(Received for publication, July 10, 1995; and in revised form, November 22, 1995)
From the
Previously we defined a binding site for high molecular weight
kininogen (HK) in the A1 domain of factor XI (FXI). Since thrombin can
activate FXI and HK inhibits the activation of FXI by thrombin, we have
identified a thrombin binding site in FXI. Both the recombinant A1
domain (Glu-Ser
) and a synthetic peptide
(Phe
-Ser
) containing the HK binding
site inhibited FXI activation by thrombin. Both a monoclonal antibody,
5F7, recognizing the A1 domain, and the rA1 domain were shown to be
competitive inhibitors of thrombin-catalyzed FXI activation. The
peptides Ala
-Arg
and
Val
-Arg
acted synergistically to
inhibit FXI activation by thrombin. Mutant rA1 domain constructs
(Val
Ala and Ile
Ala), which do
not inhibit FXI binding to HK, retain full capacity to inhibit FXI
activation by thrombin. The peptide Ala
-Arg
inhibited thrombin-catalyzed FXI activation, whereas it had no
effect on FXI binding to HK. In contrast, the peptide
Asn
-Leu
(which inhibited FXI binding to
HK) did not inhibit FXI activation by thrombin. Thus, a thrombin
binding site exists in the A1 domain of FXI spanning residues
Ala
-Arg
that is contiguous with but
separate and distinct from the HK binding site. These sites may
regulate which ligand is bound to FXI and through which pathway FXI is
activated.
Factor XI (FXI) ()is a homodimeric plasma
glycoprotein that circulates as a complex with its cofactor high
molecular weight kininogen (HK) (1, 2) and is
proteolytically activated on negatively charged surfaces by FXIIa to
give rise to
FXIa(3, 4, 5, 6, 7, 8, 9, 10) .
The mechanism, involving interactions of FXII, prekallikrein (PK), and
HK, by which contact activation is initiated and its significance in vivo have yet to be established, since individuals
congenitally deficient in any one of these contact factors (FXII, HK,
and PK) do not experience abnormal bleeding, suggesting that these
proteins are not required for coagulation in
vivo(11, 12) . In contrast, a deficiency of FXI
can result in excessive bleeding after trauma or
surgery(13, 14) . These observations suggest that FXI
may be activated in vivo by a protease(s) other than FXIIa.
The ability of thrombin, an enzyme generated late in the coagulation cascade, to activate FXI has been demonstrated(15, 16) . The site at which FXI is cleaved by thrombin is identical to that cleaved by FXIIa (16, 17) . Determination of the kinetic parameters of FXI activation by thrombin and FXIIa indicate that at a physiological concentration of FXI, in the presence of dextran sulfate, thrombin would be the more potent activator(16) . Although FXI is readily activated by thrombin in a purified system with dextran sulfate present, the reaction may not proceed as readily in plasma(15, 16, 18) , since although HK promotes the FXIIa-mediated reaction it inhibits thrombin-catalyzed activation of FXI(15, 16, 18) . These observations raise the following two related questions. Is thrombin a physiological activator of FXI in plasma? What is the mechanism by which HK can inhibit thrombin-catalyzed FXI activation?
The present study was undertaken to determine the sequence of amino acids in FXI that mediate its interaction with thrombin. Clarification of the mechanism of interaction of these two proteins might also help to elucidate the physiological importance of thrombin-catalyzed FXI activation. Four tandem repeat sequences (designated A1, A2, A3, and A4 or Apple domains) are present in the heavy chain of FXI(7) . We have previously reported evidence for the presence of an HK binding site in the A1 domain(19, 20) , a binding site for FXIIa in the A4 domain(21) , a substrate binding site for FIX in the A2 domain(22) , and recently, a specific binding site for platelets in the A3 domain(23) . Evidence for a binding site in the A1 domain of FXI that is important for interaction with thrombin is reported in the present study.
