(Received for publication, August 3, 1995)
From the
Protease nexin-2/amyloid -protein precursor (PN-2/A
PP)
is a Kunitz-type protease inhibitor which has been shown to be a
tight-binding inhibitor of coagulation factors XIa and IXa. Here we
show that PN-2/A
PP and its KPI domain also inhibited isolated
factor Xa with a K
of
10
M. On a solid phase binding assay,
PN-2/A
PP formed a complex with factor Xa. Incubation of molar
excess factor Xa to PN-2/A
PP produced a single cleavage within
PN-2/A
PP's heparin binding domain liberating a 8.2-kDa
amino-terminal peptide. PN-2/A
PP and its KPI domain equally
inhibited factor Xa in the prothrombinase complex with a K
of 1.9
10
M and 1.3
10
M,
respectively. A
PP
which does not contain the KPI
domain was a substrate of factor Xa but did not inhibit it, indicating
the PN-2/A
PP inhibition of factor Xa was not substrate inhibition.
All of the factor Xa inhibition in the prothrombinase complex by
PN-2/A
PP and its KPI domain on the chromogenic assay was accounted
for by inhibition of release of prothrombin fragment F
as determined on immunochemical assay. In the prothrombinase
complex, PN-2/A
PP inhibited factor Xa with a k
= 1.8 ± 0.7
10
M
min
similar to
antithrombin III and heparin inhibition (k
of
3.0 ± 0.2
10
M
min
). These studies indicated that
PN-2/A
PP in the assembled prothrombinase complex inhibited factor
Xa comparable to antithrombin III in the presence of heparin.
PN-2/A
PP's factor Xa inhibitory activity along with its
known inhibition of factors XIa and IXa suggest that this protease
inhibitor and related proteins could be regulators of hemostatic
reactions on membranes of cells in the intravascular compartment.
Amyloid -protein precursor (A
PP), (
)a
multidomain protein, is the parent protein of amyloid
protein, a
39-42 amino acid peptide that is deposited in senile plaques and
in the walls of cerebral blood vessels of patients with
Alzheimer's disease (Kang et al., 1987; Glenner and
Wong, 1984). The single gene for A
PP found on chromosome 21
encodes at least three distinct mRNAs produced by alternative splicing
that result in three different sized proteins (A
PP
,
A
PP
, and A
PP
) (Ponte et
al., 1988; Tanzi et al., 1988; Kitaguchi et al.,
1988). Two of these mRNAs code for proteins (A
PP
and
A
PP
) which contain a domain homologous to
Kunitz-type protease inhibitors (KPI) (Ponte et al., 1988;
Tanzi et al., 1988; Kitaguchi et al., 1988). The
secreted isoforms of A
PP containing the KPI domain are identical
to protease nexin-2 (PN-2) (Van Nostrand et al., 1989;
Oltersdorf et al., 1989).
PN-2/APP and its KPI domain
have been recognized to be potent inhibitors of trypsin, chymotrypsin,
epidermal growth factor binding protein, and the
subunit of nerve
growth factor (Van Nostrand et al., 1989, 1990b; Oltersdorf et al., 1989). PN-2/A
PP which is present in high
concentrations in platelets is a potent inhibitor of factor XIa (Van
Nostrand et al., 1990a, 1990b; Smith et al., 1990).
PN-2/A
PP also is an inhibitor of factor IXa (FIXa) in the assembly
of the tenase complex on phospholipid vesicles (PSPC), platelets, and
endothelial cells (Schmaier et al., 1993, 1995). Similarly, a
homologue of PN-2/A
PP, amyloid
-protein precursor-like
protein-2, has been shown to have inhibitory activity against
hemostatic enzymes factors XIa, IXa, and Xa (Petersen et al.,
1994; Sprecher et al., 1993; Van Nostrand et al.,
1994). These studies suggest that this family of proteins may have a
regulatory role in hemostasis. While examining the ability of
PN-2/A
PP to inhibit factor IXa on non-biologic surfaces, we found
in one assay that the degree of factor IXa inhibition could not be
fully accounted for by its inactivation alone. The present
investigation shows that PN-2/A
PP also is an inhibitor of factor
Xa alone and when assembled on PSPC in the prothrombinase complex.
