From the Division of Molecular Pathology, Department
of Neuroscience and Immunology, Kumamoto University Graduate School of
Medical Sciences, Kumamoto 860, Japan, the § Department of
Microbiology, Institute of Molecular Biology, Jagiellonian University,
31-120 Kraków, Poland, ¶ Abteilung
Strukturforschung, Max-Planck-Institut für Biochemie,
Martinsried, D-82152 Germany, and the ** Department of Biochemistry and
Molecular Biology, University of Georgia, Athens, Georgia 30602
Received for publication, July 27, 2000, and in revised form, March 13, 2001
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ABSTRACT |
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The effect of 95- (HRgpA) and 50-kDa gingipain R
(RgpB), arginine-specific cysteine proteinases from
periodontopathogenic bacterium Porphyromonas gingivalis on
human prothrombin activation was investigated. Each enzyme released
thrombin from prothrombin in a dose- and time-dependent
manner with the former enzyme, containing adhesion domains, being
17-fold more efficient than the single chain RgpB. A close correlation
between the generation of fibrinogen clotting activity and amidolytic
activity indicated that Blood coagulation is an important defense system, protecting the
body against blood loss from injured vessels. The process is initiated
by the binding of factor VII to tissue factor (1), present in tissues
surrounding vessels (2), followed by proteolytic activation of plasma
coagulation factors in a cascade pathway (3-5). Thrombin, the ultimate
product of these reactions, is an extremely potent platelet activator
(6, 7) and converts fibrinogen to a fibrin clot (8), thus plugging
damaged vessels. Besides its central role in hemostasis, thrombin also
enhances vascular permeability (9), induces leukocyte chemotaxis (10, 11), and potentiates lipopolysaccharide-stimulated interleukin-1 production by macrophages (12). These data, and the fact that prothrombin activation in vivo is known to be associated
with inflammatory conditions, implicate thrombin as a major player in inflammation.
The deposition of fibrin is a common feature at the site of bacterial
infection (13). Endotoxin can induce fibrin accumulation in
vivo through the Shwartzman reaction (14), presumably by activating monocytes to express tissue factor (15). For this reason it
is recognized as the component primarily responsible for blood
coagulation associated with bacterial infections. Proteinases from such
foreign sources are also thought to be virulence factors involved in
various inflammatory events occurring at infected sites (16). For
example, many of these enzymes present in snake venoms are known to
activate prothrombin (17, 18); however, whereas bacterial proteinases
may be able to convert prothrombin to thrombin, such a process has not
been studied in detail.
A close relationship between Porphyromonas gingivalis
(formerly Bacteroides gingivalis) and adult periodontitis
has been reported (19-21), with proteolytic enzymes that are known to
be produced in large quantity by this microorganism and have been shown
to act as important pathogenic agents (22-24). From the culture medium of P. gingivalis HG66 we have purified previously two major
forms of arginine-specific cysteine proteinases,
HRgpA1 and RgpB, formerly
referred to as high molecular mass gingipain R (95-kDa gingipain R1 or
HRGP) and 50-kDa gingipain R2 (RGP-2), respectively (24, 26). Both of
these enzymes are products of two distinct but related genes (27).
rgpA encodes a polyprotein which, after post-translational
processing/modifications, yields three different forms of the enzyme
(28, 29); the major one is a non-covalent complex containing separate
catalytic and adhesion/hemagglutinin domains (HRgpA). In contrast, the
fragment encoding the latter domain(s) is missing in the
rgpB gene structure, and its translation product is a single
protein with a primary structure essentially identical to the catalytic
domain of HRgpA (30, 31). Despite both a structural similarity and a
specificity restricted to Arg-Xaa peptide bonds, HRgpA and RgpB show
considerable differences in catalytic potency (26) which is most
profoundly manifested in their ability to activate factor X (32) and
protein C (33). In addition to activation of these members of the
coagulation cascade pathway, it was also shown that RgpB was capable of
generating kallikrein from plasma prekallikrein (16). Thus, it may be
anticipated that gingipains R could activate other coagulation cascade
proenzymes in this pathway, since each of these processes requires
cleavage of peptide bonds at the carboxyl-terminal side of specific
arginine residues (34). In the present study, we describe the results of experiments designed to investigate the ability of two forms of
gingipains R to convert prothrombin to thrombin, an enzyme known to
have multiple functions in both coagulation and pro-inflammatory processes.
Materials--
Benzoyl-L-arginine-p-nitroanilide,
tosyl-L-lysine chloromethyl-ketone (TLCK), leupeptin, and
fibrinogen were purchased from Sigma. Factor X-deficient plasma was
obtained from George King Bio-Medical, Inc. (Overland Park, KS).
Purified human prothrombin, Proteinase Purification--
RgpB and HRgpA were isolated
according to the method described by Potempa et al. (26).
The amount of active enzyme in each purified proteinase was determined
by active site titration using Phe-Pro-Arg-chloromethyl ketone (35),
with the concentration of active gingipain R being calculated from the
amount of inhibitor needed for complete inactivation of the proteinase.
