Cardiovascular Research 60 (2003) 40–48
www.elsevier.com / locate / cardiores
Review
Infections and endothelial cells
Tymen T. Keller a , *, Albert T.A. Mairuhu d , Martijn D. de Kruif b , Saskia K. Klein d , Victor
E.A. Gerdes c , Hugo ten Cate e , Dees P.M. Brandjes d , Marcel Levi c , Eric C.M. van Gorp b,d
a
Department of Vascular Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Laboratory of Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The
Netherlands
c
Department of Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
d
Department of Internal Medicine, Slotervaart Hospital, Louwesweg 6, 1066 EC Amsterdam, The Netherlands
e
Department of Internal Medicine, Academic Hospital Maastricht, P. Debyelaan 25, 6229 HX Maastricht, The Netherlands
b
Abstract
Systemic infection by various pathogens interacts with the endothelium and may result in altered coagulation, vasculitis and
atherosclerosis. Endothelium plays a role in the initiation and regulation of both coagulation and fibrinolysis. Exposure of endothelial cells
may lead to rapid activation of coagulation via tissue factor (TF) expression and the loss of anticoagulant properties by impairment of
antithrombin III, TF pathway inhibitor (TFPI) and the protein C system. Endothelial-derived plasminogen activator inhibitor (PAI) is
essential for the regulation of fibrinolysis and impaired endothelial function leads to imbalance in fibrinolysis, resulting in a procoagulant
state. The interaction between inflammation and coagulation, soluble adhesion molecules and circulation endothelial cells is important in
the pathogenesis of an unbalanced haemostatic system. Rather than being a unidirectional relationship, the interaction between
inflammation and coagulation appears to be significant. In the crosstalk, the endothelium is playing a pivotal role.
 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Endothelium; Infection; Coagulation; Atherosclerosis; Protein C
1. Introduction
The vascular endothelium exerts a number of important
functions in order to maintain adequate blood supply to
vital organs. These functions include prevention of coagulation, regulation of vascular tonus, orchestration of the
migration of blood cells by the expression of adhesion
molecules, regulation of vasopermeability and production
of chemoattractant compounds. Injury or activation of
endothelial cells in response to inflammation could grossly
alter these functions. During severe inflammation leading
to shock, the role of endothelial cells in the process of
increased vascular leakage and thrombopathy seems to be
crucial [1]. The endothelial cell surface changes from an
*Corresponding author. Tel.: 131-20-56-62-377; fax: 131-20-69-68833.
E-mail address: t.t.keller@amc.uva.nl (T.T. Keller).
anti- into a prothrombotic surface and the thromboresistance of the cardiovascular system is further decreased by
the impairment of endothelium-dependent relaxation [2].
Endothelial cells are able to express a wide variety of
cytokines and factors that alter the haemostatic balance in
a prothrombotic state. In addition to the indirect effects of
adhesion molecules, growth factor and cytokines, endothelial cells interfere directly with the initiation, regulation
and modulation of coagulation (Fig. 1).
Tissue factor (TF), as principal initiator of this inflammation-induced thrombin generation [3,4] is expressed by
endothelial cells [5], which in addition to the decreased
production of antithrombin and protein C, leads to a
prothrombotic state [6]. Furthermore, during sepsis the
activated coagulation system and formation of fibrin is not
counteracted by fibrinolysis, because of an impaired endo-
Time for primary review 36 days.
0008-6363 / 03 / $ – see front matter  2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016 / S0008-6363(03)00354-7
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Received 2 December 2002; accepted 18 March 2003
T.T. Keller et al. / Cardiovascular Research 60 (2003) 40–48
41
thelial fibrinolytic system [7]. Thus an activated or defective endothelium influences all three major pathways of the
coagulation system: tissue factor mediated thrombin generation, dysfunction of anticoagulant pathways and blocked
fibrinolysis. In this review we will discuss current insights
into the major mechanisms involving the endothelium for
the initiation and regulation of the coagulation system in
acute and chronic infectious diseases.
2. Clinical aspects
2.1. Acute infections and altered coagulation system
During acute infections the endothelium may become
activated by pathogens or indirectly via inflammatory
mediators. Depending on the pathogen, there are several
mechanisms by which the endothelium is challenged [1].
