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Prepublished online as a Blood First Edition Paper on April 17, 2002; DOI 10.1182/blood-2001-12-0237.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Section of Medical Entomology, Laboratory of
Parasitic Diseases, National Institute of Allergy and Infectious
Diseases, National Institutes of Health, Bethesda, MD, and Center for
Vector-Borne Disease, University of Rhode Island, Kingston.
Saliva of the hard tick and Lyme disease vector, Ixodes
scapularis, has a repertoire of compounds that counteract host
defenses. Following sequencing of an I scapularis salivary
gland complementary DNA (cDNA) library, a clone with sequence homology
to tissue factor pathway inhibitor (TFPI) was identified. This cDNA
codes for a mature protein, herein called Ixolaris, with 140 amino
acids containing 10 cysteines and 2 Kunitz-like domains. Recombinant
Ixolaris was expressed in insect cells and shown to inhibit factor VIIa
(FVIIa)/tissue factor (TF)-induced factor X (FX) activation with an
inhibitory concentration of 50% (IC50) in the
picomolar range. In nondenaturing gel, Ixolaris interacted
stoichiometrically with FX and FXa but not FVIIa. Ixolaris behaves as a
fast-and-tight ligand of the exosites of FXa and Following tissue injury, exposition of
membrane-bound tissue factor (TF) is a crucial step in the initiation
of blood coagulation.1 TF binds to blood coagulation
factor VIIa (FVIIa), and the binary FVIIa/TF complex then activates
factor X (FX) to factor Xa (FXa), leading to thrombin generation and
fibrin formation.1 Initiation of blood coagulation by
FVIIa/TF is under control of tissue factor pathway inhibitor
(TFPI),2-5 a 34- to 43-kd multidomain protein with an
acidic amino terminus, 3 typical Kunitz-type inhibitor domains, and a
basic carboxy terminus.6,7 The second Kunitz domain binds
and inhibits FXa,8 and the first Kunitz domain binds to
FVIIa/TF through formation of a final quarternary inhibitory complex
consisting of FVIIa/TF/TFPI/FXa.9-13 In addition to TFPI and other physiologic inhibitors of blood coagulation (eg, antithrombin III), a number of exogenous coagulation inhibitors from the salivary gland of blood-sucking invertebrates have been
characterized,14 including nitrophorin-2 from
Rhodnius prolixus15 and anophelin from
Anopheles albimanius,16,17 among
others.14 Nitrophorin-2 inhibits the intrinsic FX
activating complex,15 whereas anophelin is a cysteine-free
molecule obtained by peptide synthesis16 and shown to
tightly bind thrombin.17
Ticks, such as Ixodes scapularis, are ectoparasites that
feed for several days with their mouthparts embedded in their
vertebrate hosts. A number of pharmacologic properties have been
described in I scapularis saliva including inhibitors of
neutrophil function18 and complement
activation,19 in addition to anti-inflammatory and
immunosuppressive components.20 Antihemostatic compounds have also been molecularly characterized in the soft tick
Ornithodoros moubata, including a platelet
To understand the complexity of I scapularis saliva,
with a primary focus on antihemostatic molecules, massive sequencing of
a complementary DNA (cDNA) library of the salivary gland of I
scapularis has been performed. Together with a complementary functional approach, a clone with sequence homology to TFPI has been
expressed as an active molecule and its anticoagulant mechanism studied. The recombinant protein has the same properties found in
saliva. This molecule, called Ixolaris, binds to FX in addition to FXa
and is characterized here as a specific inhibitor of FVIIa/TF in the
presence of zymogen or enzyme as scaffolds.