To determine whether the A1 domain peptides
(Glu,
Phe
-Ser
,
Ala
-Arg
) inhibit the activity of
-thrombin, the peptide was incubated with
-thrombin for 5 min
at 37 °C, and the mixture was assayed using the chromogenic
substrate S-2238 (H-D-Phe-pip-Arg-pNA, Kabi Vitrum, Stockholm, Sweden)
at concentrations of 3 µM, 10 µM, and 1
mM.
To determine whether the A1-derived peptides inhibit
the enzymatic activity of thrombin in the conversion of fibrinogen to
fibrin, an established procedure (31) was employed in which 0.1
ml of human fibrinogen (Sigma), 1.5 mg/ml was diluted in 0.8 ml of 0.05 M Tris-HCl, 0.1 M NaCl, pH 7.4, to which was added
0.1 ml of -thrombin (5 units/ml) that had previously been
incubated with A1 domain peptides
Glu
-Ser
,
Phe
-Ser
, or
Ala
-Arg
. The increase in turbidity was
recorded for 10 min.
Figure 1:
Effects on the rate of activation of
FXI (60 nM) by thrombin (1.25 nM) of the presence of
various concentrations of monoclonal antibodies (A) or heavy
chain-derived peptides (B). FXI was incubated with either
buffer or various concentrations of the antibody solution or peptides
prior to use in the assay. The rate of FXIa formation was determined as
described under ``Materials and Methods.'' A, data
shown are those obtained with monoclonal antibodies, 5F7
(-
), 3C1 (
-
), 1F1
(
-
), and 5F4 (
-
).
Antibody 5F7 is immunologically reactive to the A1 domain of
FXI(19) . Antibody 3C1 is heavy chain-specific, is
immunologically cross-reactive with the A2 domain(36) , and is
a competitive inhibitor of FIX activation by
FXIa(24, 33) . Antibody 5F4 is light
chain-specific(24, 33) . Antibody 1F1 is also directed
against the heavy chain of FXI(24) . B, data shown are
those obtained with Gly
-Lys
(
-
), Ala
-Ala
(
-
), Asn
-Arg
(
-
), rA2,
Ser
-Ala
(
-
),
Phe
-Ser
(
-
),
and rA1, Glu
-Ser
(
-
).
Figure 2:
Effects on the rate of activation of FXI
(60 nM) by thrombin (1.25 nM) of the presence of
various concentrations of recombinant peptides. Thrombin was incubated
with either buffer or various concentrations of peptides prior to
addition to the assay. The rate of FXIa formation was determined as
described under ``Materials and Methods.'' Data shown are
those with Gln-Ser
(
-
), Glu
-Ser
(Ile
Ala
)
(
-
), Glu
-Ser
(Val
Ala
)
(
-
) and Ser
-Ala
(
-
).
Previously we have
identified specific amino acid residues within the A1 domain involved
in binding HK(19, 20, 37) . Utilizing
mutational analysis we have determined that the binding of FXI to HK is
mediated at least in part by Val and Ile
in
the A1 domain of FXI(37) . Therefore, we examined the effects
of mutations of these two residues on the capacity of the rA1 domain to
inhibit thrombin-catalyzed FXI activation. We found that mutant rA1
domain constructs (Val
Ala and Ile
Ala), which have lost the capacity to inhibit FXI binding
to HK(37) , retain the full capacity of the rA1 domain
(Glu
-Ser
) to inhibit thrombin-catalyzed
FXI activation (Fig. 2). Therefore, the binding sites for HK and
thrombin in the A1 domain, although contiguous, are apparently separate
and distinct. Another experiment that supports this conclusion is that
after reduction and alkylation, the rA1 domain
(Glu
-Ser
) retains the capacity to
inhibit FXI binding to HK (IC
10
M)(19, 20, 37) , whereas it is
unable to inhibit thrombin-catalyzed FXI activation (data not shown).