Direct activation by bacteria, viruses and other pathogens
or indirect activation of the endothelium may result in
altered coagulation and fibrinolytic systems [8]. In sepsis
with gram negative bacteria, activation by endotoxins is
characteristic. Invasion of endothelial cells by a number of
viruses is common, for example (para-)influenza, adenoviruses, herpes simplex virus (HSV), polio virus, ech-
ovirus, measles virus, mumps virus, cytomegalovirus
(CMV), human T-cell leukaemia virus type-1 (HTLV-1)
and human immunodeficiency virus (HIV) [8,9]. Infection
of endothelial cells has been demonstrated for haemorrhagic fevers (HF) caused by Dengue, Marburg, Ebola,
Hantaan and Lassa HF as well [8]. HIV infection alters the
coagulation system by impairment of heparin co-factor II
[10]. Not endothelium-related procoagulant actions of HIV
are the prothrombinase complex assembly on the viral
surface [11] and a reduced protein S system [12]. Leptospirosis, especially Weil’s syndrome, may be presented with
haemoptysis, epistaxis, intestinal bleeding, adrenal bleeding, haematuria, and even subarachnoid haemorrhage [13].
The pathogenesis may be either primary activation of
coagulation or diffuse vasculitis, resulting in bleeding or
ischaemia of the vascularized tissue. The enhanced
coagulability of CMV may be mediated by the prothrombinase complex assembly on the CMV surface [14], but
this action is not endothelial dependent. This observation
suggests that the CMV surface contains the necessary
procoagulant phospholipids for assembly of the coagulation enzyme complex leading to thrombin generation.
Infectious agents have been linked with coronary artery
disease and myocardial infarction. Different authors have
suggested a link between Coxsackie B virus and coronary
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Fig. 1. Endothelial cells (EC) and infection. Under normal conditions endothelial cells contains anticoagulant agents, such as thrombomodulin, heparan
sulphate (HP) and plasminogen activator. Stimulation by pathogens and / or cytokines activates the endothelial cells. Tissue factor (TF) and von Willebrand
factor (vWF) are expressed. Subsequently coagulation factors are activated and fibrin is formed. Furthermore normal antithrombotic properties are lost:
antithrombin (AT), activated protein C (APC) and fibrinolysis. Activation of endothelial cells results in a procoagulant state.
42
T.T. Keller et al. / Cardiovascular Research 60 (2003) 40–48
heart disease [15,16]. In acute coronary syndromes the
levels of circulation inflammatory markers, such as CRP,
serum amyloid A and interleukin (IL)-6, are elevated
[17–19]. A recent investigation of a large number of
randomly selected patients demonstrated an increase in
enterovirus-specific antibodies at the time of diagnosis of
myocardial infarction, thus suggesting that this virus may
trigger acute coronary syndromes [20].
2.1.2. Vasculitis
Vasculitis, which may be triggered by infection, is
characterized by local or more generalized vascular
changes, resulting from infarction secondary to occlusion
by thrombi of the lumen of small blood vessels in the
upper part of the dermis. Vasculitis has been documented in
association with bacterial and viral infections [25]. Coronary arteritis was shown during experimental infections
with Coxsackie B4 virus in mice [26]. Different viruses
have been associated with graft arteriopathy [27]. Recent
research has demonstrated a link between viral infections,
particularly adenovirus, and coronary vasculopathy leading
to graft loss after heart transplantation [28]. HIV infection
is linked with polyarteritis nodosa, Henoch–Schonlein
purpura and leukocytoplastic vasculitis [29]. Polyarteritis
nodosa may occur also in hepatitis B and C [30,31] and
CMV infections [32]. Vasculitis-like syndromes, including
Kawasaki disease, polyarteritis nodosa, and Wegener’s
granulomatosis has been associated with Parvovirus B19
[33,34]. None of these syndromes, DIC or vasculitis, are
specific for a certain pathogen, and their occurrence
depends on factors such as virulence of the pathogen, the
patient’s prior condition, source and size of the inoculums,
and availability of antimicrobial treatment.