Activation of Proteinases--
Each P. gingivalis
proteinase was activated with 10 mM cysteine in 0.2 M HEPES buffer, pH 8.0, containing 5 mM
CaCl2 at 37 °C for 10 min. The activated proteinase (2 µM) was diluted with 10 mM Tris-HCl, pH 7.3, containing 150 mM NaCl (TBS) and 5 mM CaCl2 prior to use.
Determination of Prothrombin Concentration--
The molar
concentration of purified prothrombin was calculated using
A Clotting Assay--
The fibrinogen clotting activity of released
thrombin was measured by incubating 90 µl of prothrombin (90 µg/ml)
with 10 µl of a given proteinase at 37 °C for 3 min. One hundred
µl of fibrinogen (3 mg/ml), prewarmed to 37 °C, was then added to
the mixture, and the clotting time was measured with a Coagulometer KC
1A (Amelung, Lemgo, Germany). For plasma clotting time assays, 90 µl
of factor X-deficient plasma supplemented with 4 µM
factor X-specific inhibitor (DX-9065a) or the same plasma reconstituted
with 10 µl of factor X (100 µg/ml) in the absence of DX-9065a were
prewarmed to 37 °C, and then 10 µl of a proteinase was added and
the clotting time measured. For activated partial thromboplastin time
(APTT) assay, 90 µl of citrated plasma was mixed with 90 µl of
PTT-LT® (cephalin 1.2 mg/ml, silica 1 mg/ml) (Roche Molecular
Biochemicals) and preheated to 37 °C in a plastic cell for 1 min.
Then, 20 µl of HRgpA was added, and the mixture was incubated at
37 °C for 2 min. After adding 100 µl of 25 mM
CaCl2, the clotting time was measured with a Coagulometer
KC 1A (Amelung, Lemgo, Germany).
Kinetic Analysis of Prothrombin Activation--
Prothrombin,
dissolved in 50 µl of 0.1 M Tris-HCl, pH 7.6, containing
0.15 M NaCl and 5 mM CaCl2, was
incubated with the same volume of either gingipain R (0.1 nM HRgpA or 0.4 nM RgpB final concentration)
dissolved in the same buffer supplemented with 80 µg/ml phospholipids
at 37 °C for 30, 60, 90, or 120 s. Then, 50 µl of 6 µM leupeptin in the same buffer was added, to inhibit completely the cysteine proteinase activity. At this concentration leupeptin does not affect the amidolytic activity of thrombin. To this
mixture 50 µl of a thrombin-specific substrate, Boc-Val-Pro-Arg-MCA (0.4 mM), in the same buffer was added. Substrate cleavage
and the release of AMC by thrombin was monitored by the relative
fluorescence increase at 440 ± 20 nm after excitation at 380 ± 20 nm, using a microplate fluorescence spectrophotometer (CytoFluor
Series 4000, Perspective Biosystems). To calculate concentrations of thrombin produced by either gingipain R, the amidolytic activity of
purified SDS-Polyacrylamide Gel Electrophoresis and Western Blot
Analysis--
Eighteen microliters of activated HRgpA or RgpB (3.6 pmol) were added to 162 µl of prothrombin (3.68 nmol in 0.1 M Tris-HCl, pH 7.6, 150 mM NaCl, 5 mM CaCl2, and 0.5 mM benzamidine),
and the mixture (20 nM and 20 µM final
concentration of HRgpA or RgpB and prothrombin, respectively) was
incubated at 37 °C. At 0.5 mM concentration, benzamidine
inhibits thrombin activity but not that of gingipains, and it was
included into the assay buffer to avoid autocatalytic
cleavage. At specific time intervals, aliquots were withdrawn, and 1.5 µl of D-Phe-Phe-Arg-chloromethyl ketone (10 mM) was added to terminate the reaction. Samples were
boiled in reducing treatment buffer and applied for SDS-PAGE with 10% slab gels, according to the method of Laemmli (39).
For Western blot analysis 2-µl aliquots of the prothrombin/gingipain
incubation mixture were transferred to 8 µl of HEPES, pH 7.6, containing 10 µM biotinylated Phe-Pro-Arg-chloromethyl ketone, incubated for 10 min at room temperature, and boiled in reducing treatment buffer. After SDS-PAGE the separated protein fragments were electroblotted onto a polyvinylidene difluoride membrane
(Hybond-P membrane from Amersham Pharmacia Biotech). The membrane was
incubated with streptavidin-horseradish peroxidase conjugate, and bands
were developed by enhanced chemiluminescence (Amersham Pharmacia Biotech).
Amino-terminal Sequence Analysis--
Automatic sequence
analysis was performed with a pulse liquid-phase sequencer (model 477A
Protein Sequencer, PerkinElmer Life Sciences/Applied Biosystems Inc.).
