(Received for publication, April 3, 1995; and in revised form, September 6, 1995)
From the
Triabin, a new thrombin inhibitor, has been purified from the
saliva of Triatoma pallidipennis, a blood-sucking triatomine
bug. It forms a noncovalent complex with thrombin at a molar ratio of
1:1, inhibits thrombin-induced platelet aggregation, and prolongs
thrombin clotting time and activated partial thromboplastin time.
However, it only minimally suppresses the amidolytic activity of
thrombin, as measured by a chromogenic peptide substrate assay. It
completely blocks trypsin-catalyzed cleavage of thrombin, probably via
protection of the anion-binding exosite and inhibits the effect of
thrombomodulin on thrombin in a dose-dependent fashion. These results
indicate that the inhibitor is directed toward the anion-binding
exosite of thrombin. The protein was partially sequenced and the
information used to isolate cDNA clones from a T. pallidipennis salivary gland library. Four slightly polymorphic variants coding
for mature proteins of 142 amino acids preceded by a putative leader
sequence were obtained. The recombinant protein expressed in the
periplasmic space of Escherichia coli has a biological
activity similar to that of salivary triabin, as tested in a
thrombin-induced platelet aggregation assay. In addition, recombinant
triabin inhibits thrombin-catalyzed hydrolysis of fibrinogen with a K of about 3 pM.
Evolution has provided hematophageous animals with specific mechanisms to interfere with blood coagulation. Factor Xa inhibitors were isolated from the leech Haementeria officinalis(1) and the tick Ornithodoros moubata(2) . Likewise, several proteins which inhibit thrombin have been described. The most widely known is hirudin, which was identified almost 40 years ago in the leech Hirudo medicinalis(3) . Thrombin is a key enzyme in the blood coagulation cascade and a strong inducer of platelet aggregation. Three functional domains of thrombin have been described: an active site, a heparin-binding site, and a so-called anion-binding exosite mediating the binding to fibrinogen(4) , the platelet receptor(5) , and thrombomodulin(6) .
The triatomine bug Rhodnius prolixus has been reported to contain a Kazal-type inhibitor which seems to be specifically directed to the active site of thrombin(7) . We investigated the related triatomine bug Triatoma pallidipennis, which also belongs to the family of Reduviidae or assassin bugs, and recently described the purification, cloning, and expression of a protein called pallidipin which specifically inhibits collagen-induced platelet aggregation(8) . Now we have identified a thrombin inhibitor in the saliva of this insect and report its purification and characterization, and the cloning and expression of the corresponding cDNA. In contrast to the inhibitor from R. prolixus, the protein we isolated from T. pallidipennis and called triabin appears to be directed at the anion-binding exosite of thrombin.
Aggregation of washed platelets was measured as described earlier(8) . Briefly, thrombin (human from Sigma, 1 nM) and triabin or hirudin preincubated for 1 min at 37 °C were added to washed platelets and aggregation was monitored in an aggregometer (Micron).
To assay amidolytic activity, 20 µl of thrombin (human from Sigma, 45 pM final concentration) were incubated with 80 µl of 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 100 µl of inhibitor or buffer for 2 min at 37 °C in microtiter plates followed by the addition of 100 µl of H-D-Phe-Pip-Arg-p-nitroaniline (S2238, Kabi Vitrum, 0.5 mM). The absorbance at 405 nm was recorded for 60 min in a microplate reader and the initial rate of p-nitroaniline liberation determined.
For thrombin clotting time assays, 100 µl of thrombin (Behring, 3 IU/ml) and 100 µl of inhibitor or diethylbarbiturate acetate buffer (Behring) were incubated for 1 min at 37 °C. After addition of 100 µl of plasma (control plasma N from Behring), the time until a clot formed was measured. The same method was used to determine reptilase activity but 200 µl of reptilase (Sigma, 5 µg/ml) was added instead of 100 µl of thrombin.
To determine activated partial thromboplastin time (APTT),
100 µl of pooled citrated human plasma, 10 µl of inhibitor, and
100 µl of APTT reagent (Pathromtin, Behring, Germany) were
incubated for 3 min at 37 °C. After addition of 100 µl of
prewarmed 25 mM CaCl, the time until a clot formed
was measured in a fibrometer (Sarstedt Biomatic B10).
To measure factor Xa and trypsin activities, triabin was incubated with factor Xa (American Diagnostica Inc.) or with trypsin (Sigma) up to a molar ratio of 43:1 for 2 min at 37 °C followed by the addition of N-benzoyl-Ile-Glu-Gly-Arg-p-nitroaniline (S2222, Kabi Vitrum, final concentration 0.167 mM). After 20 min at 37 °C, the absorption at 405 nm was measured and compared to the control value without triabin.