2.2. Atherosclerosis
The clinical consequences of chronic infections for the
development of thrombotic complications, atherosclerosis
3. (Patho)physiology of endothelial activation during
inflammation
Endothelial cells have a primary function in combating
blood clotting and thrombosis (Table 1). In pathological
conditions prothrombotic properties are of major importance. During inflammation, endothelial cells are involved
in all coagulation mechanisms leading to a procoagulant
state: TF mediated initiation of the coagulation cascade,
disturbed regulation mechanisms and impaired fibrinolysis.
3.1. Tissue factor pathway
TF expression on the surface of activated endothelial
cells, monocytes and macrophages can be induced in vitro
by a number of proinflammatory mediators and several
other compounds, including cytokines, C-reactive protein
and advanced glycosilated endproducts [5]. Some evidence
is emerging that endothelial cells also play an important
role in the generation of TF during severe infection. The
main route for activation of the coagulation cascade during
sepsis is the TF-factor VIIa pathway [41] (Fig. 1). In
primates, antibodies directed against TF or factor VIIa or
treatment with TF pathway inhibitor (TFPI) prevent activation of the common pathway [7]. Factor VIIa, in a complex
with TF catalyzes the conversion of factors IX and X [42].
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2.1.1. Disseminated intravascular coagulation
Endothelial injury by endotoxines [21] is considered to
be the primary event of disseminated intravascular coagulation (DIC), an acquired disorder in which the coagulation
system is activated. Furthermore, the release of endotoxines activates the extrinsic route of the coagulation system
[22]. Several clinical conditions, i.e. bacterial and severe
viral infections leading to DIC, may alter the coagulation
system by activation of TF-mediated pathways [23,24].
The infection results in activation of endothelium and
platelets and the conversion of fibrinogen to fibrin. Consumption of platelets, coagulation factors and activation of
the fibrinolytic system are hallmarks of the clinical
syndrome. Patients with DIC may present with manifest
thrombo-embolic disease or the clinically less apparent
microvascular thrombosis. Bleeding, thrombosis, or both
may dominate symptoms and signs.
and cardiovascular complications are highly significant.
Recent research has focussed on unravelling the mechanisms of chronic infection leading to atherosclerosis.
Pathogens, including viruses such as H. simplex (HSV),
hepatitis A (HAV) and cytomegalo virus (CMV) and
bacteria such as C. pneumoniae and H. pylori and peridontitis [35] can be linked with vascular pathology. The
process of the influx of blood leukocytes and plaque
formation has been described for several pathogens, i.e. C.
pneumoniae and H. pylori [36,37]. In chronic infections
activation of the endothelium may result in long-term
complications such as atherosclerosis and subsequent
cardiovascular disease [38]. Serologically, several infectious agents have been associated with cardiovascular
disease, including bacteria such as C. pneumoniae and H.
pylori and viruses such as CMV, Coxsackie B virus,
enteroviruses, HSV and Epstein–Barr virus. Furthermore,
there is some evidence that the presence of antibodies
directed against C. pneumoniae and CMV are associated
with an increased risk of future cardiovascular events [39].
Increased titres of antibodies against C. pneumoniae have
been used as a predictor of further adverse events in
patients who have had a myocardial infarction [40]. In fact,
a cumulative ‘pathogen burden’ seems to lead to
atherogenesis and cardiovascular disease. Nevertheless
epidemiological studies on the relationship between
chronic infections and atherosclerosis have yielded mixed
results and the pathogenesis has not yet been clarified.
