Current Cardiology Reviews, 2010, 6, 325-332
1573-403X/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.
Tissue Factor/Factor FVII Complex Inhibitors in Cardiovascular Disease.
Are Things Going Well?
Gianluca Petrillo, Plinio Cirillo*, Greta-Luana D’Ascoli, Fabio Maresca, Francesca Ziviello and
Department of Internal Medicine, Cardiovascular and Immunological Sciences (Division of Cardiology) University of
Naples "Federico II", Italy
Abstract: Blood coagulation is a complex biological mechanism aimed to avoid bleeding in which a highly regulated and
coordinated interplay of specific proteins and cellular components respond quickly to a vascular injury. However, when
this mechanisms occurs in the coronary circulation, it has not a “protective” effect, but rather, it plays a pivotal role in de-
termining acute coronary syndromes. Coagulation recognizes Tissue Factor (TF), the main physiological initiator of the
extrinsic coagulation pathway, as its starter.
Since TF:VIIa complex is the critical point of the blood coagulation cascade, it is a pharmacological attractive issue for
the development of agents with anti thrombotic properties that can exert their activity by inhibiting complex formation
and/or its catalytic activity. In fact, it is intuitive that an antithrombotic agent able to inhibit this initial step of the coagula-
tion pathway has several theoretical, extremely important, advantages if compared with drugs active downstream the co-
agulation pathway, such as FXa or thrombin. The present report gives a brief overview of TF pathophysiology, highlight-
ing the most recent advances in the field of inhibitors of the complex TF/VIIa potentially useful in cardiovascular disease.
Keywords: Blood coagulation, cardiovascular disease, factor VIIa, tissue factor.
aimed to avoid bleeding in which a highly regulated and
coordinated interplay of specific proteins and cellular
components respond quickly to a vascular injury. How-
ever, when this mechanism occurs in the coronary circula-
tion, it has not a “protective” effect, but rather, it plays a
pivotal role in determining acute coronary syndromes .
Coagulation recognizes Tissue Factor (TF), the main
physiological initiator of the extrinsic coagulation path-
way, as its starter. Indeed, several experimental and clini-
cal studies indicate that TF plays a pivotal role in the
pathophysiology of acute coronary syndromes: it triggers
the formation of intracoronary thrombi following endothe-
lial injury [2-5]. TF is an integral transmembrane protein
expressed on the surface of several cell types located in
subendothelial structures throughout the vasculature, and it
is normally not in contact with circulating blood, where
other coagulation factors are present in their inactivated
forms. In this respect, cells normally not exposed to the
flowing blood, such as smooth muscle cells, constitutively
express TF on their surface [6, 7], while cells exposed to
the blood stream, such as endothelial cells, express TF on
their membrane only when activated after exposure to spe-
cific stimuli, such as LPS, certain cytokines , and oxy-
gen free radicals .
Blood coagulation is a complex biological mechanism
*Address correspondence to this author at the Department of Internal
Medicine, Cardiovascular and Immunological Sciences (Division of Car-
diology), University of Naples “Federico II”, Via Sergio Pansini 5, 80131
Naples, Italy; Tel: +39-081-7462216; Fax: +39-081-7462223;
point of the blood coagulation cascade, it is an attractive tool
for the development of agents with anti thrombotic properties,
which could inhibit complex formation and/or its catalytic ac-
tivity. Indeed, it is intuitive that an antithrombotic agent able
to inhibit this initial phase of the coagulation pathway has sev-
eral theoretical, extremely important, advantages if compared
with drugs active downstream the coagulation pathway, such
as FXa or thrombin, since this pathway is specifically blocked
right from the beginning.
Since TF/VIIa complex formation represents the critical
physiology, highlighting the recent advances in the field of in-
hibitors of the TF/VIIa complex useful in cardiovascular dis-
The present report gives a brief overview of TF patho-
Tissue Factor Physiology and Extrinsic Coagulation
CD142, is a glycosylated transmembrane protein consisting of
a single polypeptide chain (MW: 45,000) . This glycopro-
tein is a type I integral membrane protein, and is a member of
the class 2 cytokine receptor superfamily . The extracellu-
lar part of TF is made up of two fibronectin type III domains,
and membrane anchoring of TF has been demonstrated to be
essential to support full proteolytic activity by FVIIa  (Fig.