Protein C activation was measured in
96-well plates in an assay diluent (20 mM Tris-HCl, pH 7.4,
0.1 M NaCl, 0.1% NaN, 0.5% bovine serum albumin,
2.5 mM CaCl
) as described previously(14) .
Triabin, thrombomodulin (35 ng/ml, kindly provided by Dr. John Morser,
Berlex Biosciences), thrombin (Sigma, 0.44 units/ml), and protein C
(Genzyme, 3 µM) were incubated for 1 h at 37 °C. The
reaction was quenched with hirudin (American Diagnostica Inc., 0.16
units/ml) and 100 µl of D-Val-L-Leu-L-Arg-p-nitroaniline
(S2266, Kabi Vitrum, 1 mM) were added. The absorption at 405
nm was recorded for 15 min in a microplate reader.
The saliva of T. pallidipennis was found to inhibit not only platelet aggregation induced by collagen but also aggregation mediated by thrombin. These two inhibitory activities were separated by gel filtration although the two proteins have similar molecular masses. This was achieved by using a Superose 12 column with a buffer containing only low salt concentrations which led to a retardation of pallidipin but not of the thrombin inhibitor. The thrombin inhibitor was further purified by affinity chromatography on thrombin Sepharose. 11 µg of purified inhibitor, which we named triabin, were recovered from 10 mg of dried saliva. The purity of this preparation was ascertained by SDS-polyacrylamide electrophoresis, which showed a single band with an apparent molecular mass of 21 kDa under reducing conditions and 18 kDa under nonreducing conditions (Fig. 1, lanes 7 and 8).
Figure 1: Polyacrylamide gel electrophoresis of triabin and thrombin. Thrombin (lane 1), triabin (lane 2), thrombin and triabin in a molar ratio of 1:1 (lane 3), and thrombin and triabin in a molar ratio of 1:1.5 (lane 4) were electrophoresed on a nondenaturing 15% polyacrylamide gel. SDS-polyacrylamide gel electrophoresis was performed with thrombin (lane 5), triabin and thrombin in a molar ratio of 1:1 (lane 6) and triabin (lane 7) after reduction. Nonreduced triabin was also analyzed (lane 8). Molecular mass standards are given in kDa. Proteins were stained with Coomassie Brilliant Blue.
In order to investigate the triabin-thrombin complex, we incubated triabin with thrombin at different molar ratios and analyzed the probes by nondenaturing polyacrylamide gel electrophoresis. As shown in Fig. 1, the inhibitor was totally bound in a complex with thrombin when the molar ratio of triabin to thrombin was 1:1, whereas free triabin was detected at a ratio of 1.5:1 (lanes 3 and 4). This complex was not covalently linked since it could be separated by SDS-polyacrylamide gel electrophoresis (lane 6).
Triabin inhibited thrombin-induced platelet aggregation in a dose-dependent fashion (Fig. 2). If platelet aggregation was induced by 1 nM thrombin, total inhibition was observed at 1 nM triabin. The dose-response curve was almost identical to that of hirudin. In contrast, the COOH-terminal tail of hirudin, hir(54-65), also called hirugen, exhibited a nearly 500 times lower activity (Fig. 2). No effect of triabin was noted on the aggregation induced by collagen or ADP (not shown). Triabin inhibited not only platelet aggregation but also interfered with the other known activities of thrombin. A 4-fold prolongation of thrombin-clotting time was measured with 22 nM triabin (Fig. 3). Clotting of plasma can also be catalyzed by reptilase, an enzyme from the snake venom of Bothrops atrox(22) . Triabin did not inhibit this enzyme (data not shown). Activated partial thromboplastin time was prolonged 4-fold by about 600 nM triabin and about 400 nM hirudin. While hirudin already showed an effect at 10 nM, triabin was only active at concentrations higher than 500 nM (Fig. 3). In contrast to its strong inhibitory effect in the assays described above, triabin hardly affected thrombin activity in an amidolytic assay using a synthetic peptide substrate (Fig. 4). Only a 35% inhibition was achieved with a 3-fold molar excess of triabin. Higher concentrations up to a 67-fold molar excess did not lead to an increase of this inhibitory effect. This is in contrast to hirudin, which totally blocked the amidolytic activity of thrombin at low concentrations (Fig. 4). Other serine proteases, e.g. trypsin and factor Xa, were not inhibited in an amidolytic assay even when using a 45-fold molar excess of triabin (data not shown).