T.T. Keller et al. / Cardiovascular Research 60 (2003) 40–48
43
Table 1
Endothelial antithrombotic properties
Action
Anticoagulant properties
Fibrinolysis
Antiplatelet aggregation
Antithrombin binding to heparan sulphate
Synthesis and release of protein S
Thrombomodulin expression
Tissue factor pathway inhibitor (TFPI)
Tissue-type plasminogen activator (tPA)
Urokinase plasminogen activator (uPA)
Induced synthesis of plasminogen activator inhibitor (PAI-1)
Prostacyclin synthesis
Synthesis of nitric oxide (NO)
Surface heparan sulphate
Yes
Yes
Yes
Yes
–
–
–
–
–
Yes
–
–
–
–
Yes
Yes
Yes
–
–
–
–
–
–
–
–
–
–
Yes
Yes
Yes
3.2. Regulatory properties of the endothelium
Under normal conditions endothelial cells exhibit major
antithrombotic properties. Several mechanisms are responsible for this homeostasis (Fig. 1). Antiplatelet function
seems to be intrinsic to the plasma membrane of endothelium. When platelets are activated, aggregation to intact
endothelium is inhibited by prostacyclin and nitric oxide.
Their synthesis in endothelial cells is stimulated by ADP,
thrombin, cytokines and other factors produced during
coagulation [46]. Thrombin generation is limited by antithrombin, the proteins C system and TF pathway inhibitor
(TFPI). The fibrinolytic system is another key element in
the regulation of fibrin deposition. Fibrinolysis is activated
by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) after their synthesis and release
from the endothelial cell system. These activators initiate
the conversion of plasminogen to plasmin, which hydrolyzes polymerized fibrin strands into soluble fibrin degradation products. Infection may lead to an imbalance
between platelet function and the regulatory mechanisms
of the coagulation cascade and fibrinolysis, resulting in
bleeding, thrombosis, or both.
3.2.1. Antithrombin
Antithrombin is the main inhibitor of thrombin and
factor Xa. During severe infection, antithrombin levels are
very low due to consumption, impaired synthesis primarily
as a result of endothelial dysfunction and degradation by
elastase from activated neutrophils [47,48]. A reduced
antithrombin function is further mediated by a decreased
availability of glycosaminoglycan at the perturbed endothelial surface. Under normal conditions, glycosaminoglycans act as physiological heparin-like co-factors that
catalyze antithrombin [49]. Decreased concentration of
glycosaminoglycans inactivates less thrombin, Xa and IXa.
This results in a procoagulant state.
3.2.2. Protein C
The involvement of endothelial dysfunction in the
impaired functioning of the protein C system is even more
apparent. Under physiological conditions, protein C is
activated by thrombin bound to thrombomodulin [50]. This
complex together with protein S cleaves factors Va and
VIIIa. So thrombomodulin and protein S generate a
negative feedback via protein C to the generation of
thrombin [51]. In cell cultures proinflammatory agents,
such as tumor necrosis factor (TNF)-a and IL-1, reduce
the concentration of thrombomodulin [52]. As a result,
they compromise thromboresistance to factors Va and
VIIIa. In sepsis, in addition to the already low levels of
protein C, the main cause of protein C system dysfunction
is this down-regulation of thrombomodulin [53]. Furthermore, endothelial cells, primarily from large blood vessels,
express an endothelial protein C receptor (EPCR) that
enhances the activation of protein C on the cell surface
[54]. During sepsis this EPCR is reduced, causing further
impairment of the protein C anticoagulation pathway [55].
3.2.3. Tissue factor pathway inhibitor
A third inhibitory mechanism of thrombin generation
involves tissue factor pathway inhibitor (TFPI), which is
synthesized and stored in endothelial cells. This molecule
inhibits TF-factor VIIa complex by forming a quaternary
complex in which factor Xa is the fourth component. The
relevance of TFPI in the coagulopathy of severe inflammation is demonstrated in several experimental studies
[56,57]. A recent study in healthy human volunteers
confirmed the potential of TFPI to block the procoagulant
pathway triggered by endotoxines [58]. However, clinical
studies in septic patients have not provided clues to its
clinical importance, because in the majority of the patients
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Factors IXa and Xa enhance the activation of factor X and
prothrombin, respectively. Although contact activation
does not seem to play an essential role in activation of the
coagulation cascade, blocking of the contact activation
system by the administration of monoclonal antibodies
against factor XIIa could prevent lethal hypotension [43].
This effect is most probably generated by kinins [43] and
the nitric oxide mediated vasodilatation [44]. Moreover,
the contact system seems to play an independent role in the
activation of the fibrinolytic system [45].