To determine the amino-terminal sequence of prothrombin-derived
fragments, the mixture was separated by SDS-PAGE and transferred to
ImmobilonTM polyvinylidene difluoride transfer membrane
(Millipore Co., Ltd., Bedford, MA). The transferred proteins were
visualized by staining with Coomassie Brilliant Blue R-250. Excised
bands were placed on a Polybrene-treated glass filter prior to sequence analysis.
Activation of Human Prothrombin by Gingipains R--
In order to
determine whether gingipains R activate prothrombin, each bacterial
proteinase was incubated with the human zymogen, and the release of
thrombin activity was measured. Both proteinases caused prothrombin
activation in a dose- and incubation time-dependent manner
(Fig. 1, A and B),
with HRgpA being nearly 17 times more potent. At conditions used for
SDS-PAGE analysis of the prothrombin degradation pattern (Fig. 3), the
thrombin activity released from 20 µM prothrombin by 20 nM of either gingipain transiently reached a peak after 10 min of incubation and then slowly disappeared during prolonged enzyme
exposure (Fig. 2). Significantly, no
generation of thrombin activity was observed if prothrombin was
incubated with TLCK-treated gingipains R (Fig. 1).
Due to autoproteolytic cleavage or fragmentation by other proteolytic
enzymes, thrombin in vitro can occur in three major forms
referred to as
The prothrombin activation assays in vitro based on
generation of amidolytic and/or fibrinogen clotting activities do not reflect the complexity of reactions in blood plasma where a multitude of other proteins could hinder the interaction of gingipains R with
prothrombin. Therefore, to determine if gingipains R can produce a
significant amount of
In addition to the ability to generate directly Cleavage of Prothrombin by Gingipains R--
To elucidate
further the mechanism of prothrombin activation, the zymogen was
incubated with gingipains R for various times, and the products
obtained were analyzed by SDS-PAGE. The major polypeptide bands were
then subjected to amino-terminal amino acid sequence analysis. From
both the electrophoretic mobility and amino-terminal sequence of each
prothrombin-derived fragment, bands of apparent molecular mass of
55, 45, 38.5, 34, 33, 23, and 15 kDa were found and indicated as being
prethrombin 1 (fragment 2/A-chain/B-chain), fragment 1·2, prethrombin
2 (A-chain/B-chain), B-chain of
Despite large differences in the efficiency of prothrombin activation
by HRgpA and RgpB, the zymogen digestion pattern was similar as
determined by laser densitometry analysis (data not shown) of the gels
shown in Fig. 3A. With HRgpA digestion it is clear that the
first major cleavages occurred specifically at the
Arg271-Thr272 and/or
Arg320-Ile321 peptide bonds, releasing
simultaneously fragment 1·2, prethrombin 2, and
The initial accumulation of prethrombin 2 during 15 min of incubation
(Fig. 3A, lanes b-d and i-k)
indicates that the peptide bond
(Arg320-Ile321) at the junction between the A-
and B-chains in prethrombin 2 is relatively refractory to cleavage by
gingipains. The lack of conversion of prethrombin 2 into
Significantly, RgpB was able to convert
The active site labeling of the active thrombin forms generated during
prothrombin incubation with gingipains fully substantiates the
conclusion drawn from the SDS-PAGE analysis that Effect of Phospholipids and Ca2+ on Prothrombin
Activation by Gingipains R--
Phospholipids and Ca2+ are
important cofactors accelerating the proteolytic cascade reaction of
coagulation factors. Therefore, we studied their effect on prothrombin
activation by gingipains R. Thrombin production by HRgpA
increased in phospholipid in a concentration-dependent
manner, with the effect reaching a plateau at concentrations above 40 µg/ml. At this point phospholipids augmented prothrombin activation
by HRgpA about 1.5-fold over the control. Interestingly, phospholipids
did not affect prothrombin activation by RgpB. Phospholipids also did
not increase prothrombin activation by HRgpA in the absence of
Ca2+, and the prothrombin activation by the two proteinases
was not affected by Ca2+ in the absence of phospholipids
(data not shown).
Kinetics of Prothrombin Activation by Gingipains R--
To
investigate the kinetics of prothrombin activation by gingipains R, the
values of Km and kcat were
measured in the presence of phospholipids (40 µg/ml) and determined
as 0.26 ± 0.01 µM and 0.32 ± 0.02 s With the exception of staphylocoagulase which causes human plasma
coagulation through formation of an active molecular complex with
prothrombin (42), surprisingly little is known as to how bacteria-derived proteins interact with this zymogen. Indeed, until now
the only bacterial proteinase that had been shown to activate
prothrombin by limited proteolysis was a metalloproteinase from
Staphylococcus aureus (43). Our data indicate, however, that
the activity of this proteinase was several thousand-fold lower than
that of either of the gingipains R (data not shown). The present work,
therefore, demonstrates that gingipains R of P. gingivalis
are the most potent proteolytic human prothrombin activators of
bacterial origin yet described.
