© 1999 American Heart Association, Inc.
Thrombosis |
Structural Requirements for TFPI-Mediated Inhibition of Neointimal Thickening After Balloon Injury in the Rat
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Abstract |
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Abstract—The intimal thickening that follows vascular injury is inhibited by periprocedural tissue factor pathway inhibitor (TFPI) treatment in animal models. TFPI is a multivalent Kunitz-type protease inhibitor that inhibits factor Xa via its second Kunitz domain and the factor VIIa/tissue factor (TF) complex via its first Kunitz domain. The basic C-terminus of TFPI is required for the binding of TFPI to cell surfaces and cell-bound TFPI mediates the internalization and degradation of factor X and the down regulation of surface factor VIIa/TF activity. The C-terminus of TFPI is also required for its reported direct inhibition of smooth muscle cell proliferation in vitro. To examine the structural requirements for the inhibition of neointimal formation by TFPI, several TFPI-related proteins were tested in the rat carotid angioplasty model: 1) XK1, a hybrid protein containing the N-terminal portion of factor X and the first Kunitz domain of TFPI that directly inhibits factor VIIa/TF; 2) TFPIWT, the full-length TFPI molecule that inhibits factor Xa and factor VIIa/TF and binds cell surfaces; 3) TFPIK36I, an altered form of TFPI that inhibits factor Xa, but not factor VIIa/TF, and binds cell surfaces; 4) TFPI13–161, a truncated form of TFPI that inhibits factor VIIa/TF but interacts with factor Xa poorly and does not bind to cell surfaces. Seven day infusions of XK1, TFPIWT, and high levels of TFPIK36I begun the day before balloon-induced vascular injury produced a significant reduction in the intimal hyperplasia measured 28 days after angioplasty. The infusion of high concentrations of TFPI13–161 was ineffective in this model. These in vivo results directly mirror the ability of each TFPI-related protein to inhibit tissue thromboplastin-induced coagulation in rat plasma: XK1
TFPIWT>TFPIK36I>>TFPI13–161.
The studies confirm the important role of TF-mediated coagulation in
the smooth muscle proliferation and neointimal thickening
that follows vascular injury and suggest that the anticoagulant effect
alone of TFPI and TFPI-related proteins is sufficient to explain their
therapeutic action.
Key Words: tissue factor pathway inhibitor • angioplasty • rat
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanisms underlying the intimal thickening and associated restenosis that follow arterial injury are complex, but recent data suggest that activation of the coagulation cascade plays an important role in the process.1 2 3 4 5 6 Tissue factor (TF) is an integral membrane protein that serves as an essential cofactor for plasma factor VII(a) in the initiation of coagulation. TF is present at high levels in atherosclerotic plaques, and TF expression is induced in vascular smooth muscle cells after balloon injury.7 8 9 The factor VIIa/TF catalytic complex activates factor X, which in turn produces thrombin generation. Both factor Xa and thrombin have been shown to be potent mitogens for vascular smooth cells in vitro.4 5 6 Moreover, anticoagulant agents that inhibit thrombin (hirudin), factor Xa (tick anticoagulant protein, TAP), or factor VIIa/TF (inactivated factor VIIa [VIIai], tissue factor pathway inhibitor [TFPI]) reduce postangioplasty intimal hyperplasia in animal models.10 11 12 13 14
TFPI is a trivalent Kunitz-type proteinase inhibitor,
which circulates at trace concentrations (2 nmol/L) in human plasma.
The in vivo infusion of heparin and other polyanions increases the
circulating levels of TFPI in plasma 2- to 4-fold, and the source of
this additional TFPI is thought to be the endothelium.