Figure 2: Inhibition of thrombin-induced platelet aggregation. Thrombin (1 nM) preincubated for 1 min at 37 °C with triabin (solid circles), recombinant triabin (crosses), hirudin (open circles), or hirugen (triangles) was added to washed platelets and maximum aggregation was measured. Results are expressed as percentages of control measurements using buffer alone.
Figure 3:
Inhibition of thrombin clotting time and
APTT. Thrombin preincubated with triabin for 1 min was added to human
plasma and the time until a clot formed was measured (triangles). Human plasma, APTT reagent and triabin (solid
circles), or hirudin (open circles) were preincubated
before starting the reaction with CaCl and measuring the
APTT.
Figure 4: Effect of triabin and hirudin on the amidolytic activity of thrombin. Thrombin (45 pM) was preincubated with triabin (solid circles) or hirudin (open circles) for 1 min before adding S2238. The absorbance at 405 nm was monitored. Data are presented as the percent of thrombin amidolytic activity without inhibitors.
These results demonstrate that triabin is specific for thrombin. The difference in its inhibitory activity using either fibrinogen or a small synthetic peptide as substrate indicates that it may exert its inhibitory effect by binding to the anion-binding exosite rather than to the catalytic site of thrombin.
Limited trypsin
proteolysis converts -thrombin into
-thrombin through
cleavage of the Arg
-Asn
bond within the
anion-binding exosite(23) . Preincubation of thrombin with
triabin prevented the cleavage by trypsin as analyzed by
SDS-polyacrylamide gel electrophoresis (data not shown). Since triabin
did not inhibit trypsin, the effect was probably achieved by binding of
triabin to the exosite of thrombin.
Thrombomodulin is known to exhibit its effect on thrombin by binding to the anion-binding exosite(6) . The resulting change of substrate specificity enables thrombin to activate protein C. This activity can be tested by incubating thrombomodulin and thrombin with the inactive preform of protein C. This leads to activation of protein C which is detectable by its amidolytic activity. Triabin neither showed a thrombomodulin-like activity, i.e. it did not modulate thrombin, nor did it activate protein C. However, it was able to antagonize the effect of thrombomodulin in the protein C activation assay in a dose-dependent manner (Fig. 5). This observation further corroborates our hypothesis that triabin binds to this exosite of thrombin.
Figure 5: Protein C activation assay. Triabin, thrombomodulin, thrombin, and protein C were incubated for 1 h at 37 °C. After quenching the reaction with hirudin, S2266 was added and the generation of p-nitroaniline was monitored. Results are expressed as percentages of protein C activity without inhibitor.
Twenty
amino-terminal amino acids of triabin were sequenced and additional
internal sequences were determined after cleavage with endoproteinase
Lys-C and separation of the fragments by high performance liquid
chromatography. Degenerate primers were made based on the protein
sequence information and used to amplify a specific tag from T.
pallidipennis salivary gland first-strand cDNA. The probe was
employed to screen approximately 1 10
primary phage
clones from a T. pallidipennis salivary gland cDNA library.
DNA sequencing revealed that four different types of triabin cDNA had
been isolated (Tr5, Tr12, Tr28, and Tr45). The clone with the longest
insert is depicted in Fig. 6. It exhibited an open reading frame
potentially coding for a 160-amino acid long protein. Hydrophilicity
analysis showed that the 18 NH
-terminal residues displayed
the hallmarks characteristic of a signal peptide(24) . The
following position was occupied by the alanine residue representing the
NH
terminus of triabin isolated from T. pallidipennis saliva. Mature triabin is therefore a 142-amino acid long protein
with a predicted relative molecular mass of 15,948, slightly less than
that of the saliva protein which is probably glycosylated. Indeed a
potential N-glycosylation site was detected in the deduced
amino acid sequence. The stretches previously determined by protein
sequencing were retrieved in the amino acid sequence deduced from the
cDNA but two discrepancies were noted at positions 8 and 77 where
leucine and aspartic acid found in the protein were replaced by
isoleucine and glycine, respectively. The untranslated region
downstream of the open reading frame contained a sequence almost
exactly matching a polyadenylation signal (25) in the
appropriate position upstream of a poly(A) tail.
Figure 6: cDNA and deduced protein sequences of triabin. The insert sequence of the Tr5 clone is shown. The vertical arrow points to the cleavage site between leader peptide and mature protein. The potential N-glycosylation site is underlined twice. The stretches previously determined by protein sequencing after Lys-C cleavage are highlighted with a dashed line. The nine amino acid positions where variations were found are underlined. The polyadenylation signal is overlined. Converging arrowheads delimitate the polymerase chain reaction fragment used for the screening.