Materials
Ticks and tick saliva
Salivary gland cDNA construction This was done as detailed before.19 Briefly, the Micro-FastTrack messenger RNA (mRNA) isolation kit (Invitrogen, San Diego, CA) was used to isolate the mRNA. The I scapularis salivary gland mRNA (200 ng) was reverse transcribed to cDNA followed by double-strand synthesis and ligated into a Lambda Triplex2 vector; the resulting ligation reaction was packed using Gigapack gold III from Stratagene/Biocrest (Cedar Creek, TN). The library obtained was plated by infecting log-phase XL1-blue cells. Randomly picked clones from this library were sequenced exactly as described before.19 After identifying a cDNA with high similarity to TFPI following the Basic Local Alignment Search Tool X (BLASTX) program of the cDNA against the National Center for Biotechnological Information (NCBI) nonredundant database,27 an aliquot (~100 ng) of Ixolaris polymerase chain reaction (PCR) sample was reamplified, and the entire cDNA was fully sequenced using custom primers.Ixolaris expression vector For expression of Ixolaris, full-length cDNA was used as a template to amplify only the cDNA that begins at the initial methionine and ends at the first stop codon. A Kozak consensus sequence (ANNATGG) was added and the DNA amplified (forward primer: 5'-AAA ATG GGC GCT GTT TCC TGC TTC-3'; reverse primer: 5'-GGA TGA TCA GTT AAT AGT GAC ATT TAC-3') and cloned into the vector pIB/V5-His TOPO (Invitrogen, San Diego, CA) following manufacturer's specifications.Expression of Ixolaris in insect cell line BTI-TN-5B1-4 (High Five) High Five cells (Invitrogen), cultivated in High Five serum-free medium supplemented with 10 µg/mL gentamycin were used for transfection with Ixolaris and antisense (control) constructs plasmid as indicated by the manufacturer. After 2 days of incubation, the supernatant (5 mL) was collected and stored for further analysis.Sequence analysis Sequence similarity searches were performed using the BLAST27 program. Cleavage site predictions of the mature proteins used the SignalP28 program. Alignments of protein sequences were done with the ClustalW program version 1.7.29 The molar extinction coefficient of Ixolaris at 280 nm was obtained at http://www.mbshortcuts.com, yielding a value of 280 nm = 20 260
M 1.cm1; A 280 nm/cm (1 mg/mL) = 1.287. Other calculated parameters are: Mr, 15738.35; pI, 4.56. Detection of N-linked glycosylation
sites was obtained at
http://molbio.info.nih.gov/molbio/gcglite.
Chromatographic procedures and purification of recombinant Ixolaris Supernatants of transformed cells were filtrated with a 50-kd cutoff Centriprep filter (Millipore, Bedford, CA). The filtrate was concentrated 20-fold using a Centriprep (3-kd cutoff). One milliliter of concentrated supernatant (~8 mg) was diluted to 5 mL with water and applied to a 5 × 100-mm Pharmacia MonoQ eluted with a gradient from 25 mM Hepes pH 7.2 to 1 M NaCl in the same buffer, for 1 hour, at 0.5 mL/min. Active fractions were pooled and applied to a 10 × 250-mm Vydac 218TP510 octadecyl-silica column (The Separation Group, Hesperia, CA) eluted at 1.5 mL/min with a 60-minute gradient from 10% to 80% acetonitrile in water containing 0.1% trifluoroacetic acid (TFA). Active fractions were pooled, diluted with water to 8 mL, and chromatographed again in a 2.1 × 250-mm Macrosphere octadecylsilica column (Altech, Deerfield, IL) eluted at 0.2 mL/min for 55 minutes, and at 0.1 mL/min afterward.Polyacrylamide gel electrophoresis of recombinant Ixolaris A sample of purified recombinant Ixolaris (0.25 µg) in the absence (native conditions) or presence of sodium dodecyl sulfate (SDS) and dithiothreitol (DTT; denaturing conditions) was loaded into a 12% NU-polyacrylamide gel electrophoresis (PAGE) gel (MOPS buffer). Gels were silver stained (Bio-Rad, Hercules, CA).Estimation of Ixolaris concentration Concentration of Ixolaris (corrected for 280 nm)
was estimated by the area of absorbance at A280 nm (calibration with bovine serum albumin (BSA)) of the peak containing Ixolaris
activity obtained in the last purification step.