1). It is intuitive that TF is normally not exposed to circulating
blood to avoid its improper interaction with other circulating
coagulation factors. Vice versa, vascular injury, through
physical damage of the endothelial layer of the blood vessel,
causes the exposure of TF to circulating blood, making it ac-
cessible to circulating factor VII (FVII) [13-16]. This coagula-
Tissue Factor (TF), also known as thromboplastin or
326 Current Cardiology Reviews, 2010, Vol. 6, No. 4 Petrillo et al.
Fig. (1). Schematic representation of Tissue Factor. This glyco-
protein is a type I integral membrane protein. The extracellular
part of TF binds FVII with very high affinity and specificity.
Once bound to TF, FVII is rapidly converted to its activated form
(FVIIa) via limited proteolysis. Membrane anchoring of TF has
been demonstrated to be essential to support full proteolytic activ-
ity by FVIIa.
tion factor binds to TF with very high affinity and specific-
ity . Once bound to TF, FVII is rapidly converted to its
activated form (FVIIa) via limited proteolysis [18-19] (Fig.
2). Activated FVII binds Factor X (FX), that, in turn, is
converted in its activated form (FXa). Then, the extrinsic
pathway continues, leading to thrombin activation and clot
formation . Many coagulation proteases such as factors
IXa, Xa, XIIa, thrombin and plasmin [18-24] are able to
amplify this activation process since they cause the direct
activation of FVII to FVIIa. More important, the TF:VIIa
complex can itself catalyze the activation of FVII bound to
TF, via an auto activation reaction [25-26], in which is in-
volved FXa too. Although the FVII in plasma circulates as
a zymogen, it has been demonstrated that normal indivi-
duals might have low levels of activated factor VII (FVIIa)
in their plasma (about 1% or less of the total factor VII)
with an unknown role .
FVIIa is an extremely weak serine protease on its own,
but its enzymatic activity is enhanced dramatically when it
binds to TF . Specifically, TF/FVIIa binding signifi-
cantly increases FVIIa ability to catalyze the hydrolysis of
small peptidyl amide and ester substrates from 20- to 100-
fold, and this phenomenon is closely dependent upon the
substrate [28, 29]. Substrate hydrolysis by serine proteases
is known to be a multi-step process, and any of the steps
along the reaction pathway might be affected in the allos-
teric activation of FVIIa by TF. Because TF is an integral
membrane protein, the TF/VIIa complex is always tethered to
the membrane surface. This has two important consequences:
first, the coagulation cascade is activated only where it is
needed, i.e. at sites of vascular injury; second, binding of
FVIIa to TF activates a number of intracellular signals that
culminate in cell proliferation and new gene expression, in-
cluding inflammatory genes [30-32].
cells that express TF on their surface is significantly lower if
compared with the activity measurable in the same cells when
damaged, lysed, or treated with calcium ionophore . In-
deed, although TF is present on the surface of such cells, it be-
comes fully active only when the membrane properties of the
cell are altered [34, 35]. In particular, it has been described a
phenomenon called “TF encryption”. It is known that the dis-
tribution of aminophospholipids (such as the negatively
charged phosphatidylserine) is restricted to the inner leaflet of
the plasma membrane of the cells. Negatively charged phos-
pholipids are required for substrate molecules such as factors
IX or X to bind to the membrane, so their sequestration limits
the activity of TF on cell surface. When cells are lysed, dam-
aged or treated with calcium ionophore, this phospholipid
asymmetry is lost. Moreover, in some cell types, TF may asso-
ciate with caveolae, which are areas of the cell surface with
altered lipid composition. Again, it has been proposed that di-
merization or oligomerization of TF in the membrane may re-
duce its activity, and that damage or lysis of cells may promote
the formation of active TF monomers.