Although the catalytic domains of both gingipains R are essentially
identical (30), these proteinases activated prothrombin with distinctly
different kinetics. Taking into account that during prothrombin
incubation with either gingipain the first active product was
In pathophysiological conditions in vivo where the substrate
concentration is set, the affinity of Rgps for prothrombin
(Km) may be a factor limiting the ability of
gingipains R to activate efficiently this zymogen. The
Km value of HRgpA (0.26 µM) for
prothrombin is 4-fold lower than the normal plasma concentration of
this protein (around 80 µg/ml, 1.1 µM) (34) and
comparable to the Km value of the prothrombinase
complex (Table IV), supporting the likelihood that this gingipain may
activate prothrombin in vivo. In contrast, the high
Km value of 6.6 µM for RgpB
interaction with prothrombin does not favor activation of this
coagulation factor. In addition, RgpB activates prothrombin about 20 times slower than HRgpA explaining together why only the later
gingipain R can clot plasma. Significantly, however, in comparison to
fibrinogen, plasma clotting occurred at much higher gingipain
concentrations. This discrepancy is predominantly due to the fact that
in fibrinogen clotting experiments designed to prove that The superior ability of HRgpA in comparison to RgpB to activate
prothrombin directly is most likely related to the presence of the
hemagglutinin/adhesion domain in the former proteinase. It is
conceivable that this domain participates in the initial binding of
prothrombin to HRgpA, enforcing the proper orientation of the zymogen
for proteolytic attack at the Arg271-Thr272
and Arg320-Ile321 peptide bonds, which is
required for The significant difference between prothrombin activation by
prothrombinase complex or OSV-PTA and gingipains R is the lack of
prethrombin 2 conversion by the bacterial proteinases, as is apparent
from transient accumulation of this form of partially processed zymogen
under in vitro conditions (Fig. 3A). In
vivo, however, in the presence of phospholipids any released
prethrombin 2 may become instantly processed to Taken together, the data presented here indicate that at a P. gingivalis-infected periodontitis site gingipains R, present in
significant amount in the gingival crevicular fluid (45), are able to
release locally In addition to the potential role in the pathogenesis of periodontitis,
our data provide a logical rationalization for the emerging
relationship between periodontitis and cardiovascular disease (51, 52).
First, gingipains would have an indirect role as factors
aggravating and/or sustaining chronic inflammation. Second, they
may contribute more directly to cardiovascular complications because
P. gingivalis has been immunohistochemically in
atherosclerotic plaque shoulders and macrophage-rich infiltrate has
been associated with ulcer and thrombus formation (53). If these
bacterial cells still express gingipains, it is conceivable that these
proteinases could affect local homeostasis through effective and
uncontrolled activation of both proteinase-activated
receptors2 and coagulation factors.
-thrombin was produced by gingipains R, and
this was confirmed by SDS-polyacrylamide gel electrophoresis, thrombin
active site labeling, and amino-terminal sequence analysis of
prothrombin digestion fragments. Significantly, the catalytic
efficiency of HRgpA to generate thrombin
(kcat/Km = 1.2 × 106 M
1
s
1) was 100-fold higher than that of RgpB
(kcat/Km = 1.2 × 104 M
1
s
1). The superior prothrombinase activity of
HRgpA over RgpB correlates with the fact that only the former enzyme
was able to clot plasma, and kinetic data indicate that prothrombin
activation can occur in vivo. At P. gingivalis-infected periodontitis sites HRgpA may be involved in
the direct production of thrombin and, therefore, in the generation of
prostaglandins and interleukin-1, both have been found to be associated
with the development and progression of the disease. Furthermore, by
taking into account that the P. gingivalis bacterium has
been immunolocalized in carotid atherosclerotic plaques at thrombus
formation sites (Chiu, B. (1999) Am. Heart J. 138, S534-S536), our results indicate that bacterial proteinases may
potentially participate in the pathogenesis of cardiovascular disease
associated with periodontitis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-,
-, and
-thrombins, and
biotinylated Phe-Pro-Arg-chloromethyl ketone were purchased from
Hematologie Technologies, Inc. (Essex Junction, VT).
t-Butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide (Boc-Val-Pro-Arg-MCA) was obtained from the Peptide Institute (Minoh,
Japan); p-nitrophenyl-p'-guanidinobenzoate was a
product from Nacalai Tesque (Kyoto, Japan), and DX-9065a, a specific
factor Xa inhibitor, was obtained from Daiichi Pharmaceutical Co., Ltd. (Tokyo, Japan). The purified human factor X was purchased from Enzyme
Research Laboratories, Inc. (South Bend, IN). Factor X-, IX-, and
XI-deficient plasmas and Platelin® (rabbit brain phospholipids) were
obtained from George King Biomedical (Overland Park, KS), and from
Organon Teknika (Durham, NC), respectively. Normal human plasma was
prepared by centrifugation of a mixture of 9 volumes of freshly drawn
blood from healthy volunteers and 1 volume of 3.8% (w/v) sodium citrate.