Binding of Ixolaris to FX, FXa, and FVIIa Ixolaris (20 nM) was preincubated with FX (20 nM), FXa (20 nM), or FVIIa (20 nM) in 5 mM Hepes, pH 7.4, for 10 minutes. The sample was loaded into 8% precast PAGE and proteins were then transferred to polyvinylidene difluoride (PVDF) membrane. Primary antibody (polyclonal antibody anti-FX, 10 µg/mL, or monoclonal antibody anti-FVII, 5 µg/mL) was incubated for 1 hour in Tris-buffered saline, with 0.05% Tween and 5% nonfat milk (TBS-T). After 3 washes with TBS-T, the membranes were incubated for 30 minutes with appropriate AP-coupled secondary antibody (1:10 000). Reactions were developed with Western Blue Stabilized substrate for AP (Promega, Madison, WI).Kinetic assay of FXa production by FVIIa/TF This was performed as described. Ixolaris was incubated for 15 minutes at 37°C with FX, followed by addition of FVIIa/TF (1 nM/0.2 pM) previously incubated for 15 minutes at 37°C in buffer containing 50 mM Hepes, 0.1 M NaCl, 5 mM CaCl2, 0.5% BSA, pH 7.4 (buffer A). Chromogenic substrate (S2222, 250 µM) hydrolysis was detected using a Versamax microplate enzyme-linked immunosorbent assay (ELISA) reader (Molecular Devices, Sunnyvale, CA) equipped with a microplate mixer and heating system.17 Reactions were continuously recorded at 405 nm for 1 hour at 37°C. The total volume of the reactions was 200 µL. Care was taken to ensure that substrate was less than 20% hydrolyzed. To have an estimation of FXa production, smoothing of the raw data (readings every 12 seconds) was performed and the background (~0.042 U OD/405 nm) discounted using Excel 2000 software (MicroSoft Excel Analysis Tools, Seattle, WA). Then, data were transformed as the A405/min as
described.9,10 Briefly, A405 nm absorbance readings at times 1.00, 1.12, 1.24 up to 59.36, 59.48, and 60.0 minutes were respectively subtracted from absorbance readings at times 0, 0.12, 0.24 up to 58.36, 58.48, and 59.0 minutes, enabling determination of
FXa concentrations at each time point. The FXa concentrations were
interpolated from a standard curve relating A405/min and known FXa
concentrations.9,10 Amidolytic activity by FVIIa/TF alone
on S2222 alone was not detected. In some experiments, data were plotted
as Vs/Vo against Ixolaris concentration, where Vs is the velocity of
substrate hydrolysis in the presence of inhibitor and Vo in its
absence.30 Enzymes, Ixolaris, and chromogenic substrate
were diluted in buffer A.
Preparation of DEGR-FX and des-Gla-DEGR-Xa Briefly, DEGR (40 µM) was added to des-Gla-FXa (2 µM) in 100 mM Tris, 100 mM NaCl, pH 7.4 and incubated overnight at room temperature.31 des-Gla-DEGR-FXa was extensively dialyzed against 1 liter TBS (3 times) and shown to be devoid of amidolytic activity with S2222. FX (2 µM) was incubated with DEGR (400 µM) as described above, dialyzed, and shown to be devoid of amidolytic activity (S2222) when incubated with FVIIa/TF. The rationale for using DEGR-FX is based on experiments showing that serine catalytic site mutated FX (FXs195A) has been used as an effective scaffold for NAPc2, a TFPI-like molecule from Ancylostoma caninum.32FVIIa/TF amidolytic activity Ixolaris only, or Ixolaris/DEGR-FXa was incubated for 15 minutes at 37°C with FVIIa/TF (1 nM/1 nM) followed by addition of S2288 (1 mM).9,10Activation of FIX This was performed as described by Komiyama et al.33 In all experiments, plasma- and albumin-free recombinant FIX34 was used (BeneFIX, 250 U diluted at 1 U/µL in distilled water). In a final volume of 25 µL, FVIIa (1 nM)/TF (1 nM) was incubated for 15 minutes with preformed Ixolaris/scaffold complexes followed by addition of recombinant FIX (0.02 U/µL, ~1.2 µM). After 90 minutes at 37°C in a thermocycler, reactions were stopped by addition of Laemmli buffer and boiling. Proteins were separated by 4% to 12% NU-PAGE (MES buffer). Reactants were incubated in 50 mM Hepes, 100 mM NaCl , 5 mM CaCl2, 0.01% BSA. Gels were scanned (Scan Jet 4p) to perform band densitometry.Human umbilical vein endothelial cell culture and generation of FXa by HUVECs Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (San Diego, CA). After trypsinization, cells were grown to confluence in 96-well plates in 200 µL endothelial cell basal medium-2 (EBM-2) supplemented with endothelial cell growth medium (EGM-2) in a humidified incubator at 37°C with 5% CO2. On the day of the experiment, EBM-2 was replaced by 200 µL fresh EBM-2, and lipopolysaccharide (LPS; 10 µg/mL) was added for 4 hours to induce TF expression as described.35 Subsequently, cells were rinsed 3 times with Hepes buffer (10 mM Hepes, 135 mM NaCl, 4 mM KCl, 