Pathophysiology of coagulation is tightly regulated by an-
other important protein known as Tissue Factor Pathway In-
hibitor (TFPI), that is the endogenous inhibitor of the extrinsic
coagulation pathway; specifically, TFPI is a potent inhibitor of
the TF/FVIIa complex, and its action is related to the presence
of FXa  (Fig. 2). TFPI is composed of three Kunitz-type
protease inhibitor domains: the first Kunitz domain reacts with
the active site of FVIIa in the TF:VIIa complex , while the
second Kunitz domain reacts with the active site of FXa. Once
the TFPI:Xa complex forms, it binds with higher affinity to
TF:VIIa than does the TFPI molecule alone; this results in the
formation of a fully inhibited tetramolecular complex
TF:VlIa:TFPI:Xa [37, 38]. Much of the circulating TFPI is
bound to lipoproteins [39-40]; this form represents about 50%
to 60% of the total circulating TFPI, whereas carrier-free TFPI
represents about 20% of the total. A third pool of TFPI is con-
fined to platelets, which carry approximately 10% of the total
TFPI . The in vivo infusion of heparin increases the circu-
lating levels of TFPI in plasma 2- to 4-fold [42, 43]. The
source of this additional TFPI is thought to be the endothe-
lium, at the surface of which TFPI is bound. The TFPI released
by heparin in vivo represents the carrier-free molecule, which
might be biologically most active . TFPI also promotes the
internalization and degradation of TF:VIIa complexes on the
surface of monocytes , thus subtracting these complexes to
It has been described that procoagulant activity of intact
TF/FVIIa in Cardiovascular Disease
demonstrated that the complex TF/FVIIa is the key initiator of
the coagulation cascade in cardiovascular disease .
Several experimental and clinical studies have clearly
TF/FVII Inhibitors and Cardiovascular Disease Current Cardiology Reviews, 2010, Vol. 6, No. 4 327
relationship existing among several chemical mediators
and the pathophysiology of coronary artery disease as well
as of its main complication represented by acute coronary
syndromes. In fact, it has been demonstrated that these
mediators might exert their effects by inducing TF expres-
sion. Specifically, inflammatory markers such as C-
Reactive Protein or neopterin as well as smoke-derivative
substances might play an important role in acute coronary
syndromes since it has been demonstrated that they are ac-
tive partaker in triggering coronary TF-mediated coagula-
tion [15, 45, 46]. In addition, molecules involved in patho-
physiology of other cardiovascular co-morbidity such as
urotensin II or angiotensin have been associated with TF
expression to explain their mechanism of action [13, 47].
TF expression represents the link explaining the
ated upon post-ischemic reperfusion, induce TF-mRNA
transcription and expression of TF procoagulant activity.
As seen in in ex vivo and in vivo hearts subjected to ische-
mia and reperfusion, a condition associated with a produc-
tion of oxygen free radicals in large amounts, a marked in-
crease in TF activity occurred. This increase was accom-
panied by a significant impairment of coronary flow during
reperfusion and possibly contribute to the occurrence of
reperfusion injury .
Moreover, oxygen free radicals, endogenously gener-
TF can be detected in several cell types, such as mono-
cytes, foam cells, and fibroblasts isolated from human
Immunohistochemistry studies have demonstrated that
atherosclerotic coronary and carotid plaques [3, 48]. Interest-
ingly, TF of human atherosclerotic plaques retains its full pro-
coagulant properties . Moreover, in patients with clinical
evidence of acute coronary syndromes, TF antigen levels and
TF procoagulant activity measured in human atherectomy
specimens were significantly higher that those measurable in
specimens obtained from patients with stable angina .
Conversely, TF was rarely detected in patients with restenosis
lesions even if the resulting clinical presentation was an unsta-
ble coronary syndrome. Tissue Factor was readily detected in
de novo lesions in patients with unstable coronary syndromes,
suggesting a role for TF in the pathogenesis of this disease
process . Again, Randi et al, analyzing gene expression in
coronary plaques from patients with stable or unstable angina
using gene arrays, demonstrated higher TF expression in un-
stable angina samples . Furthermore, plasma TF activity
seems to have impact on prognosis in patients with ACS.
Steppich et al demonstrated that systemic TF activity in acute
myocardial infarction has an unfavorable prognostic value and,
as a marker for altered coagulation, it might predict the athero-
thrombotic risk .
the coagulation cascade. Bozzini et al. showed that polymor-
phisms in the factor VII gene promoter on activated factor VII
levels may modulate the risk of myocardial infarction in males
with advanced coronary artery disease . In addition, a re-
cent study has demonstrated that C-reactive protein plasma
levels were related with FVII concentration in patients with
coronary artery disease . Moreover, activity of FVII may
Plasma levels of FVIIa seem to be an another key point of
Fig. (2). Schematic representation of Extrinsic coagulation pathway: FVII bound to TF is rapidly converted to its activated form (FVIIa).