TFPI directly inhibits factor Xa and, at
physiological concentrations, produces factor
Xa-dependent inhibition of the factor VIIa/TF complex. The TFPI
molecule contains an acidic amino-terminal region followed by three
tandem Kunitz-type domains and a basic carboxy-terminal region. The
second Kunitz-domain of TFPI is responsible for factor Xa inhibition,
but other parts of the molecule are involved in its interaction with
factor Xa. The basic carboxy-terminal region of TFPI is required for
rapid inhibition of factor Xa and the enhancement of TFPI-mediated
factor Xa inhibition produced by heparin and other polyanions. Factor
Xa-dependent inhibition of the factor VIIa/TF complex by TFPI involves
the formation of a quaternary complex containing factor Xa-TFPI-factor
VIIa/TF in which the second Kunitz domain of TFPI binds factor Xa and
the first Kunitz domain binds factor VIIa. The requirement of factor Xa
for the inhibition of factor VIIa/TF by TFPI is not absolute, and at
high, therapeutic concentrations TFPI inhibits the factor VIIa/TF
catalytic complex in the absence of factor Xa.15 16 17 18 19 20
Besides its direct anticoagulant effects on factor Xa and factor VIIa/TF, TFPI possesses additional properties that may be related to its in vivo action. TFPI binds to the surface of cells through a process that requires its basic carboxy-terminus and this cell-bound TFPI dramatically enhances the cellular binding, internalization, and degradation of factor Xa.21 Moreover, in a process that involves a glycosyl-phosphatidyl-inositol-linked membrane protein that may represent the cell surface binder for TFPI, quaternary factor Xa-TFPI-factor VIIa/TF complexes are translocated to caveolae, thereby decreasing cell surface TF.22 23 In addition, TFPI reportedly inhibits TF-induced aortic smooth muscle cell migration24 and neonatal aortic smooth muscle cell proliferation.25 Whether these cellular effects of TFPI contribute to its in vivo action is not known.
Because recent studies have shown that recombinant, full-length TFPI reduces intimal thickening after vascular injury in the rabbit atherosclerotic femoral artery11 and the carotid artery of the cholesterol-fed minipig,10 we conducted the present study to evaluate the structures within the TFPI molecule that are important for its effect on neointimal formation. Accordingly, we tested a number of TFPI congeners in the rat carotid model of vascular injury.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Proteins
Human recombinant (r)TFPIWT, rTFPIK36I, and rTFPI13–161 were expressed in Escherichia coli and refolded in vitro as previously described.26 rXK1 was expressed in baby hamster kidney cells and isolated as previously described.27 Each of the proteins appeared >90% pure by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Due to limitations in their solubility, TFPIWT (4.7 mg/mL) and TFPIK36I (6 mg/mL) were reconstituted in 0.3 mol/L arginine-HCl, 20 mmol/L sodium phosphate, pH 7.4 (arg-phos buffer). TFPI13–161 (28 mg/mL) and XK1 (3 mg/mL) were reconstituted in 0.1 mol/L NaCl, 0.05 mol/L sodium phosphate, pH 7.4 (phosphate-buffered saline [PBS] buffer).
Delivery of TFPI Forms
Osmotic pumps with 2-mL capacity and a delivery rate of 10 uL/h
(2 ML1, Alza Corp) were used to infuse the test reagents
intravenously for 1 week. Following the manufacturer's
instructions, pumps were loaded with protein solution or buffer
controls and incubated at 37°C for 6 hours before implantation into
anesthetized (ketamine 75 mg/kg, xylazine 10 mg/kg,
acepromazine 5 mg/kg, IP) 300 to 350 g female Sprague-Dawley rats.
A ventral incision was made in the neck and the left external jugular
vein freed using blunt dissection. The rostral end of the vein was
ligated and a PE60 polyethylene catheter (Becton Dickinson) connected
to the osmotic pump was inserted proximally. The pump was placed in a
subcutaneous pocket fashioned under the skin of the dorsal neck. To
double the infusion rate of TFPIK36I in some of
the rats, an additional pump was connected to the right jugular vein in
the same fashion. The ventral incision was then closed with clips.