1 mM MgCl2, 4 mM CaCl2, 11 mM D-glucose, and 0.5% BSA). Hepes buffer was removed and a mixture of 180 µL containing FX (200 nM), and Ixolaris (0-4 nM) previously incubated at 37°C for 15 minutes was added to the cells. This was followed by addition of 20 µL FVIIa (1 nM, final concentration) to start reactions. After 30 minutes, 100 µL was removed and added to 100 µL S2222 (500 µM) diluted in buffer A. Absorbance readings at 405 nm were followed for 1 hour and FXa concentration was estimated using a standard curve.Prothrombin time Ixolaris (0-10 nM) was incubated with prewarmed (37°C) plasma (100 µL), followed by addition of 200 µL prewarmed Thrombomax with calcium reagent (Sigma). Clot formation was detected by visual inspection.Specificity of Ixolaris Ixolaris was preincubated with different enzymes, followed by addition of the appropriate chromogenic substrates as indicated in Table 1.
Statistical analysis, curve fitting, and data handling Data are presented as the mean ± SE, using SigmaPlot 5.0 Graphing software, curve fitting and statistical modes (Jandel Scientific, San Rafael, CA).
A salivary gland cDNA library of the tick I
scapularis was randomly cloned and sequenced, identifying a cDNA
with high similarity to rabbit TFPI. The full-length nucleotide and
deduced amino acid sequences of Ixodes TFPI-like protein are
shown in Figure 1A. The translated
protein has a short hydrophobic sequence of 25 amino acids typical of
signal peptide, according to Signal P software for prediction of
N-terminus of proteins.28 The mature protein, herein
called Ixolaris, contains 140 amino acids (15.7 kd) including 10 cysteines, and a pI of 4.56. Ixolaris is similar to other members of
the Kunitz family of proteins including human TFPI precursor (e value
= 4e
The Ixolaris sequence suggests the existence of a salivary anticoagulant directed toward the extrinsic pathway. Thus, we tested saliva of I scapularis separated by gel filtration in an assay measuring activation of FX by FVIIa/TF. Inhibition of FX activation was detected in fractions eluted between 11 and 17 minutes of retention time (not shown). To determine whether Ixolaris accounts for the inhibitory activity identified in the saliva, the full-length cDNA of Ixolaris was expressed in insect cells as described in "Materials and methods." The concentrated supernatant was applied to a gel-filtration column under conditions identical to those described for saliva and the inhibitory activity was found with the same retention time (not shown). No activity was found with the supernatants of cells transfected with the control plasmid. These data indicate that Ixolaris and the salivary TFPI-like molecule have similar chromatographic properties on gel filtration. To obtain larger amounts of purified Ixolaris, the supernatants of transfected cells were purified by 3 chromatographic steps (see "Materials and methods"). In the final purification step, one single peak at A280 nm was obtained and the corresponding fraction analyzed by PAGE under denaturing and nonreducing conditions. A major band of approximately 24 kd in addition to a minor component of about 15.5 kd were silver stained (not shown). Under reducing conditions, a band of about 27 kd and a minor component of about 15.5-kd were detected (not shown). PAGE of the sample under native conditions shows that only one major band has been stained (not shown). The mechanism of inhibition of blood coagulation by Ixolaris was then
studied. In these experiments, Ixolaris (0-10 nM) and FX (200 nM) were
incubated at 37°C for 15 minutes, followed by addition of FVIIa (1 nM)/TF (0.2 pM) and chromogenic substrate for FXa (S2222). The progress
curves were characterized by a lag phase, followed by significant
hydrolytic increase between 5 and 20 minutes, and a linear augmentation
thereafter showing accumulative production of FXa that was dose
dependently inhibited by Ixolaris (Figure
3A), or recombinant full-length clone
human TFPI (Figure 3A, inset). To estimate the production of FXa at
each minute,
To detect Ixolaris binding to FX and FXa, reactants were incubated in
equimolar concentrations (20 nM) for 15 minutes at 37°C followed by
PAGE under nondenaturing conditions. Proteins were transferred to PVDF
membrane. Experiments were performed using antihuman FX polyclonal
antibodies. Results indicate complex formation between Ixolaris and FX
(Figure 4A) and FXa (Figure 4B). On the other hand, complex formation between Ixolaris and FVIIa was not detected using antihuman FVIIa monoclonal antibody (Figure 4C), even
when 10 times molar excess Ixolaris was used.