FVIIa binds FX, that is converted in its activated form (FXa). Many coagulation proteases such as factors VIIa, IXa, amplify this activation
process. TFPI modulates the TF/FVIIa complex activity. In presence of FXa, it forms a complex which, in turn, binds with high affinity to
TF/VIIa thus causing the formation of a fully inhibited tetra-molecular complex TF/VlIa/TFPI/Xa.
328 Current Cardiology Reviews, 2010, Vol. 6, No. 4 Petrillo et al.
be considered as an independent cardiovascular risk factor.
Specifically, Karatela et al. have recently demonstrated
that raised FVII and leptin levels in coronary artery disease
(CHD) patients were independently associated with insulin
resistance; this was not observed among the non-CHD sub-
tance of TF:FVIIa as one of the main determinant of hu-
man atherosclerotic plaque thrombogenicity .
Taken together, these data clearly underline the impor-
TF/VIIa Complex Inhibitors in Cardiovascular Disease
Tissue Factor Pathway Inhibitor
tivity of the TF:FVIIa complex (Fig. 2), recombinant hu-
man TFPI might be useful in patients with acute coronary
syndromes. Thus, this protein has been successfully ex-
pressed in a variety of hosts, including bacteria, and has
been shown to be effective in preventing thrombus forma-
tion in a variety of experimental models. Haskel et al. 
for the first time demonstrated that administration of phar-
macological doses of human recombinant TFPI was asso-
ciated with lack of reocclusion after discontinuation of t-
PA in a canine model of coronary thrombolysis. Other ex-
perimental studies have also shown that recombinant TFPI
was effective in inhibiting intravascular thrombosis, but
this effect was achieved at doses far higher than those
physiologically measurable in plasma . Not surpris-
ingly, therefore, considering also the FXa inhibitory effects
of TFPI, Oltrona et al.  found that systemic administra-
tion of recombinant TFPI led to a marked prolongation in
PT, suggesting that recombinant TFPI at doses effective in
preventing arterial thrombosis might be associated with a
substantial risk of bleeding. St. Pierre et al.  demon-
strated that recombinant TFPI administration after balloon
overstretch insult to the carotid arteries in pigs reduced TF
expression, FXa activity, and attenuated accumulation of
thrombus at the site of insult. Finally, in a clinical trial in
patients with sepsis, recombinant TFPI showed promise in
reducing mortality in critically ill sepsis patients .
However, several clinical trials aimed to evaluate TFPI
systemic administration in patients with acute coronary
syndromes have been interrupted for ethical reasons since
the risk of bleeding increased significantly. Fortunately,
the rapid progress of in vivo gene transfer technologies has
created powerful new tools to transfer foreign genes into
the cells of a variety of organs, including the vascular wall.
Different vectors have been developed to efficiently trans-
fect target cells, including retroviral, adenoviral, and direct
DNA transfer. Therefore, giving the feasibility of transfect-
ing the arterial wall with foreign genes, it is of no surprise
that TFPI has been the focus of several studies in this field.
The main theoretical advantage of increasing local TFPI
concentrations by gene transfer to the arterial wall is that
therapeutic TFPI levels can be achieved only where they
are needed, i.e., at the damaged arterial site where TF is
exposed, without concomitant, potentially dangerous sys-
temic effects. Thus, starting from these considerations,
several studies have clearly demonstrated the antithrom-
botic efficacy of arterial TFPI gene transfer in different
models of intravascular thrombosis [60, 61]. However, to
Since TFPI has an important role in modulating the ac-
date, considering ethical concerns regarding “gene therapy” in
humans, this kind of pharmacological approach to inhibit
TF/FVIIa complex seems to be far.
site of FVIIa is blocked with a covalent inhibitor, such as
chloromethylketone (known as rFVIIai), has been produced
and successfully used to block TF:VIIa procoagulant activity.
rFVIIai retains its TF binding capacity but is enzymatically
inactive. This molecule exerts its antithrombotic effect by
competing with native factor VIIa (FVIIa) for TF binding.