Recognition that PN-2/A
PP's inhibits factor Xa enlarges its
role as a regulator of hemostasis.
The equilibrium
inhibition constants (K) presented for
PN-2/A
PP and its KPI domain were calculated as previously reported
(Van Nostrand et al., 1990b) by the procedure of Bieth(1984)
for tight-binding inhibitors using the following equation: K
=
{[(I)/(1-a)] - (E)}/(1/a), to yield an apparent K
where (I) is the inhibitor concentration, (E) is the factor Xa concentration, and a is the
residual factor Xa activity after incubation with the inhibitor. The
actual K
was calculated using the subsequent
equation: K
= K
/1 +
([S]/K
), where [S] is the
concentration of the substrate, factor II, and K
is the Michaelis constant for the factor Xa-factor II
(protease-substrate) reaction (Bieth, 1984). The second-order
association rate constants (k
) for each of the
inhibitors were calculated using the integrated second-order rate
equation: k" = [(1/I-E)
ln E(I - EI)/I(E - EI)]/t, where E is the FXa
concentration, I is the inhibitor concentration, EI is the
concentration of the FXa-inhibitor complex, and t is the time
of incubation in minutes (Gigli et al., 1970).
Figure 1:
Solid phase
binding assay between PN-2/APP and FXa. Top, purified
PN-2/A
PP (250 ng) was linked to microtiter plate cuvette wells
(see ``Experimental Procedures''). After blocking with bovine
serine albumin, purified FXa (50 ng), antibody to Factor X, and a
second antibody to detect the antibody to Factor X was added in
sequential order. In various wells, one of the components of the
complex assay was excluded. No APP represents wells where no
PN-2/A
PP were linked to the cuvette wells; No FXa indicates wells where no FXa was added; No AntiFXa represents wells were no primary antibody to Factor X was added; No 2nd Ab indicates wells where no alkaline
phosphatase-conjugated second antibody were added; and ALL represents cuvette wells were all components were added. The data
presented represents the mean ± S.E. of nine experiments. Bottom, purified FXa (50 ng) was linked to microtiter plate
cuvette wells (see ``Experimental Procedures''). After
blocking with bovine serine albumin, purified PN-2/A
PP (250 ng),
antibody to PN-2/A
PP, and a second antibody to detect the antibody
to PN-2/A
PP was added in sequential order. In various wells, one
of the components of the complex assay was excluded. NoFXa represents wells where no Factor Xa was linked to the
cuvette wells; No APP indicates wells where no PN-2/A
PP
was added; and No AntiAPP represents wells were no primary
antibody to PN-2/A
PP was added. The data presented represents the
mean ± S.E. of nine experiments.
Additional investigations showed that PN-2/APP was
a substrate of FXa (Fig. 2). When PN-2/A
PP was incubated
with increasing concentrations of FXa (1-16-fold molar excess),
there was a decrease in the large, dark 124-kDa band of the starting
material on an immunoblot of a nonreduced 6% SDS-PAGE and the
appearance of 3 new bands at 116, 97, and 90 kDa, respectively, that
migrated further into the gel (Fig. 2A). At a presumed
1:1 molar ratio of FXa to PN-2/A
PP some cleavage in PN-2/A
PP
occurred (Fig. 2A). As the concentration of FXa to
PN-2/A
PP increased from 4:1 to 16:1, all of the 124-kDa starting
material was converted into lower molecular mass bands at 116, 97, and
90 kDa. Four-fold molar excess FXa to PN-2/A
PP liberated a single
8.2-kDa peptide which was detected on a reduced 18% SDS-PAGE (data not
shown). The amino terminus sequence of this peptide was LEVPTDGNAG . .
. which is the known amino-terminal sequence of PN-2/A
PP after
cleavage of its signal peptide at alanine 17 (Fig. 3). The
liberated peptide was a single peptide because on multiple gel
electrophoreses using different percentage acrylamide gels
(15-22%), only this single amino-terminal sequence was obtained.
These data indicated that FXa liberated an amino-terminal peptide from
PN-2/A
PP.