Human TFPI inhibits the catalytic site of FXa.8 To
determine whether the binding of Ixolaris to FXa was accompanied by a
change in the amidolytic activity of FXa, inhibitor, and chromogenic substrate (S2222) were incubated for 15 minutes at 37°C, followed by
addition of FXa (125 pM). Ixolaris, at a concentration similar to the
enzyme, instantaneously increased FXa activity (Figure 5A) indicating that it is a
tight-and-fast ligand of FXa, with an effective dose (ED50)
of 159 ± 3.8 pM (Figure 5B). In contrast, full-length human TFPI
behaved as a typical slow-binding inhibitor of FXa (Figure 5A), as
reported.8 Similar results were obtained with des-Gla-FXa
(Figure 5C) whose proteolytic activity increased 1.98-fold in the
presence of Ixolaris (control, 0.4 nM des-Gla-FXa: 1.4 ± 0.12 U
Vmax; 0.4 nM des-Gla-FXa plus 1.6 nM Ixolaris: 2.78 ± 0.1 units of
Vmax; triplicate determination). Similar results were obtained for FXa
but not for thrombin (Figure 5C).
Because both FXa and FX interact with Ixolaris as determined by native PAGE, we determined the relative affinities of Ixolaris for FX and FXa by studying the effects of FX derivatives on Ixolaris-mediated increase of FXa amidolytic activity. Figure 5D shows that when Ixolaris was added to and incubated with a preformed mixture of FXa and increasing concentrations of DEGR-FXa, or des-Gla-DEGR-FXa, followed by addition of S2222, increase of FXa amidolytic activity by Ixolaris was inhibited. However, this effect was not observed when Ixolaris was added to a mixture containing FXa and increasing concentrations of FX (up to 16 nM; see "Discussion"). When Ixolaris was preincubated with FX, DEGR-FXa, or des-Gla-DEGR-FXa and S2222, followed by addition of FXa, inhibition of Ixolaris-mediated increase of FXa amidolytic activity was attained in all cases with comparable IC50s (Figure 5D, inset; see "Discussion"). It has been shown that human TFPI at high concentrations blocks the
catalytic activity of FVIIa/TF, inhibition being remarkable in the
presence of FXa.1 To test the effects of Ixolaris only, or
Ixolaris in the presence of DEGR-FXa in the catalytic activity of
FVIIa/TF, amidolytic assays were performed using chromogenic substrate
(S2288). Figure 6A shows that Ixolaris at
high concentrations (0-4.8 µM) dose dependently inhibits FVIIa/TF
amidolytic activity. However, low concentrations of Ixolaris (5 nM) in
the presence of DEGR-FXa (0-5 nM), effectively blocked FVIIa/TF
amidolytic activity (Figure 6B). A transformation of the data as Vs/Vo
yields an IC50 of 0.41 ± 0.04 nM; an almost complete
(> 95%) inhibition was observed at DEGR-FXa concentration of 1 nM
(Figure 6C). The finding that Ixolaris inhibits FVIIa/TF activity in an
FXa-dependent manner was confirmed by increasing the concentration of
Ixolaris (0-5 nM) at one concentration of DEGR-FXa (5 nM; Figure 6D);
this result is compared with Ixolaris only (up to 4.8 µM; Figure 6D).
