vivo models. However, Gram-positive bacteria are increasing in prevalence in sepsis patients, and not all complications that occur in Gram-negative sepsis can be explained by the effect of endotoxin.172-175 An increasing knowledge of the role of microbial products in hemostasis may lead to novel treatments in life-threatening infectious diseases.
Submitted March 27, 2000; accepted May 22, 2000.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Heiko Herwald, Department of Cell and Molecular Biology, Section for Molecular Pathogenesis, Lund University, PO Box 94, S-221 00 Lund, Sweden; e-mail: heiko.herwald{at}medkem.lu.se.
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Top
Introduction
Current view on hemostasis
2-antiplasmin and 
B and activator protein 1, whereas TF expression
induced by serum, phorbol esters, and VEGF is controlled by a promoter
region containing overlapping Sp1/endothelial growth factor 1 sites.
, interleukin 1
(IL-1