Figure 2:
PN-2/APP is a substrate of FXa. Panel A, PN-2/A
PP (1.0 µg or 8.3 pmol) were incubated
with equal to 16-fold molar excess FXa in 0.05 M Tris-HCl,
0.15 M NaCl, pH 7.4, in the presence of 2 mM Ca
for 1 h at room temperature. The reactions
were stopped with sample buffer and applied nonreduced to a 6%
SDS-PAGE. The samples were electroblotted onto nitrocellulose and an
immunoblot was performed using monoclonal antibody P2-1. The
immunoblot was detected by chemiluminescence. The figure is a
photograph of an autoradiogram. PN-2 represents the
immunoblot of 8.3 pmol of PN-2/A
PP being applied to the SDS-PAGE.
Ratio 1:1 to 1:16 represent the ratio of PN-2/A
PP to FXa (mol/mol)
in the incubation mixture. Panel B, the figure represents a
Coomassie-stained 6% SDS-PAGE of reduced 83 pmol of PN-2/A
PP alone (PN2) or 332 pmol of FXa and 83 pmol of PN-2/A
PP, ratio
4:1 of FXa to PN-2/A
PP (Xa PN2). The numbers and
stained bands to the right of the figure represent molecular mass
standards (M
standards) in kilodaltons. Panel
C, the figure represents a photograph of a Coomassie-stained
reduced 6% SDS-PAGE of 83 pmol of A
PP
alone (695) or
332 pmol of FXa and 83 pmol of A
PP
, ratio of 4:1 of
FXa to A
PP
(Xa 695). The numbers and
stained bands to the right of the figure represent molecular mass
standards (M
standards) in
kilodaltons.
Figure 3:
The FXa cleavage site in PN-2/APP.
The figure is the amino-terminal sequence of PN-2/A
PP using the
single letter code for each amino acid. The arrow after
alanine 17 represents the cleavage site for the single peptide for
PN-2/A
PP (Ponte et al., 1988). The arrow after
arginine 102 represents the FXa cleavage site in
PN-2/A
PP.
Further investigations sought the FXa cleavage site
on the amino-terminal side of PN-2/APP. When PN-2/A
PP (a
major band at 124 kDa and two minor bands at 105 and 98 kDa) was
cleaved by 4-fold molar excess FXa, three new corresponding lower molar
mass bands were detected (a major one at 115 kDa and two minor bands at
97 and 90 kDa) when the sample was reduced and electrophoresed on a 6%
SDS-PAGE, as detected by a Coomassie Blue staining (Fig. 2B). The amino-terminals of each of these three
bands were sequenced and a single amino acid sequence (KQCKTHPHFV . . .
) was found for all three of these bands (Fig. 3). These data
indicated that molar excess FXa to PN-2/A
PP cleaved PN-2/A
PP
at a single site after arginine 102. Additional investigations showed
that A
PP
, a form of A
PP which does not contain
the KPI domain and does not inhibit FXa, also was a substrate of the
enzyme (Fig. 2C). These data indicated that inhibition
of FXa was independent of the inhibitor being a FXa substrate. Further
isolated KPI domain was not cleaved by FXa (data not shown).
Figure 4:
Substrate/velocity plot (A) and
double reciprocal plot (B) of FII activation by FXa. End point
rates of FII activation (mean ± S.E.) were determined as
described under ``Experimental Procedures'' at each of the
various concentrations of added FX as indicated in the graph. The graph
is the result of FII activation by FXa (1.0 nM) in the
presence of PSPC (25 µM), FVa (5 nM), and 2
mM Ca. The plotted results at each point are
the mean ± S.E. of four independent
experiments.
Investigations were next performed to determine the comparative
inhibitory abilities of PN-2/APP versus antithrombin III
and heparin on isolated FXa and FXa in the prothrombinase complex (Table 4). When PN-2/A
PP was incubated for 5 min with
isolated FXa in the presence of polylysine, its second-order
association rate constant (k
) (1.4 ±
0.4
10
M
min
) for FXa inhibition was 5-fold higher than
that of antithrombin III and heparin (k
= 3.0 ± 3.3
10
M
min
).
Interestingly if heparin were present in the reaction mixture with
PN-2/A
PP, no inhibition of FXa was detected (data not shown).