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T.T. Keller et al. / Cardiovascular Research 60 (2003) 40–48
the levels of TFPI are not diminished compared with
normal subjects [59].
3.3. Interaction between coagulation and inflammation:
the crosstalk
The impairment of the endothelial anticoagulant mechanism by proteases that affect the inflammatory response
follows similar pathways to those of coagulation proteases.
Several endothelial derived mediators of blood coagulation
have a major impact on the inflammatory host response
[65]. Factor Xa, thrombin and factor VIIa–TF complex
have each been shown to elicit pro-inflammatory activities
[66,67]. Thrombin stimulated endothelial cells express IL6, IL-8 and monocyte chemotactic protein-1 (MCP-1) [68].
Exposure of cultured endothelial cells to factor Xa stimulated the production of MCP-1, IL-6 and IL-8, and
increased expression of adhesion molecules that mediated
adhesion of leukocytes [69]. Furthermore, the coagulation
system of endothelial cells is an important feedback
activator of systemic infection. The in vivo anti-inflammatory properties of activated protein C and antithrombin are
well known [67,70,71].
3.3.1. Cytokines
During sepsis, endothelial cells are the target organs for
bacteria, and viruses and cytokines. On the endothelial
surface, these cytokines regulate the expression of several
proteins involved in coagulation and fibrinolysis [62]. The
derangement of coagulation and fibrinolysis is mediated by
IL-6 and IL-8 [72]. Cytokines such as, TNF-a induces the
expression of thrombomodulin on endothelial cells [73]
and influences indirectly the activation of coagulation
because of its effects on IL-6. Furthermore IL-6 is the
pivotal mediator of the deregulation of endothelial anti-
3.4. Soluble adhesion molecules
In the endothelial process of transformation into a
procoagulant state, surface adhesion molecules are of
major importance [79]. Endothelial cells express several
adhesion molecules on their surface, including E-selectin
(ELAM-1), intercellular adhesion molecule-1 (ICAM-1),
the vascular cell adhesion molecule-1 (VCAM-1) and
P-selectin, presumably in response to stimulation with
cytokines or thrombin [80]. Soluble ICAM-1, VCAM-1
and platelet endothelial cell adhesion molecules-1
(PECAM-1) play an essential role in the binding of
leukocytes [81], since P-selectin [82] and E-selectin are
expressed by endothelial cells [83] and function as
leukocytes-binding elements (Fig. 2). The subsequent
influx of leukocytes results in a local inflammatory response, endothelial damage, and plasma leakage and shock
[8]. Blockage of these adhesion molecules with antibodies
improves sepsis-associated organ dysfunction [84].
During shock and cardiovascular disease elevated levels
of adhesion molecules could be a sign of endothelial
damage. In patients with septic shock elevated levels of
adhesion molecules are found [85]. In peripheral artery
disease and coronary artery disease, the levels of ICAM-1
are elevated. These levels could be predictors of thrombotic disorders [86] and ischemic heart disease [87], but
results are still inconclusive. The levels of sVCAM-1 and
sPECAM-1 do not predict an adverse outcome in these
diseases [88]. Still the finding of increased plasma concentrations of these endothelial surface adhesion molecules
is thought to reflect the level of activation and perhaps
damage of the endothelial cell [89,90]. In the clinical
setting, measurement of E-selectin may be of diagnostic or
prognostic value for diseases associated with endothelial
pathology, since it is expressed only by vascular endothelial cells and not by any other cell types [91].
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3.2.4. Fibrinolysis
Endothelial cells are involved in the fibrinolytic process
by release of urokinase plasminogen activator (uPA), tissue
plasminogen activator (tPA) and its modulator tissue
plasminogen activator inhibitor 1 (PAI-1). Fibrinolysis is
activated during severe infections, demonstrated in an
experimental setting [7], mainly by activation of endothelial cells by cytokines [60] and in response to fibrin
formation as well. Fibrinolysis, triggered by coagulation
activation in severe inflammation [61], is shut off rapidly
by the release of relatively large amounts of PAI-1. After
infection of TNF-a and lipopolysaccharide (LPS) in
healthy volunteers, activation of the coagulation cascade is
preceded by a transient activation of fibrinolysis, which is
reflected by increased levels of circulating tPA and urokinase plasminogen activator followed by an increase in PAI-I
[62–64]. The elevated levels of PAI-1 depress the fibrinolytic system, resulting in a procoagulant state [7].