To study the effects of FX and FXa as scaffolds for Ixolaris in the
inhibition of FVIIa/TF, FIX activation assays33 were performed. This assay is particular useful for these studies because FIX is a physiologic substrate for FVIIa/TF.24,25 To
completely rule out the presence of contaminating coagulation factors
that could operate as scaffolds for Ixolaris in this assay, experiments were performed with recombinant FIX34 (BeneFIX). FIX
activation proceeds through two consecutive steps33 and is
dependent on both catalytic site and FVIIa/TF
exosite.37-40 In the first step, FIX (~62 kd, apparent
molecular weight) cleavage at the Arg145-Ala146 peptide bond generates
the intermediate product termed FIX
In another set of experiments, we have attempted to characterize the
kinetics of the interaction between FVIIa/TF and Ixolaris/FXa as slow
or fast, by means of 2 independent approaches. Accordingly, Figure
8A shows that when FVIIa/TF was added to
a mixture containing preincubated Ixolaris/DEGR-FXa and S2288, a
partial and typical slow-type inhibition was attained. We have also
tested the macromolecular substrates FIX, in reactions initiated by
FVIIa/TF. Accordingly, when FVIIa/TF was added to a mixture containing
preincubated Ixolaris/DEGR-FXa and FIX, complete inhibition was
observed (Figure 8B). Identical results were obtained with DEGR-FX
(Figure 8C; see "Discussion").
In an attempt to study the effects of Ixolaris in a cell-mediated
initiation of blood coagulation,35 HUVECs were stimulated to express TF after 4 hours of incubation with LPS. Then a mixture containing FX (200 nM) and Ixolaris (0-4 nM) previously incubated for
15 minutes was added to the cells, followed by addition of FVIIa (1 nM). After 30 minutes, an aliquot was taken to measure FXa using
chromogenic substrate for FXa (S2222). Figure
9 shows that Ixolaris dose dependently
inhibits FXa production, with an IC50 of about 500 pM.
Table 1 shows that Ixolaris is a specific inhibitor for FVIIa/TF, and
does not affect the catalytic activity of other enzymes. We have also
tested the effects of Ixolaris on the prothrombin time and no
inhibition was detected (control, 12.6 ± 0.3 seconds; Ixolaris up to
10 nM, 12.3 ± 0.3 seconds). Figure
10 shows a model proposing the
mechanism of action of Ixolaris (see figure legends).
The recombinant tick salivary protein, called Ixolaris in this paper, potently inhibits FVIIa/TF-induced FX activation with an IC50 in the picomolar range. Ixolaris is functionally and structurally distinct from its endogenous counterpart, human TFPI1; although the 6 cysteines that characterize the first Kunitz domain of TFPI1 are conserved in Ixolaris, only 4 of 6 cysteines present in the second Kunitz domain of human TFPI are present.6,7 Also, whereas the sixth and the first cysteines that, respectively, terminate and initiate the first and second Kunitz domains in human TFPI are separated by 20 amino acids,6,7 only 7 amino acids separate the corresponding cysteines in Ixolaris. Additionally, the Kunitz-type domain 2 in Ixolaris is unusual by containing 4 additional amino acids between the fourth and fifth cysteine residues, making this loop longer than most Kunitz-type family members. Also, the presumed P1 reactive-site residue of the first domain in Ixolaris is Glu, whereas Lys occupies this position in TFPI.6,7 Finally, Ixolaris has a short and basic carboxy terminus but, unlike TFPI, it has only 14 amino acids where the positively charged amino acids are not organized as a cluster. In human TFPI, this basic carboxy terminus has been consistently shown to increase its anticoagulant activity41 and to shorten its half-life.42,43 The Ixolaris cDNA also encodes 3 putative N-linked glycosylation sites, at Asn65, Asn98, and Asn136. Consistent with a calculated mass of 15.7 kd for the carbohydrate-free protein, we could detect a band of about 15.5 kd in the gels loaded with recombinant Ixolaris; however, an intense smear was observed in PAGE of Ixolaris at a molecular weight range of about 24 kd. Accordingly, it is likely that these Asn residues are indeed glycosylated, and this is the most abundant form (> 95%) of the secreted recombinant molecule. It remains to be determined whether both forms of Ixolaris are equally effective as anticoagulants. Ixolaris and TFPI are unrelated in many functional aspects.