Pumps were explanted 1 week after implantation. All protocols involving
animals were approved by the Animal Studies Committee at Washington
University.
Balloon-Induced Vascular Injury
Balloon injury of the left common carotid artery was produced
the day after pump implantation. Rats were anesthetized as
described above, the ventral incision opened, and the left external
carotid artery identified. A 2 French balloon embolectomy catheter
(Baxter) was introduced via the external carotid artery and advanced to
the aorta. The balloon was partially inflated and the catheter
withdrawn along the full length of the common carotid artery and then
deflated. This procedure was repeated 3 times before the catheter was
removed, the external carotid artery ligated, and the ventral neck
incision closed. Four weeks later, the animals were exsanguinated by
perfusion through the heart with PBS followed by 4% formaldehyde. The
left carotid artery was harvested (2.5 cm) and immersion-fixed in
4% formaldehyde solution for histological
examination.
Histologic Examination of Carotid Arteries
Fixed tissue was dehydrated and paraffin embedded according to
standard protocols. The entire length of the carotid artery was step
sectioned (100 µm) and tissue sections stained with Verhoff's
Van Gieson stain. Twenty consecutive sections around the greatest
extent of neointimal thickening were selected for
morphometric analysis using the ImagePro system (Media
Cybernetics). The neointimal cross-sectional area was
calculated by subtracting the cross-sectional area of the lumen from
that demarcated by the internal elastic lamina. The area of the media
was determined by subtracting the area inside the internal elastic
membrane from that delineated by the external elastic membrane. The
ratio of the intimal area to the medial area was calculated for each of
20 sections, and an average ratio was determined for each carotid.
Statistical analysis was done with unpaired Student's
t test to evaluate 2-tailed levels of significance between
treated group and control group, respectively.
Immunoassay of TFPI Forms
Blood was drawn from the retroorbital sinus of the rats into
ethylenediamine-tetraacetic acid–coated capillary tubes at the
time of angioplasty, and plasma was obtained by
centrifugation. For assay, test plasma or normal rat
plasma containing various concentrations of the specific TFPI-related
protein were diluted 5-fold into 0.1 mol/L NaCl, 0.05 mol/L Tris-HCl,
pH 7.4 containing 1 mg/mL bovine serum albumin. To determine
levels of TFPIWT, TFPIK36I
and TFPI13–161, 20 uL of diluted sample or
standard was added to 20 uL 0.5% (wt/vol) latex beads coated with the
monoclonal anti-TFPI antibody MC2B12 (100 µg/mL), which recognizes
the second Kunitz domain of TFPI, in a well of a Fluorocon microtiter
plate (Baxter). In the case of XK1, 20 uL of
diluted plasma sample or standard was added to 20 uL 0.5% (wt/vol)
latex beads coated with the monoclonal antifactor X antibody MC1066
(100 µg/mL), which recognizes the light chain of factor X. After the
addition of 20 uL fluorescein isothiocyanate-labeled
monoclonal anti-TFPI antibody MC2H8, which recognizes the first Kunitz
domain of TFPI, the mixtures were incubated for 30 minutes at room
temperature before the beads were washed and the latex bead associated
fluorescein isothiocyanate determined using a
Fluorescence Concentration Analyzer (Baxter).
Factor VIIa/TF Activity Inhibition
The relative ability of TFPIWT,
TFPIK36I, and TFPI13–161
to inhibit factor VIIa/TF in the presence of factor Xa was determined
using a commercially available kit (Actichrome TFPI, American
Diagnostica Inc). This is an end-point assay in which
factor VIIa, tissue factor, a small quantity of factor X(a), and the
inhibitor are first incubated for 30 minutes. Factor X is
then added, and the extent of factor Xa generated in 15 minutes by the
remaining factor VIIa/TF activity determined by the addition of
ethylenediamine-tetraacetic acid (to stop the reaction) and the
chromogenic substrate Spectrazyme Xa (American
Diagnostica, Inc).