In addition to Tr5, cDNAs coding for three variant forms of triabin were also isolated. The deduced mature proteins had the same length but differed slightly in their sequence. The degree of conservation was, however, very high since there was over 94% amino acid sequence identity between the mature forms. In total, there were only 9 positions where all four proteins were not identical and in four of these positions the amino acid exchange was conservative (Tr12: 8L, 77D, 86G, 114I, 127F, and 139N; Tr28: 8L 32D, 77D, 86G, 114I, 127F and 139N; Tr45: 8L, 32D, 72A, 74K, 77D, 86G, 127F, and 139N). It can therefore be assumed that the biological function of the variants is identical. Unlike the product of Tr5, the three additional forms exactly matched the partial sequences directly determined on the natural salivary protein. A search in the SwissProt data base did not reveal any significant homology between triabin and other known proteins (the best score was 19% identity for an 83-amino acid long overlap).
A periplasmic expression construct
based on the pKK233 plasmid and harboring the
isopropyl-1-thio--D-galactopyranoside-inducible trc promoter was devised. The coding sequence of mature triabin was
fused to the signal peptide coding part of secreted bacterial
cyclophilin, modified so as to optimize the leader peptidase cutting
site(18, 26) . The Tr28 cDNA was selected for these
studies because the protein it encoded showed no difference to the
protein sequence data and most closely corresponded to a consensus
triabin sequence inferred from the four variants. The periplasmic
fraction of transformed E. coli was prepared. The fibrinogen
clotting test indicated the presence of an inhibitory activity in this
fraction, which was not observed with mock-transformed bacteria.
Recombinant triabin was purified using a two-step procedure
involving a separation on DE-52 cellulose and affinity chromatography
on a thrombin-Sepharose column. The purity of the protein was verified
by SDS-polyacrylamide gel electrophoresis and a single band
corresponding to a molecular mass of about 18 kDa, comparable to that
of saliva triabin was observed (not shown). The identity was further
checked by determination of the NH-terminal sequence and
the protein was found to start with the expected alanine residue,
indicating that correct processing had taken place.
The biological
activity was evaluated in a platelet aggregation assay (Fig. 2).
A dose-response study showed that recombinant triabin inhibited
aggregation induced by 1 nM thrombin in a dose-dependent
manner, the IC being 0.82 nM. A kinetic analysis
of thrombin-catalyzed fibrinogen cleavage was performed by measuring
fibrinopeptide A release, and the K
was found to
be 14.0 ± 3.7 µM. The inhibitory effect of
recombinant triabin was assessed in this test, and the K
measured. Since in this analysis, the amount of inhibitor bound
by the enzyme was no longer negligible, the graphical method of Dixon (21) was used as it avoids the assumption that the total
concentration of inhibitor is equal to the theoretically required free
concentration and is therefore also applicable for tight-binding
inhibitors. The K
was found to be 3 pM (Fig. 7).
Figure 7:
Effect of triabin on the steady-state
velocity of thrombin. Assays were performed with 75 pM thrombin and 10 µM fibrinogen as described under
``Experimental Procedures.'' Using the graphical method of
Dixon(21) , the K was
determined.
Blood-sucking insects employ various mechanisms to interfere
with the blood coagulation of their host species. The saliva of T. pallidipennis inhibits collagen-induced platelet
aggregation by pallidipin (8) and ADP-mediated aggregation by
an apyrase. ()Now we report a third anticoagulatory
mechanism, thrombin inhibition by a protein which we named triabin.
Triabin forms a complex with thrombin, probably in a molar ratio of
1:1. This complex is noncovalent, hence it is formed via a mechanism
different from the physiological heparin-antithrombin III-thrombin
interaction. Other inhibitors from hematophageous animals also form
noncovalent complexes with thrombin but they differ in their mechanism
of action. Hirudin binds to the active site and with its COOH-terminal
tail to the anion-binding exosite(27) , whereas rhodniin seems
to affect mainly the active site(7) . Bothrojaracin, isolated
from the venom of Bothrops jararaca(28) , exhibits its
inhibitory effect exclusively via binding to the anion-binding site.