Ixolaris is a highly specific inhibitor that, unlike human TFPI, does
not inhibit trypsin or chymotrypsin.44 Ixolaris
immediately increases the amidolytic activity of FXa toward chromogenic
substrates (Figure 5A); therefore, it behaves as a fast ligand of FXa,
in contrast to TFPI, a typical FXa slow-binding
inhibitor.8 Additionally, Ixolaris binds to des-Gla-FXa
and to a tripeptidyl chloromethylketone covalently occupied catalytic
site of FXa (DEGR-FXa), 2 properties not shared by
TFPI.2,10 This indicates that Ixolaris binds at a site
distinct from the active center of FXa, or exosite,45-47 and implies that It has been shown that TFPI at high concentrations (µM range) inhibits FVIIa/TF amidolytic activity, inhibition being remarkable in the presence of FXa.1,2 Figure 6A shows that Ixolaris at high concentrations also blocks the amidolytic activity of FVIIa/TF. This indicates that the inhibitor interacts directly with the active site of the enzyme or sterically prevents access of the substrate to the active site. As described for TFPI,6,7 we suggest that Ixolaris interaction with FVIIa/TF occurs via its first Kunitz domain that is typical11 for catalytic site recognition. Because this effect occurs at [I] >>> [E], Ixolaris does not behave as a tight inhibitor of FVIIa/TF, a contention that is in accordance with no complex formation being detected between Ixolaris and FVIIa in native gel experiments (Figure 4). Remarkably, in the presence of Ixolaris/DEGR-FXa, a stoichiometric blockade of FVIIa/TF amidolytic activity was achieved at [I] ~ [E] (Figure 6). It is also clear from the FIX activation assays that both FX zymogen (FX) and enzyme (FXa) operate as efficient scaffolds for the inhibitor, with formation of a tight complex composed by FVIIa/TF/Ixolaris/FX(a). The finding that Ixolaris binds to the zymogen, and this complex inhibits FVIIa/TF, is one of the most remarkable observations of this paper and it clearly distinguishes this inhibitor from TFPI.1,2 This inhibitory strategy seems to be especially effective because Ixolaris/FX may inhibit FVIIa/TF in vivo before and independently of FXa production. Although the true Ki of Ixolaris/scaffolds for FVIIa/TF and the type of inhibition (competitive versus noncompetitive) remain to be determined, relative affinities are comparable as determined in Figure 7. On the other hand, des-Gla-DEGR-FXa was shown to be a completely ineffective scaffold in both amidolytic (not shown) and FIX activation assays (Figure 7). This indicates that the anionic Gla domain of FX(a), known to play a crucial role in the interaction of FXa with procoagulant membrane surface50 and TF,39 is directly involved in Ixolaris/FX(a) interactions with FVIIa/TF complex. To study the kinetics of Ixolaris/FXa interaction with FVIIa/TF, 2 independent and complementary approaches have been used. Accordingly, when FVIIa/TF was used to initiate reactions containing Ixolaris/FXa and small chromogenic substrate, a typical slow-type inhibition was attained (Figure 8A). It is likely that the interaction of the (first) Kunitz domain of Ixolaris/FXa with FVIIa/TF requires an induced fit to dock productively to the enzyme catalytic site. However, and notably, production of FXa was immediately blocked when FVIIa/TF was added to a mixture containing Ixolaris/FX and FX (Figure 3B), characterizing a fast-type inhibition. On the other hand, TFPI was shown in the experiments described here (Figure 3B, inset) and elsewhere,9 to behave as a slow-type inhibitor in similar experimental conditions. Of interest, our results have also demonstrated that FIXa generation by FVIIa/TF was completely prevented when enzyme/cofactor was added to a mixture containing Ixolaris/FX(a) and