Since it has a significantly higher affinity to TF than native
FVIIa, it avoids that coagulation cascade could proceed down-
stream [62-64] (Fig. 3).
Recently, human recombinant FVIIa in which the active
can be detected at site of arterial injury in vessel sections ob-
tained from animals 24 h following rFVIIai administration,
despite at this time the compound was completely eliminated
from the circulation. In different animal models, rFVIIai effi-
ciently prevented TF-induced arterial thrombosis, without any
concomitant potentially hazardous systemic effects [65-67].
Interestingly, and in line with histological observations,
rFVIIai-dependent inhibition of arterial thrombosis could be
observed despite plasma concentrations were undetectable,
thus witnessing that this molecule exerted its effect only at site
of injury .
The potential role of rFVIIai in cardiovascular disease has
been demonstrated in ischemia/reperfusion too . Recently,
it has been demonstrated that this rFVIIai effect is primarily
due to rFVIIai ability to reduce inflammation-related lethal I/R
injury. Inhibition of toll-like receptor-4 (TLR-4) and of nuclear
factor-kappaB (NF-kB) mediated cell signalling might be in-
volved. Specifically, levels of NF-kB and of NF-kB-dependent
protein such as TF and IL-6, usually increased after ische-
mia/reperfusion were significantly reduced after rFVIIai ad-
Immunohistochemical studies have evidenced that rFVIIAi
single doses of rFVIIa up to 400 mcg/kg to 64 healthy subjects
did not affect the safety of the subjects nor the hemostatic
function, except for the expected prolongation of the
prothrombin time (PT) . On the basis of these findings, a
multicenter, double-blind, dose-escalation, randomized trial
evaluating the efficacy and safety of rFVIIa in patients under-
going elective or urgent PCI was performed , and in asso-
ciation with this trial, a substudy was designed to evaluate the
antithrombotic effect of FFR-rFVIIa in an ex vivo perfusion
flow chamber connected directly to the patients’ blood streams
. These studies demonstrated that FFR-rFVIIa has a potent
antithrombotic effect at different shear rates and severe arterial
injury conditions, suggesting a potential use for this molecule
in this clinical setting.
Other TF/FVIIa Inhibitors
A phase I clinical study has reported that administration of
plex is represented by antibodies directed against TF: binding
of these antibodies prevents FVII interaction with its natural
ligand. One of the first antibody able to interfere with TF/
FVIIa complex was AP-1, a monoclonal antibody raised
against rabbit TF. AP-1 has been proven to block TF-procoa-
Another potential option to interfere with TF/FVIIa com-
TF/FVII Inhibitors and Cardiovascular Disease Current Cardiology Reviews, 2010, Vol. 6, No. 4 329
gulant activity in vitro and in vivo at very low concentra-
tions  (Fig. 3). In particular, administration of AP-1 to
rabbits with recurrent thrombosis of the carotid artery was
associated with a complete inhibition of thrombosis with-
out a concomitant prolongation in systemic hemostatic pa-
rameters or an alteration in platelet aggregation . The
same agent has also been shown to accelerate the throm-
bolytic properties of t-PA and prevent reocclusion after its
discontinuation in a rabbit model of carotid artery throm-
bosis and thrombolysis .
Recently, it has been developed a chimeric mouse-
human monoclonal antibody directed against TF and
known as ALT836. This antibody binds to TF at its FX
binding site. In patients with stable coronary artery disease
enrolled in the PROXIMATE-TIMI 27 trial, this antibody
had an interesting dose-dependent anticoagulant effect
without any significant side effect such as bleeding .
XK1. It is a chimeric protein which consists of the light
chain of FXa linked to the first Kunitz domain of TFPI
. Other hybrid proteins with increased affinity for
TF/FVIIa complex have also been genetically engineered
to inhibit activation of coagulation cascade [76, 77], and a
version with even greater potency for FVIIa has been
created by linking a modified Kunitz-type inhibitor with a
mutated form of soluble TF . In addition, NAPc2, an
inhibitor of the TF/VIIa complex, has been cloned from
hookworms . This molecule exerts its effects by bind-
ing to FXa and has an inhibitory mechanism resembling
Another potent TF:VIIa complex inhibitor is known as
that of TFPI. The antithrombotic effect of NAPc2 has recently
been demonstrated in a dose-finding study on the prevention of
venous thromboembolism in patients undergoing total knee
As reported above, TF/FVIIa inhibitors have been successfully
tested in vivo after parenteral administration, but research of an
orally bio-available drug still remains an undiscovered field.