-thrombin, which had been titrated with
p-nitrophenyl-p'-guanidinobenzoate (37), was used
as a standard. The initial velocity of thrombin production at various
prothrombin concentrations (final concentrations: 50, 100, 150, 200, 300, 400, 600, and 1000 nM for HRgpA and 1, 2, 3, 4, 5, 7, and 10 µM for RgpB) was determined by the best fit line
after incubation for various periods. The values for Km and Vmax were extracted by
direct fit of the Michaelis-Menten equation to experimental data using
non-linear curve fitting employing the method of least squares with
Taylor expansion (38). Moreover, because the values generated in this
way were very similar to the ones obtained by three transformations of
the Michaelis-Menten equation ([S]0/v
versus [S]0, 1/v versus
1/[S]0 and v versus
v/[S]0, where v and
[S]0 denote the catalytic rate and the initial substrate concentration, respectively), the means ± S.D. derived from four independent experiments and four different transformations of the
Michaelis-Menten equation were calculated and presented in Table IV.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activation of human prothrombin by HRgpA and
RgpB. Ninety µl of human prothrombin (90 µg/ml dissolved in
0.1 M Tris-HCl, pH 7.6, 150 mM NaCl, and 2.5 mM CaCl2) was supplemented with 10 µl of a
proteinase and incubated at 37 °C (final prothrombin concentration,
1.12 µM). Activation was quenched with 500 µl of the
above buffer containing 1.5 µM antipain. Ten microliters
of Boc-Val-Pro-Arg-MCA (10 mM) was then added to the
solution, and 7-amido-4-methylcoumarin released was measured at
37 °C. A, proteinases of various concentrations incubated
with prothrombin for 3 min. The final concentrations of each
proteinase in the reaction mixture are shown. , HRgpA;
, RgpB;
, a mixture of TLCK-inactivated HRgpA and RgpA. B,
proteinase (0.05 nM, final concentration) was incubated
with prothrombin at 37 °C for various periods.
, HRgpA;
,
RgpB;
, TBS instead of a proteinase solution.
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Fig. 2.
Time course of thrombin generation and
degradation by gingipains R. Eighteen microliters of activated
HRgpA (3.6 pmol) was added to 162 µl of prothrombin (3.68 nmol), and
the mixture (20 nM and 20 µM final
concentration of HRgpA and prothrombin, respectively) was incubated at
37 °C in 0.1 M Tris-HCl, 150 mM NaCl, 5 mM CaCl2, pH 7.6, containing 1 mM
cysteine. At the given time intervals 10-µl aliquots were removed to
985 µl of the same buffer supplemented with 2 mM
antipain, and thrombin activity released by HRgpA ( ) and RgpB
(
) was measured using
H-D-Phe-Pipecolyl-Arg-p-nitroanilide as
substrate.
-,
-, and
-thrombin. All of these enzymes have
amidolytic activity, but only
-thrombin is capable of clotting fibrinogen. Therefore, to determine if amidolytic activity released by
gingipains from the zymogen is at least partially due to the presence
of
-thrombin, which is the more important form of this proteinase,
the samples of prothrombin incubated with gingipains were examined for
clotting activity. As summarized in Table
I both gingipains induced fibrinogen
clotting activity from prothrombin in a time- and
concentration-dependent manner. However, due to the
apparent progressive cleavage of the
-thrombin B-chain and the
creation of
- and/or
-thrombin, a correlation between clotting and amidolytic activity was observed only at short preincubation times.
Indeed, after prolonged incubation the former activity decreased much
faster than the latter activity (data not shown). The same process of
excessive
-thrombin cleavage in the presence of increased
concentrations of gingipains most likely skewed a concentration-dependent release of fibrinogen clotting
activity from prothrombin. This is particularly apparent in the case of RgpB where the doubling of enzyme concentration resulted only in the
moderate shortening of the fibrinogen clotting time (Table I). In
comparison to HRgpA, an ~5-fold higher concentration of RgpB was
necessary to induce clotting activity from prothrombin, and
significantly, clotting times determined after the same preincubation time were 3-4 times longer. Taken together, these results indicate that HRgpA is about 20 times more efficient than RgpB in
-thrombin generation. This is in keeping with zymogen activation as measured with
an amidolytic substrate in which HRgpA was shown to be 17-fold more
efficient than RgpB (Fig. 1).
Fibrinogen clotting by prothrombin incubated with gingipains R
-thrombin in plasma, we measured the clotting
time of factor X-deficient plasma incubated with gingipains. In order
to evaluate interference from any residual factor X, which may still
exist in deficient plasma, the assay was performed in the presence of a
factor Xa-specific inhibitor in comparison to the deficient plasma
reconstituted with the physiological concentration of factor X. The
results obtained are summarized in Table
II and show that only HRgpA was able to
clot factor X-deficient plasma in a dose-dependent manner,
clearly indicating the generation of
-thrombin. The clotting time of
the plasma reconstituted with factor X and incubated with HRgpA was
basically the same, apparently due to the fact that in the absence of
phospholipids and Ca2+ factor Xa is a very poor activator
of prothrombin. This result indicates that the presence of factor X
does not affect prothrombin activation by HRgpA. Significantly, in this
assay HRgpA was shown to be at least 10-fold more efficient than RgpB.