FIX (Figure 8B,C). Although FIX activation assays do not allow us to estimate FVIIa/TF inhibition by Ixolaris/scaffold in the first seconds of the assay, these results suggest that enzyme activity was blocked very shortly after the initiation of the reactions. Accordingly, it is plausible to suggest that interactions of Ixolaris/FX(a) with FVIIa/TF can be better described as fast when macromolecular (FX or FIX), instead of small chromogenic substrates (S2288), are appropriately used to determine the kinetics of the interaction involving enzyme and inhibitor. Presumably, this step is mediated by the exosite formed by FVIIa/TF39 and the exosite recognition domains from both FX and FXa37,38; this interaction is strengthened by the docking of Ixolaris to the catalytic site of FVIIa/TF (Figure 8A), as described for bifunctional protein.51,52 Of interest, it has been recently proposed that the exosite formed by FVIIa/TF37-39 plays a predominant role in determining the affinity and kinetics of the interaction with FX40 (Km ~200 nM).24,40 Because the affinity of Ixolaris/FX for FVIIa/TF is significantly higher (pM range), it should operate as an effective anticoagulant in vivo even if only a fraction of FX has bound Ixolaris. In fact, due to the high affinity interaction, inhibitor may form a stable complex with the zymogen in the blood; this inhibitory complex is effective under conditions such as coagulation initiated by HUVEC-expressing TF (Figure 9). Similar conclusions have been obtained for NAPc2 that differ from Ixolaris, however, in a number of structural and kinetic aspects.32,53 In fact, NAPc2 is an 84-amino acid non-Kunitz molecule with an FXa cleavage site32 that is absent in Ixolaris. Finally, novel molecules that block initiation of blood coagulation, such as Ixolaris, may be useful as inhibitors to prevent or ameliorate a number of pathologic conditions leading to cardiovascular diseases provoked or amplified by abnormal expression of TFPI.54-62
We are grateful to Drs Robert W. Gwadz, Thomas J. Kindt, and Louis H. Miller for encouragement and support. The authors thank Brenda Rae Marshall for editorial support.
Submitted December 13, 2001; accepted January 16, 2002.
Prepublished online as Blood First Edition paper, April 17, 2002; DOI 10.1182/blood-2001-12-0237.
This work was presented at the 42nd Annual Meeting of the American Society of Hematology, December 1-5, 2000, San Francisco, CA.
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: José M. C. Ribeiro, SME, LPD, NIAID, NIH, 4 Center Dr, Rm 4/126, MSC 0425, Bethesda, MD 20892-0425; e-mail: jribeiro{at}nih.gov.
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Top
Abstract
Introduction
-carboxyglutamic
acid domainless FXa (des-Gla-FXa), increasing its amidolytic activity.
At high concentration, Ixolaris attenuates the amidolytic activity of FVIIa/TF; however, in the presence of DEGR-FX or DEGR-FXa (but not
des-Gla-DEGR-FXa), Ixolaris becomes a tight inhibitor of FVIIa/TF as
assessed by recombinant factor IX (BeneFIX) activation assays. This
indicates that FX and FXa are scaffolds for Ixolaris in the inhibition of FVIIa/TF and implies that the Gla domain is necessary for
FVIIa/TF/Ixolaris/FX(a) complex formation. Additionally, we show that
Ixolaris blocks FXa generation by endothelial cells expressing TF.
Ixolaris may be a useful tool to study the structural features of
FVIIa, FX, and FXa, and an alternative anticoagulant in cardiovascular diseases.
(Blood. 2002;99:3602-3612)
IIb
3-integrin antagonist
) 3 nM; (
) 15 nM; (
) 40 nM; (
) 75 nM; (
) 100 nM; (
)
140 nM); (
) 175 nM; (
) 200 nM (n = 3). Inset: Vs and Vo were
plotted against TFPI concentration at different FX concentrations:
(