Anticoagulant Activity of TFPI Forms
The TFPI forms were tested for their ability to inhibit the
coagulation of rat plasma induced by tissue factor (rat brain
thromboplastin) and the factor X-coagulant protein (XCP) of Russells
Viper venom, which directly activates factor X.
Rat Thromboplastin-Induced Coagulation
Rat thromboplastin was prepared as a crude saline extract of rat
brain (Pel Freez Biologicals) as previously described.28
In a fibrometer (BBL), 50 uL of rabbit brain cephalin (1:8
dilution of stock suspension, Sigma), 50 uL of
CaCl2 (50 mmol/L), 50 uL rat thromboplastin
(1:5 dilution), and 50 uL of various concentrations of test
inhibitor are incubated at 37°C. After 30 seconds, 50 uL
normal rat plasma (Pel Freez) is added and the time of clot formation
determined. Apparent inhibition of thromboplastin-induced coagulation
is determined by comparison of clotting times with a standard curve
constructed using various concentrations of thromboplastin.
XCP-Induced Coagulation
In a fibrometer, 60 uL CaCl2 (50
mmol/L), 60 uL XCP (45 pM) in rabbit brain cephalin (1:8 dilution), and
60 uL of various concentrations of test inhibitor are
incubated at 37°C. After 30 seconds, 60 uL of normal rat plasma is
added and the time to clot formation determined. Apparent inhibition of
XCP-induced coagulation is determined by comparison of clotting times
with a standard curve constructed using various concentrations of
XCP.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Characterization of TFPI-Related Molecules
Four TFPI-related molecules were evaluated: TFPIWT, TFPIK36I, TFPI13–161, and XK1. TFPIWT is the native full-length molecule that inhibits factor Xa, factor VIIa/TF, and binds to cell surfaces. In TFPIK36I, the P1 residue at the inhibitory cleft within the first Kunitz domain of TFPI has been changed from a lysine to an isoleucine. This molecule does not inhibit factor VIIa/tissue factor (Figure 1
) but retains the ability to inhibit
factor Xa and bind to cells. TFPI13–161 is a
truncated form of TFPI that lacks the third Kunitz domain and the
remainder of the carboxy-terminus of the native molecule.
TFPI13–161 inhibits factor Xa, albeit much less
avidly than TFPIWT,16 and inhibits
the factor VIIa/TF complex (Figure 1). Lacking the
carboxy-terminal basic region of TFPI,
TFPI13–161 does not bind to cell surfaces.
Finally, XK1 is a chimeric protein that contains
the amino-terminal light-chain of factor X linked to the first Kunitz
domain of TFPI. XK1 directly inhibits factor
VIIa/TF27 but does not inhibit factor Xa.
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The relative anticoagulant activities of the TFPI forms in rat
plasma were tested in 1-stage coagulation assays in which coagulation
was induced by the factor X-coagulant protein (XCP) from Russells Viper
venom or rat brain thromboplastin (Table 1). As anticipated based on their
antifactor Xa activity, TFPIWT and
TFPIK36I are potent inhibitors
(IC50=4.0±0.3 and 4.4±0.4 nmol/L, respectively)
and TFPI13–161 and XK1
poor inhibitors (apparent IC50 >625
nmol/L) of XCP-induced coagulation. Thromboplastin-induced coagulation
was potently inhibited by TFPIWT
(IC50 4.2±0.4 nmol/L) and
XK1 (IC50 3.0±0.2 nmol/L),
less inhibited by TFPIK36I
(IC50 16.0±1.5 nmol/L), and very poorly
inhibited by TFPI13–161
(IC50=625±20 nmol/L).
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Effect of TFPI-Forms on Intimal Thickening After Vascular
Injury
The relative efficacy of the various forms of TFPI in the
inhibition of intimal thickening after vascular injury was tested in
the rat carotid balloon injury model.29
Representative histological sections of
carotid arteries obtained 28 days after injury from each treatment
group are shown in Figure 2.