Comforting results observed in preclinical models and clinical
trials [81, 82] in which small protein and antibody-based in-
hibitors of the TF/FVIIa pathway have been tested, have
stimulated several future studies aimed to develop orally active
TF/FVIIa inhibitors and to perform a tailored anti-thrombotic
therapy. Probably, the main limitation that should be overcome
is that an effective oral drug will require a careful balance
between optimal inhibitor characteristics and drug-like or
pharmacokinetic properties, that is a challenge which often
does not have an easy solution.
in preventing other TF-mediated phenomena, such as inflam-
mation and cell proliferation, these molecules should be tai-
lored to exert their effects only where they are needed, without
affecting the physiological haemostasis.
Moreover, since TF/FVIIa inhibitors might be useful also
could be the engineering of a balloon coated with an inhibitor
of TF/FVIIa, such as rFVIIai able to permit local drug release
at site of injury/thrombosis, during percutaneous transluminal
coronary angioplasty (PTCA). Alternatively, a TFPI-coated
In interventional cardiology area, particularly attractive
Fig. (3). Examples of TF/VIIa complex inhibitors. Anti-TF monoclonal antibody binds TF and prevent FVII binding. rFVIIai is enzymati-
cally inactive but it has high TF binding affinity. Thus, it exerts its antithrombotic effect by competing with FVIIa for TF binding and conse-
quently impeding TF/FVIIa activity.
330 Current Cardiology Reviews, 2010, Vol. 6, No. 4 Petrillo et al.
balloon might be used in the same clinical setting, in order
to increase local TFPI concentrations and obtain a local
antithrombotic effect. Finally, a fascinating hypothesis
might be that of a balloon coated with genes codifying for
TFPI, to directly obtain gene transfer to the arterial wall, in
order to facilitate the stabilization of active atherosclerotic
plaque before stent deployment.
extrinsic pathway of blood coagulation. Thus, it is intuitive
why it has become an attractive tool for the development of
newer antithrombotic agents able to prevent complex for-
mation or to inhibit its catalytic activity. This kind of anti-
thrombotic therapy has several theoretical advantages if
compared with other interventions directed against other
“downstream” components of the coagulation cascade,
such as heparin and its derivatives or direct antithrombin
Indeed, although potent synthetic inhibitors of
TF/FVIIa had been discovered and tested in animal
models, any of these have advanced into clinical trials. The
systemic effects observed and, specifically, the marked
elongation of bleeding time observed in experimental stud-
ies have highlighted that safety, effective dose and route of
administration are the main issues to resolve. On the other
side, while the local delivery of genes or drugs during in-
terventional procedures seems very intriguing, the feasibil-
ity of this approach needs to be demonstrated.
TF:VIIa complex represents the “critical point” of the
complex still remains a challenging and important target to
study and develop future generations of antithrombotic
However, in spite of these considerations, TF:FVIIa
FVIIa = Factor VII activated
Factor X activated
Factor IX activated
FXIIa = Factor XII avtivated
TFPI = Tissue factor pathway inhibitor
Coronary artery disease
Monoclonal antibody raised against
rFVIIai = Recombinant FVIIa with active site blocked
with a covalent inhibitor
Percutaneous transluminal coronary
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Table 1. TF/VIIa Complex Inhibitors: Mechanism of Action, Effects on Bleeding and Trials in Cardiovascular Disease
MECHANISM RISK OF BLEEDING TRIAL IN CARDIOVASCULAR DISEASE
rTFPI Inhibition of TF/FVIIa complex via FXa/rTFPI complex
rFVIIai Competition with native FVIIa for TF binding
ALT-836 Chimeric antibody against TF
XK1 TFPI-like mechanism
NAPc2 TFPI-like mechanism
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Received: Jan 28, 2010
Revised: June 17, 2010 Accepted: June 22, 2010