Figure 3:
Effects on the rate of activation of FXI
(60 nM) by thrombin (1.25 nM) of the presence of
various concentrations of heavy chain-derived peptides. Thrombin was
incubated with either buffer or various concentrations of peptides
prior to addition to the assay. The rate of FXIa formation was
determined as described under ``Materials and Methods.'' A, data shown are those with Phe-Gly
(PK) (
-
),
Phe
-Ser
(FXI)
(
-
),
Ala
-Arg
(C) (
-
),
and the following peptides were added in combination at equimolar
mixtures: Ala
-Arg
(C) plus
Phe
-Ser
(
-
). B, data shown are those with Val
-Arg
(Glu
Ala
)
(
-
),
Val
-Arg
(C)
(
-
),
Ala
-Arg
(C) (
-
),
and the following peptides were added in combination at equimolar
mixtures: Ala
-Arg
(C) plus V59-R70(C)
(
-
). C, data shown are those with
Ala
-Arg
(C) (Glu
Ala) (
-
), Ala
-Arg
(
-
),
Ala
-Arg
(C) (Asp
Ala) (
-
), and
Cys
-Cys
(C)
(
-
).
Prekallikrein, a protein with 58% sequence identity to
FXI, also binds HK in the A1 domain within the homologous amino acid
sequence Phe-Gly
(38) . This
stretch of amino acids displays 65% homology with a comparable sequence
in FXI. Therefore, we tested the PK Phe
-Gly
peptide for its ability to inhibit thrombin-catalyzed FXI
activation. Unlike the FXI Phe
-Ser
peptide, the PK Phe
-Gly
peptide
did not inhibit thrombin-catalyzed FXI activation (Fig. 3A). It has been reported that this sequence of
amino acids in PK (Phe
-Gly
) binds HK (38) as does the homologous sequence of
FXI(19, 20) . Thus, the amino acid sequences involved
in FXI and PK interaction with HK are not involved in binding thrombin.
Figure 4:
Double-reciprocal plots of the activation
of FXI by thrombin in the presence of various concentrations of 5F7
monoclonal antibody (A) and rA1 domain peptide
Glu-Ser
(B). A, FXI
was incubated with various concentrations of antibody 5F7 at 37 °C
and added to the assay buffer (TBSA). Final concentration of thrombin
was 25 nM. FXI was used in the range of 16-50
nM, and the rate of FXIa formation was determined as described
under ``Materials and Methods.'' The results shown are
double-reciprocal plots in the absence (
-
) and
in the presence of antibody at concentrations of 3
10
M (
-
), 9
10
M (
-
), 1.2
10
M (
-
), and 1.8
10
M (
-
). The inset is the secondary plot of I
peptide
concentration versus substrate concentration as described by
Cha(35) . B, thrombin (25 nM) was incubated
with various concentrations of peptide for 15 min at 37 °C. FXI was
used in the range of 16-50 nM as shown on the abscissa, and the rate of FXIa formation was determined as
described under ``Materials and Methods.'' The results are
shown as double-reciprocal plots in the absence
(
-
) and in the presence of peptide
Glu
-Ser
at concentrations of 2.8
10
M (
-
), 2.8
10
M (
-
),
2.8
10
M (
-
), and 2.8
10
M (
-
). The inset is
the secondary plot of I
peptide concentration versus substrate concentration.
It is possible to activate FXI in the absence of contact proteins in the presence of the serine protease thrombin(15, 16, 18) . Consequently, several laboratories have attempted to determine the physiological conditions required for the activation of FXI by thrombin or other proteases(16, 18, 39) . Although FXI is activated by thrombin in a purified system, it is suspected that this reaction may not proceed in plasma(18, 39) . It is well known that HK enhances FXIIa-mediated activation of FXI in vitro(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) . However, HK (500 nM) inhibits thrombin-mediated activation of FXI (15, 16, 18, 39) . Thus, thrombin may not be a suitable activator of FXI in plasma. To understand the physiological importance of thrombin-mediated FXI activation, we have examined the interaction of FXI with thrombin and identified the amino acid sequence in FXI that interacts with thrombin.