coagulant pathways and the fibrinolytic defect [72]. In vivo
studies have shown that treatment of primates with anti
IL-6 after the administration of low-dose endotoxin prevents activation of the coagulation cascade but does not
affect fibrinolysis [74]. The administration of anti IL-1
results in inhibition of both the coagulation cascade and
fibrinolysis in baboons with lethal bacteraemia and patients
with the sepsis syndrome [75,76]. Anti-inflammatory cytokines, such as IL-10, may modulate the activation of
coagulation: administration of recombinant IL-10 to
humans completely eliminated endotoxin-induced effects
on coagulation [77]. Taken together, a number of coagulation proteases are able to induce pro-inflammatory mediators of endothelial cells that again have procoagulant
effects. In particular vascular endothelial cells seem to play
a pivotal mediatory role in the coagulation response to
systemic inflammation and the interaction between coagulation and inflammation [78].
T.T. Keller et al. / Cardiovascular Research 60 (2003) 40–48
45
3.5. Circulating endothelial cells
4. Conclusion
Activated endothelial cells, thrombocytes and monocytes release small particles from their outer membrane
[92–94]. These so-called microparticles (MP) expose
phospholipids, thereby providing binding sites for activated coagulating factors [95]. In vitro experiments have
shown that after activation with LPS, endothelial cells
have fewer intercellular adherent junctions [96] and that
endothelial-derived MP may have procoagulant activity
[97]. It has been demonstrated in vivo, that endotoxin
increases the number of circulating endothelial cells by
injury of the endothelial layer and detachment of endothelial cells from the basal membrane [98].
Only in patient with severe sickle cell disease, MP
derived from endothelial cells are a source of TF [99].
Further evidence of the role of endothelial MP cells during
inflammation is still not present. Thrombocytes and monocytes derived MP with a procoagulant activity are found in
the blood during severe meningococcal sepsis [100]. And
recently it was shown that in the circulation of patients
with systemic lupus erythematosus (SLE) elevated levels
of MP are found [101]. Measuring MP by immunofluorescence or immunomagnetic separation may provide additional information about activity of vascular endothelium
[99].
Endothelium plays a key role in the pathogenesis of
coagulation disorders in infectious diseases, although the
precise mechanisms are not yet always clear. The endothelium is involved in both bacterial and non bacterial
infections and is important for the initiation and regulation
of haemostasis. Infection and / or activation of endothelial
cells lead to acute complications such as thrombosis,
haemorrhage and DIC, when chronic it may result in
atherosclerosis or vasculitis. Although direct interactions
between the infectious agent and the endothelial cells
occur, cytokines—also endothelial-derived—are believed
to be important mediators in this process.
During systemic gram-negative and gram-positive bacterial infections, activation of coagulation is mediated via
the extrinsic TF pathway. Experimental studies suggest
that, as a rule, coagulation and fibrinolysis occur independently of one another, and the overall result is
usually a procoagulant state. The latter may result in DIC
with microvascular thrombosis and organ failure. Bleeding
in infectious diseases is probably a multifactor process
resulting from a combination of endothelial damage or
vascular leakage, thrombocytopenia, consumption of clotting factors and hyperfibrinolysis. In addition, immunological mediated vasculitis may contribute to bleeding in
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Fig. 2. Endothelial cells (EC) and leukocytes during infection. Endothelial cells become activated by pathogens and / or cytokines. Several adhesion
molecules (AM) are expressed and leukocytes start rolling and adhere to the activated endothelial cell. Local inflammation and subsequently endothelial
dysfunction leads to the loss of anticoagulant agents from the endothelial cell, such as heparan sulphate (HP), thrombomodulin (TM) and plasminogen
activator (PA) and the expression of tissue factor (TF) and von Willebrand factor (vWF).
46
T.T. Keller et al. / Cardiovascular Research 60 (2003) 40–48
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