These results correlate very well with the ability of both gingipains R
to induce fibrinogen clotting by prothrombin (Table I) and confirm that out of two gingipains R, HRgpA is the predominant activator of this
zymogen.
Effect of gingipains R on factor X-deficient plasma clotting time
-thrombin from
prothrombin, gingipains R are capable of efficiently activating factors
X and IX and accelerating in this way plasma coagulation through the
cascade reaction of the clotting factors in the presence of
phospholipids and Ca2+ (32, 40). In order to understand the
relative importance of these three activities, we have directly
compared the ability of HRgpA to promote the clotting of normal plasma
versus factor X-, IX-, and XI-deficient plasmas by measuring
APTT. This assay using phospholipids, and Ca2+ enables us
to study the plasma clotting via the cascade reaction. From the data
summarized in Table III, it is also
apparent that in this assay HRgpA, in concentrations much lower than
those required in the plasma clotting assay, significantly decreased
APTT in a dose-dependent manner not only in the normal
plasma but also in plasmas deficient in factors X, IX, and XI. Although
shortening of the clotting time of factor IX-deficient plasma is most
likely due to the additive effect of activation of factor X and
prothrombin, the clotting of factor X-deficient plasma must be
predominantly triggered by generation of
-thrombin directly from the
zymogen of this key clotting factor. Taken together these results
further confirm that prothrombin is, indeed, an important pathological target for the HRgpA-mediated blood coagulation and indicate that this
process can occur in vivo.
Effect of HRgpA on APTT of plasmas deficient in a factor in the
intrinsic coagulation pathway
-thrombin, fragment 1, B-2-chain of
-thrombin, and fragment 2, respectively (Fig.
3A).
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Fig. 3.
Cleavage of prothrombin by gingipains R. A, prothrombin (20 µM) and gingipains R (20 nM) were incubated together as described in Fig. 2 legend
in the buffer supplemented with 0.5 mM benzamidine. At
specific time intervals, aliquots were withdrawn, and 1.5 µl of
D-Phe-Phe-Arg-chloromethyl ketone (10 mM) was
added to terminate the reaction. Samples were boiled in sample
treatment buffer and analyzed by SDS-PAGE. Lane a,
prothrombin alone (3.5 µl loaded); lanes b-f, prothrombin
incubated with HRgpA for 1, 2, 5, 15, and 60 min, respectively;
lanes g and n, -thrombin (1.3 µg loaded);
lanes h-m, prothrombin incubated with RgpB for 0, 1, 2, 5, 15, and 60 min, respectively; lanes o-r,
-thrombin (20 µM) incubated with RgpB (20 µM) for 0, 5, 15, and 60 min, respectively (2 µg loaded); lanes s and
t, pure
- and
-thrombin, respectively (1.3 µg
loaded). The positions of molecular mass standard markers are indicated
to the right of the gel. The major fragments of prothrombin
indicated by arrows were subjected to amino-terminal
sequence analysis and identified as follows: 1, prethrombin
1; 2, fragment 1·2; 3, prethrombin 2;
4, B-chain of
-thrombin; 5, fragment 1;
6, B-2-chain of
-thrombin; and 7, fragment 2. B, schematic diagram of prothrombin fragmentation with major
cleavage sites for HRgpA and RgpB. The size of arrowheads
indicates the relative efficiency of cleavage of specific peptide bonds
in the prothrombin polypeptide chain by the two proteinases tested.
-Thrombin is generated by cleavage at the
Arg70-Tyr71 or
Arg73-Asn74 peptide bonds in the
-thrombin
B-chain, giving rise to the B1 and B2 peptides. An additional cleavage
at Lys154-Gly155 of the B2-chain generates
-thrombin.
-thrombin,
followed by hydrolysis of fragment 1·2 at the Arg155-Ser156 peptide bond (Fig.
3B). In contrast, during the initial phases of prothrombin
degradation by RgpB two major cleavages were found to take place at the
Arg155-Ser156 and
Arg271-Thr272 peptide bonds, leading to the
initial accumulation of prethrombin 1, prethrombin 2, fragment 1 (Fig.
3A, lanes i and j), and only a minor
amount of
-thrombin. In this regard the non-effective cleavage of
the Arg320-Ile321 peptide bond in prothrombin
explains why RgpB is about 20-fold slower in
-thrombin generation
than HRgpA. Taken together, differences in the pattern of
prothrombin-derived products generated by HRgpA and RgpB can be
observed only at the initial incubation times with qualitative
difference limited to release of B-chain of
-thrombin only by HRgpA.
The variations in the rate of cleavage of the
Arg155-Ser156 peptide bond contributed only to
qualitative difference that was later annihilated by further cleavage
to the same molecular mass fragments or digestion to small molecular
peptides poorly visible in the gels.