Alternatively, if polylysine was absent from the reaction mixture, the
inhibitory abilities of PN-2/A
PP and antithrombin III were
reversed. PN-2/A
PP inhibited FXa with a k
= 3.0 ± 2.0
10
M
min
;
antithrombin III and heparin blocked FXa with a k
= 1.3 ± 0.3
10
M
min
. The data
indicated that polylysine had opposite effects on both PN-2/A
PP
and antithrombin III. In the prothrombinase complex, the biologically
relevant assay, the k
= 1.8 ±
0.7
10
M
min
for PN-2/A
PP was essentially the same
as the k
= 3.0 ± 0.2
10
M
min
seen with antithrombin III and heparin. These data indicated that
in the prothrombinase complex, PN-2/A
PP in the absence of heparin
and antithrombin III and heparin were equipotent inhibitors of FXa.
The present investigations expand the coagulation protease
inhibitory spectrum of PN-2/APP. Although first recognized as a
hemostatic inhibitor to factor XIa (Smith et al., 1990; Van
Nostrand et al., 1990b), recent investigations have shown that
PN-2/A
PP also is a potent inhibitor of factor IXa (Schmaier et
al., 1993, 1995) and its KPI domain has some inhibitory activity
to tissue factor-factor VIIa (Dennis and Lazarus, 1994a, 1994b).
Initial reports suggested that PN-2/A
PP was not an inhibitor of
FXa to any great extent (Smith et al., 1990; Van Nostrand et al., 1990b); however, other reports, consistent with
coagulant assays, suggest otherwise (Kitaguchi et al., 1990;
Petersen et al., 1994; Schmaier et al., 1993). Our
investigations indicate that PN-2/A
PP is a direct inhibitor of FXa
both as an isolated protein and in the prothrombinase complex. The
inhibitory activity of PN-2/A
PP resides completely in its KPI
domain because both the parent protein and its isolated KPI domain
inhibit FXa to the same degree. Although the stoichiometry of
inhibition appears to be 1:1, inhibition by the KPI domain does not
appear to be active site-directed. Four orders of magnitude for the KPI
domain to FXa does not reduce the FXa activity to zero, both in assays
of isolated FXa and FXa in the prothrombinase complex. These data are
different from those found with FIXa. Infinite concentrations of KPI
domain abolished FIXa activity in the tenase complex (Schmaier et
al., 1995). Nonactive site-directed FXa inhibitors also recently
have been described for the hookworm-derived inhibitor of human FXa
(Cappello et al., 1995).
We found that when using a
polylysine-based FX activation assay, some of the measured inhibition
of FIXa by PN-2/APP can be accounted for by PN-2/A
PP
inhibiting generating FXa. Since the degree of FXa generated in this
assay is small (1-2 nM), the concentration of
PN-2/A
PP or KPI domain present in the assays would have been
sufficient to inhibit both FIXa and the generated FXa (Schmaier et
al., 1993, 1995). It also was of interest to learn that polylysine
itself potentiated the degree of inhibition of FXa by PN-2/A
PP and
reduced that of antithrombin III/heparin. The mechanism for this
independent activity of polylysine is not known. Since polylysine
itself can be an independent variable contributing to
PN-2/A
PP's inhibitory ability, it should probably be avoided
in assays of FIXa.
In addition to inhibition of enzymatic activity,
we were able to demonstrate a physical interaction between
PN-2/APP and FXa. On a solid phase binding assay, specific complex
formation was detected between PN-2/A
PP and FXa. This information
suggests that PN-2/A
PP is an inhibitor of FXa of the slow, tight
class characteristic of Kunitz type inhibitors. PN-2/A
PP was also
a substrate of FXa when the enzyme was in molar excess to inhibitor. It
appears that FXa proteolyzes the major band of PN-2/A
PP at 124 kDa
and the two minor bands (105 and 98 kDa) into corresponding lower
molecular mass species (116, 97, and 90 kDa, respectively), each with
the same new amino terminus as seen on immunoblot and Coomassie-stained
gels. It is possible that FXa also cleaves PN-2/A
PP at a single
point on the carboxyl-terminal side of the protein liberating an
approximate 30-34-kDa protein. This result would explain the
intensification of the post-cleaved 90-kDa band of PN-2/A
PP seen
in Fig. 2, A and B. However, we never have
found any evidence of such a band on our Coomassie-stained gels since
it would be migrating with one of the subunits of FXa and thus be
hidden. The fact that PN-2/A
PP is a substrate to molar excess FXa
does not indicate that its mechanism of inhibition of the enzyme is
substrate inhibition. First, A
PP
is a substrate of
FXa but it is not a FXa inhibitor. Second, the isolated KPI domain of
PN-2/A
PP which does not contain the FXa cleavage site inhibits FXa
to the same degree as its parent protein. Third, the isolated KPI
domain is not cleaved by FXa. It is of interest that FXa cleaves
PN-2/A
PP through its heparin binding domain. Since heparin
neutralizes PN-2/A
PP's inhibitory activity on FXa, cleavage
through this domain may preserve the inhibitory function of
PN-2/A
PP for FXa.