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The degree of neointimal thickening was assessed by
calculating the ratio of intima area:media area within the injured
artery (Table 2). In the animals treated
with TFPIWT and XK1, there
was significant reduction of neointimal formation compared
with all other treated groups and the buffer controls
(P<0.01 to <0.001). TFPIK36I at a
high infusion rate also produced a significant reduction of the
intima:media ratio compared with buffer control, whereas
TFPI13–161 treatment did not significantly
effect the extent of neointimal thickening after vascular
injury. Hemorrhagic complications were not observed in the treated
animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A 24-hour infusion of TFPI has been shown previously to reduce neointimal thickening after balloon vascular injury in the cholesterol-fed pig,10 whereas a 3-day infusion of rTFPI was effective in decreasing neointimal thickening after air-desiccation injury in cholesterol-fed rabbits.11 TFPI has multiple functional attributes, including the inhibition of factor Xa, the inhibition of factor VIIa/TF, and cellular effects related to its ability to bind to cell surfaces.18 19 20 21 22 23 24 25 To examine the structural determinants and mechanism(s) of the therapeutic efficacy of TFPI, we tested a variety of TFPI-related agents for their ability to inhibit neointimal thickening after balloon overstretch injury of the rat carotid artery.
The relative efficacy of the TFPI-related agents in reducing
neointimal thickening mirrored their relative anticoagulant
potency in a 1-stage plasma assay of thromboplastin-induced
coagulation:
XK1~TFPIWT>TFPIK36I>>TFPI13–161
(Tables 1
and 2). These results are consistent
with previous data suggesting an important role for tissue
factor-induced coagulation in the formation of neointima
after vascular injury in rabbits and pigs,10 11 and they
occurred despite the lack of marked thrombus formation in the rat
vascular injury preparation. Moreover, the results suggest that the
anticoagulant properties of these TFPI-related agents alone can explain
their therapeutic benefit. Of the 2 most potent agents,
XK1 reduces factor Xa generation by directly
inhibiting the factor VIIa/TF complex, whereas
TFPIWT not only inhibits factor Xa generation by
factor VIIa/TF but also inhibits factor Xa itself. Plasma
concentrations achieved during the constant intravenous
infusion of these agents were several-fold higher than their
IC50's for inhibition of tissue
thromboplastin-induced coagulation (Tables 1 and 2).
TFPIK36I, which does not inhibit factor
VIIa/tissue factor but is a potent inhibitor of factor Xa,
was less effective than either XK1 or
TFPIWT in inhibiting neointimal
thickening (Table 2). However, even at the "high" dosage
level, the plasma concentration of TFPIK36I
achieved during constant infusion (13 nmol/L) was lower than its
IC50 (16 nmol/L) for inhibition of tissue
thromboplastin-induced coagulation (Table 1). We interpret the
apparent dose response found with TFPIK36I to
imply that higher levels of TFPIK36I
(>>IC50) may have been more effective, yielding
effects on neointimal thickening similar to
XK1 and TFPIWT. In
contrast, although the factor VIIa/tissue factor inhibition produced by
TFPI13–161 in an endpoint assay was comparable
with that of TFPIWT (Figure 1), this agent
was a poor anticoagulant and failed to inhibit neointimal
thickening (Tables 1 and 2).
Interestingly, although the anticoagulant effect of
TFPIK36I was equivalent to that of
TFPIWT in the XCP assay,
TFPIK36I was 4-fold less active than
TFPIWT in the tissue thromboplastin assay (Table 1). This result suggests that a significant proportion of the
anticoagulant effect of TFPIWT in a 1-stage assay
of tissue thromboplastin-induced coagulation is due to its inhibition
of factor VIIa/TF or, alternatively, that the TFPI-mediated inhibition
of the initial factor Xa generated by the factor VIIa/TF complex is
enhanced when the TFPI is able to bind simultaneously to
factor Xa and factor VIIa in a factor VIIa/TF-factor Xa tertiary
complex.30 Preliminary experiments indicate that the
latter explanation is more likely (Han and Broze, unpublished
observations, 1998).