-thrombin,
as shown by both SDS-PAGE (Fig. 3A, lanes e and
l) and the time course for the generation of thrombin
activity (Fig. 2), argues that this form of zymogen is only a minor
intermediate source of active enzyme that undergoes exhaustive
degradation by gingipains. Such an interpretation is supported by the
appearance of peptide fragments of lower molecular mass than
prethrombin 2 but bearing the same amino-terminal sequence (Fig. 3,
lanes d and k, bands between B-2-chain and
fragment 2).
-thrombin to
-thrombin by
cleavage at the Arg383-Asn394 peptide bond,
with release of both the B-1- and B-2-chains (Fig. 3A,
lanes p-r). Furthermore, the B-2-chain was slowly degraded by this gingipain, resulting in a gradual loss of enzymatic activity. In the case of HRgpA interacting with
-thrombin a similar pattern was observed, but the substrate cleavage occurred at higher rate, and
after 60 min all
-thrombin was totally degraded to lower molecular
mass fragments with similar electrophoretic mobility as those generated
by RgpB. One significant but only qualitative difference was the low
amount of accumulated B-2-chain of
-thrombin which apparently was
further degraded by HRgpA (data not shown). There may be some more
profound differences in the cleavage pattern of
-thrombin,
but weak staining of low molecular peptides in the gel excludes the
possibility to analyze this in detail.
-thrombin is,
indeed, directly released from the zymogen (Fig.
4, A and B).
Note that in Fig. 4 the blot with prothrombin-derived products generated by digestion with RgpB was exposed about 15 times longer (Fig. 4B) than the other blot (Fig. 4A), in order
to visualize the biotinylated active forms of thrombin. This provides
supplementary evidence that HRgpA is a far more potent prothrombinase
than RgpB. In addition, labeling experiments confirmed that the
-thrombin initially accumulated is the subject of further
degradation in order to yield products that still preserve activity as
indicated by incorporation of the biotinylated chloromethyl ketone.
During prolonged incubation with HRgpA, the accumulation of the labeled B-4-like chain derived from
-thrombin-like molecule was
apparent. In contrast, RgpB generated relatively minor amounts of
-thrombin-like derived peptide. Rather, it resulted in the
significant accumulation of a labeled low molecular mass peptide,
suggesting the generation of a form of thrombin with the B-chain
fragmented to smaller pieces than those in
-thrombin (Fig.
4A).
View larger version (32K):
[in a new window]
Fig. 4.
Western blot analysis of different forms of
active-site biotinylated thrombin generated during prothrombin
incubation with gingipains R. Prothrombin was incubated with HRgpA
(A) or RgpB (B) as described in the legend for
Fig. 2. At specific time intervals, 2-µl aliquots were transferred to
8 µl of 0.1 M HEPES, pH 7.6, containing 10 µM biotinylated Phe-Pro-Arg-chloromethyl ketone. After 10 min of incubation at room temperature, samples were boiled in reducing
treatment buffer and subjected to SDS-PAGE, followed by protein
electrotransfer onto polyvinylidene difluoride membranes. The blotted
membranes were incubated with streptavidin-horseradish peroxidase
conjugate, and bands were developed by enhanced chemiluminescence for
about 6 and 90 s for HRgpA and RgpB digestion products,
respectively. Lanes a-g, prothrombin preincubated with each
gingipain for 0, 0.5, 1, 2, 5, 15, and 60 min, respectively; lane
h, a mixture of -,
-, and
-thrombin; lane i,
prothrombin preincubated with TLCK-treated gingipain for 60 min.
1 for HRgpA and 6.6 ± 0.4 µM and 0.076 ± 0.005 s
1
for RgpB, the catalytic efficiency
(kcat/Km) of the former
enzyme was 100-fold higher than that of RgpB. The kinetic constants of
gingipains R for prothrombin conversion to thrombin were compared with
those for activated factor X (Xa) in the presence of activated factor V
(Va) (41), as well as with snake venom prothrombin activators from
Oxyuranus scutellatus (OSV-PTA) and Notechis scutulus
scutulus (NSSV-PTA), again in the presence of factor Va (7, 8).
The Km value of HRgpA was comparable to the values
of the physiological prothrombin activator and venom prothrombin
activators but much lower than the Km value of RgpB
(Table IV). In addition, the
kcat values of gingipains R were also less than
that of factor Xa and OSV-PTA, although higher than the value of
NSSV-PTA (Table IV). The
kcat/Km values of the
bacterial proteinases were substantially lower than those of factor Xa
and OSV-PTA, but the value for HRgpA was higher than that of NSSV-PTA
(Table IV). Significantly, the
kcat/Km value of RgpB, which
contains no adhesin domain, was the lowest of the five compared. These
data suggest that HRgpA is a more potent prothrombin activator than
RgpB and NSSV-PTA, although it is less potent than either factor Xa or
OSV-PTA.
Kinetic constants for the activation of prothrombin
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-thrombin-released by simultaneous cleavage of prothrombin at
Arg271-Thr272 and
Arg320-Ile321 peptide bonds (Fig. 3), it is
apparent that the data describe the direct generation of the
physiologically active form of thrombin. The difference in the rate of
the Arg320-Ile321 peptide bond cleavage is
most likely to be responsible for the 17-fold less efficient
prothrombin activation by RgpB than HRgpA, because it
directly contributes to the kcat value of
prothrombin activation that is 5 times lower for the former gingipain
R. On the other hand, the presence of the hemagglutinin/adhesion domain in the HRgpA molecule may be responsible for the increased affinity of
HRgpA to prothrombin in comparison to RgpB.