PN-2/APP is a potent anticoagulant of
FXa in the prothrombinase complex. In our laboratory the K
and k
/K
ratio of prothrombin activation by FXa is 0.62 µM and 10.2 µM
min
, respectively, results which are
comparable to the findings of other investigators (Krishnaswamy et
al., 1987) using a fluorescent marker instead of a chromogenic
substrate to monitor prothrombin activation. The degree of inhibition
of FXa by PN-2/A
PP and its KPI domain is to the same order of
magnitude in the prothrombinase complex as with the isolated pure
enzyme. Regardless of the order of addition of reactants, PN-2/A
PP
and its KPI domain inhibit FXa on PSPC. PSPC in the presence of FVa
must have oriented FXa such that it was susceptible to inhibition by
PN-2/A
PP or its KPI domain even though the inhibitors were
competing with 5 orders of magnitude more substrate. The finding that
PN-2/A
PP and its KPI domain inhibit FXa in the prothrombinase
complex make this class of inhibitors more important than what would be
appreciated by just examining isolated FXa inhibition. Since
PN-2/A
PP is not a plasma protein but rather a cell
surface-associated protease inhibitor, influencing FXa activity in the
prothrombinase complex suggests that this class of Kunitz-type protease
inhibitors may be important regulators of various hemostatic enzymes.
In fact, PN-2/A
PP and its homologue, amyloid
-protein
precursor-like protein-2, may constitute a new class of serine protease
inhibitors modulating hemostasis (Sprecher et al., 1993).
Although our investigations show that artificial agents can
influence the degree of FXa inhibition by PN-2/APP or antithrombin
III and heparin, in the prothrombinase complex assembly, PN-2/A
PP
and antithrombin and heparin were comparable inhibitors. In the absence
of added heparin, the degree of antithrombin III inhibition of FXa was
orders of magnitude less potent than that seen with PN-2/A
PP. In
plasma, antithrombin III would be the predominant inhibitor because its
plasma concentration is 4 µMversus the 30 nM level of PN-2/A
PP which may be achievable in plasma when
platelets are activated (Van Nostrand, et al., 1991). However,
it is not known which may be the predominant inhibitor on cell
membranes. The second-order rate constants for antithrombin III and
heparin inhibition of FXa that we obtained were 1-2 orders of
magnitude higher than that reported by other investigators (Olson et al., 1992; Ellis et al., 1982). Differences in
heparin preparations and concentrations and ionic strengths in the
buffers may account for these variations. Alternatively, in the
presence of heparin, PN-2/A
PP did not inhibit FXa. PN-2/A
PP
is known to have a heparin binding domain which allows heparin to
potentiate its inhibition of factor XIa but not factor IXa (Smith et al., 1990; Van Nostrand et al., 1990b; Schmaier et al., 1993). It is of interest that the FXa cleavage site on
PN-2/A
PP is at arginine 102 which is in the heparin binding region
of the protein (Small et al., 1994). Excess FXa could be
preventing PN-2/A
PP from associating with heparin on cell
membranes. These investigations show that PN-2/A
PP in the absence
of heparin and antithrombin III in the presence of heparin are the
naturally occurring inhibitors of FXa. Isolated KPI domain of
PN-2/A
PP is a fragment from a naturally occurring human protein
which may have potential use as an anticoagulant since its inhibitor
activity is equal to the tick anticoagulant peptide (Waxman et
al., 1990).