That an effect of TFPI related to cell-surface binding contributes to its beneficial effect after vascular injury cannot be excluded. Nevertheless, like hirudin, TAP, and VIIai, the anticoagulant actions of the TFPI-related proteins were sufficient to predict their ability to inhibit neointimal formation. The treatment of choice for this indication will likely depend on the incidence of hemorrhagic complications. Recent studies have suggested that agents targeting factor VIIa/TF (eg, VIIai, anti-TF antibodies) may induce less bleeding than agents that target coagulation at latter stages.31 32 33 Consistent with this notion, in preliminary studies, plasma levels of XK1 >100-fold its IC50 in the rat thromboplastin-induced coagulation assay did not produce hemorrhagic manifestations during the surgical manipulations required for the balloon angioplasty procedure.
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Acknowledgments |
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We wish to acknowledge the fine technical assistance of Judith Diaz-Collier, who constructed the TFPI13–161 cDNA and produced the initial expression TFPI13–161 in E coli and thank Terri Lewis for secretarial assistance. This work was supported by a grant from the Monsanto/Searle Company (to G.J.B.).
Received February 2, 1999; accepted March 19, 1999.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 A. H.M Moons, M. Levi, and R. J.G Peters Tissue factor and coronary artery disease Cardiovasc Res, February 1, 2002; 53(2): 313 - 325. [Abstract] [Full Text] [PDF]
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N. M. Caplice, C. Panetta, T. E. Peterson, L. S. Kleppe, C. S. Mueske, G. M. Kostner, G. J. Broze Jr, and R. D. Simari Lipoprotein (a) binds and inactivates tissue factor pathway inhibitor: a novel link between lipoproteins and thrombosis Blood, November 15, 2001; 98(10): 2980 - 2987. [Abstract] [Full Text] [PDF]
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 A. D. Schecter, A. B. Berman, L. Yi, A. Mosoian, C. M. McManus, J. W. Berman, M. E. Klotman, and M. B. Taubman HIV envelope gp120 activates human arterial smooth muscle cells PNAS, August 10, 2001; (2001) 181328798. [Abstract] [Full Text] [PDF]
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P. Zoldhelyi, Z.-Q. Chen, H. S. Shelat, J. M. McNatt, and J. T. Willerson Local gene transfer of tissue factor pathway inhibitor regulates intimal hyperplasia in atherosclerotic arteries PNAS, March 27, 2001; 98(7): 4078 - 4083. [Abstract] [Full Text] [PDF]
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 U. Rauch, J. I. Osende, V. Fuster, J. J. Badimon, Z. Fayad, and J. H. Chesebro Thrombus Formation on Atherosclerotic Plaques: Pathogenesis and Clinical Consequences Ann Intern Med, February 6, 2001; 134(3): 224 - 238. [Abstract] [Full Text] [PDF]
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A. D. Schecter, A. B. Berman, L. Yi, A. Mosoian, C. M. McManus, J. W. Berman, M. E. Klotman, and M. B. Taubman HIV envelope gp120 activates human arterial smooth muscle cells PNAS, August 28, 2001; 98(18): 10142 - 10147. [Abstract] [Full Text] [PDF]
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I. M. B. Francischetti, J. G. Valenzuela, J. F. Andersen, T. N. Mather, and J. M. C. Ribeiro Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex Blood, May 15, 2002; 99(10): 3602 - 3612. [Abstract] [Full Text] [PDF]
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H. Kato Regulation of Functions of Vascular Wall Cells by Tissue Factor Pathway Inhibitor: Basic and Clinical Aspects Arterioscler. Thromb. Vasc. Biol., April 1, 2002; 22(4): 539 - 548. [Abstract] [Full Text] [PDF]
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