All these inhibitors interfere with thrombin's activity on
fibrinogen and on the platelet receptor. Whereas triabin has the same
high inhibitory activity as hirudin, it is about 500 times more potent
than hirugen, the COOH-terminal tail of hirudin. Compared to
bothrojaracin(28) , the IC values for inhibition
of platelet activation are 7 times lower for triabin. In contrast to
the active site inhibitors, proteins specific for the anion-binding
exosite should not affect the cleavage of chromogenic peptide
substrates, which only interact with the active site. Indeed,
bothrojaracin and hirugen do not decrease the amidolytic activity of
thrombin. However, triabin partially suppresses this activity. Since
this effect is low compared to its inhibitory activity on fibrinogen
clotting and platelet aggregation, it is conceivable that it binds only
to the anion-binding exosite. One explanation for the only partial
inhibition of amidolytic activity may be that triabin sterically
hinders the access of small peptides to the active site. Another
possibility is that the binding of triabin to thrombin changes the
conformation of the enzyme, resulting in lower activity. Proteolysis of
thrombin within the anion-binding exosite also causes an inhibition of
fibrinogen cleavage and a small decrease of the affinity to peptide
substrates(6) . Hofsteenge et al.(6) suggested that the proteolysis leads to a small
conformational change in the thrombin molecule which affects the
peptide-binding residues.
Limited proteolysis of -thrombin by
trypsin results in the cleavage of the Arg
-Asn
bond, which is located in the anion-binding exosite, and
generates
-thrombin(23) . Triabin was able to protect this
site against cleavage by trypsin. Since a direct inhibition of trypsin
was excluded, it is probably the binding of triabin to this site which
is responsible for this effect. Similar experiments were performed with
hirudin and its COOH-terminal fragment, which likewise inhibited the
cleavage(29) , and x-ray diffraction analysis showed that
indeed these peptides bind to the anion-binding exosite(30) .
These results lend additional support to our hypothesis that triabin
binds to thrombin in a similar manner.
Binding of thrombomodulin to
thrombin causes a change in the specificity of the protease. Hofsteenge et al.(31) demonstrated that thrombomodulin inhibits
the binding of fibrinogen to thrombin but hardly affects the binding of
peptide substrates. Cleaving -thrombin at the
Arg
-Asn
bond reduces its affinity to
thrombomodulin(6) . Additionally, an antibody binding to
residues 62-73 of thrombin inhibits the activation of protein C
by
-thrombin in the presence of thrombomodulin(6) . These
results indicate that the region around Arg
, the
anion-binding exosite, is necessary for the interaction with
thrombomodulin. Therefore, it was of interest to investigate the effect
of triabin on this interaction as well. Indeed, triabin inhibited in a
dose-dependent manner the thrombomodulin-mediated activation of protein
C by thrombin. This result further corroborates the hypothesis that
triabin binds to the anion-binding exosite.
Four different cDNA clones with minor polymorphisms that probably do not affect the biological function of triabin were isolated. This diversity might reflect the existence either of a protein family or more likely of a genetic heterogeneity among the insects from which the salivary glands were collected. No sequence homology was found between triabin and the thrombin inhibitors hirudin and rhodniin. This is in keeping with the different modes of action of these proteins.
A vector for periplasmic expression was used for the production of triabin, in order to favor correct folding of the protein and facilitate the subsequent purification. Our results show that in both the fibrinogen clotting test and the thrombin-induced platelet aggregation assay, the biological activity of recombinant triabin purified from the periplasm of E. coli was similar to that of saliva triabin, suggesting that the protein has assumed its natural configuration.
The
inhibition constant of 3 pM, which was measured in the
thrombin-catalyzed fibrinogen cleavage assay, indicates a very tight
binding of triabin to thrombin. For the hirudin-thrombin complex,
dissociation constants of 63 pM(32) , 3
pM(33) , and 20 fM(34) have been
cited in the literature. All these values were determined with an
amidolytic substrate which could not be used for this study because
triabin does not seem to block the active site. However, the value for
triabin agrees well with the K found by Dodt et al.(33) . In contrast, the COOH-terminal hirudin
peptide Hir
, which, like triabin, recognizes the
anion-binding site of thrombin, has been shown to be less active in the
thrombin-catalyzed hydrolysis of fibrinogen, the K
being 144 nM(35) .
In summary, we have purified a thrombin inhibitor with a potency comparable to hirudin but with a different mode of action. The availability of recombinant triabin will now allow us to further explore its mechanism of action and carry out structure-function studies using site-directed mutagenesis in order to identify the structural elements involved. X-ray diffraction analysis should provide detailed information on the structure of the triabin-thrombin complex. Furthermore, in vivo models will be tested to assess the effectiveness of triabin as an antithrombotic.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X80246[GenBank].