-thrombin
was generated, prothrombin was first preincubated with gingipains R
before being mixed with fibrinogen, whereas in the plasma clotting test
there was no preincubation step. Second, interference from other plasma
proteins in gingipain R prothrombin activation and/or by degradation of
fibrinogen by gingipains R (44) could also add to slower plasma
clotting. In contrast, the absence of calcium in the plasma clotting
assay should have no contribution to the observed difference since it was shown that Ca2+ affects neither prothrombin activation
by gingipains R nor conversion of fibrinogen to fibrin by
-thrombin
(34).
-thrombin production. Because the
hemagglutinin/adhesion domain is involved in high affinity binding of
fibronectin and fibrinogen to HRgpA (44), these proteins, which occur
in plasma at relatively high concentrations, may compete with
prothrombin for binding to this bacterial proteinase. It is tempting to
hypothesize that when fibrinogen or fibronectin occupies the
hemagglutinin/adhesion domain of HRgpA; this complex behaves more like
RgpB in its ability to activate prothrombin.
-thrombin by
activated factor X generated in plasma by gingipains R (32). This
ability to activate factor X by HRgpA was also shown to be 10-fold more
efficient than RgpB, primarily due to a 7-8-fold acceleration of the
reaction by phospholipids in the presence of Ca2+ (32).
Because in the absence of phospholipids the catalytic potency of HRgpA
to activate factor X is only 3-fold higher than RgpB, as indicated by
the kcat/Km ratio, it is
apparent that the preferential activation of blood coagulation by HRgpA is mainly due to the stimulatory effect of phospholipids. Therefore, in
the absence of this cofactor HRgpA activates both prothrombin and
factor X with comparable efficiencies. In contrast, kinetic data
indicate that irrespective of the presence of phospholipids, RgpB acts
preferentially on factor X, as described previously (32). This
phenomenon may be explained by the fact that
-thrombin generation
from its zymogen requires a concerted cleavage at two specific peptide
bonds (Arg271-Thr272 and
Arg320-Ile321) in prothrombin, whereas that of
factor Xa needs a single cleavage at the
Arg52-Ile53 in the heavy chain of this zymogen
(34).
-thrombin by direct cleavage of prothrombin in the
reaction uncontrolled by host factors that normally keep in check
prothrombin activation. This may have significant effect on periodontal
tissues because thrombin, in addition to being a key enzyme in the
coagulation pathway, is the molecule endowed with several physiological
functions relevant to pathological changes seen at the periodontitis
site. First, it enhances vascular permeability and possesses leukocyte
chemotactic activity (9-11) and, therefore, may account for gingival
crevicular fluid formation and leukocyte accumulation. Second, it
induces the production of prostaglandins (46, 47) and interleukin-1
(12), and the elevated levels of these two factors in gingival
crevicular fluid of adult periodontitis patients (48-50) may be
connected to thrombin production by P. gingivalis
proteinases. Third, thrombin stimulates bone resorption by osteoclasts
through a prostaglandin-dependent pathway (46) and thus
could be associated with the alveolar bone resorption seen at
periodontitis sites. All together, the data described here would imply
that the conversion of prothrombin to
-thrombin is a potentially
important mechanism for the alveolar bone erosion, a practically
irreversible clinical hallmark of periodontitis.
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FOOTNOTES |
---|
Present address: IBMB-CSIC, Dept. de Biologia Molecular y
Celular, Jordi Girona, 18-26, 08034 Barcelona, Spain.
To whom correspondence should be addressed: Institute of
Molecular Biology, Jagiellonian University, Al. Mickiewicza 3, 31-120 Kraków, Poland. Tel.: 48-12-634-1442; Fax: 48-12-633-6907;
E-mail: potempa@mol.uj.edu.pl.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M006760200
This work was supported by Grant 11670219 from the Japanese Ministry of Education (to T. I.), Grant DE 09761 from the National Institutes of Health (to J. T.), and by Grant 6 P04A 047 17 from the Committee of Scientific Research, KBN, Warszawa, Poland (to J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
2 Lourbakos, A., Yuan, Y. P., Jenkins, A., Travis, J., Andrade-Gordon, P., Santulli, R., Potempa, J., and Pike, R. N. (2001) Blood, in press.
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ABBREVIATIONS |
---|
The abbreviations used are: HRgpA and RgpB, arginine-specific gingipains, products of rgpA and rgpB genes, respectively; PT, prothrombin time; TLCK, tosyl-L-lysine chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis; APTT, activated partial thromboplastin time; Boc-Val-Pro-Arg-MCA, t-butyloxycarbonyl-L-Val-L-Pro-L-Arg-4-methylcoumaryl-7-amide.
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