Our experiments support the conclusion that a sequence of amino
acids (Ala-Lys
) in the A1 domain of FXI
that contains two antiparallel
-strands connected by
-turns
comprises a surface that interacts with a substrate (FXI) binding site
within thrombin (Fig. 5). The evidence supporting this
conclusion is as follows: 1) a monoclonal antibody (5F7) that binds to
the A1 domain of FXI (19, 20, 36) can
completely block thrombin-catalyzed FXI activation with a K
5
10
M (close to the K
for 5F7 binding to FXI; see (36) and Fig. 1and Fig. 4); 2) the rA1 domain
peptide (Glu
-Ser
) inhibited the
activation of FXI by thrombin with a K
of 5
10
M ( Fig. 1and Fig. 4); 3) a molecular model of the A1 domain (Fig. 5)
predicts the presence of three peptide loop structures,
Ala
-Arg
,
Val
-Arg
, and
Asn
-Lys
, that form a solvent-exposed
surface(37) ; 4) based on this model, conformationally
constrained peptides were synthesized, two of which
(Ala
-Arg
,
Val
-Arg
) act synergistically to inhibit
thrombin-catalyzed FXI activation (Fig. 3); and, 5)
Lineweaver-Burk plots of the activation of FXI by thrombin in the
presence of either the monoclonal antibody 5F7 or the rA1 domain
peptide yielded patterns consistent with a classical competitive
inhibition (Fig. 4).
Figure 5:
Molecular model of the A1 domain of FXI.
Using the primary structure of the A1 domain and its known disulfide
linkages, a model was calculated demonstrating antiparallel
-strands connected by
-turns(20) . The colors represent the peptide loop structures (
-carbon backbone) and
amino acids postulated to comprise binding sites for thrombin (yellow) (residues 45-58), HK (blue) (residues
72-85) and a loop structure utilized for binding both (or either)
protein (red) (residues
59-71).
We have previously characterized a
binding site for HK in the A1 domain of
FXI(19, 20, 36, 37) . To perform
fine mapping of this site we prepared conformationally constrained
synthetic peptides and rA1 domain constructs(20, 37) .
To identify specific amino acid residues involved in HK binding,
conformationally constrained peptides were synthesized containing
conservative amino acid substitutions at residues suspected to contain
side chains involved in binding including Val
Ala,
Glu
A, Arg
Ala, and Ile
Ala(37) . Because abnormal results were obtained
with two of these peptides, Val
Ala and Ile
Ala, which failed to compete normally with FXI for binding HK, we
prepared two mutant rA1 domains (Val
Ala and
Ile
Ala), both of which exhibited diminished capacity to
inhibit FXI binding to HK(37) . Since the thrombin binding site
was localized to the A1 domain and found to contain amino acid
sequences overlapping the HK binding site (Fig. 1), we attempted
to identify specific amino acid residues in the A1 domain that might
bind thrombin. Our results are consistent with the following
conclusions: 1) Val
and Ile
, which are
important as contact sites for HK(37) , do not participate in
the interaction of the A1 domain with thrombin (Fig. 2); 2)
Glu
and Asp
, which are not important as
contact sites for binding HK(37) , are both apparently
important residues for binding thrombin (Fig. 3); and 3) another
important difference between the HK and thrombin binding sites in the
A1 domain is that reduction and alkylation of the A1 domain virtually
destroys the thrombin binding site while leaving the HK binding site
intact(37) .
We also examined the plasma protein PK, which
shares a high degree (58%) of sequence identity with FXI(7) ,
to determine whether homologous amino acid sequences can also inhibit
thrombin-catalyzed FXI activation. Unlike the FXI
Phe-Ser
peptide, the PK
Phe
-Gly
peptide did not inhibit
thrombin-catalyzed FXI activation. A glutamic acid is replaced by a
glycine at position 66 in PK, and this amino acid substitution
Glu
Ala
in FXI
Val
-Arg
rendered this peptide inactive (Fig. 3). It is possible that this and other amino acid
replacements in PK render PK Phe
-Gly
unable to inhibit thrombin-catalyzed FXI activation. It has also
been established that PK, like FXI, also binds HK in the A1 domain
within the homologous amino acid residues
Phe
-Gly
(38) . This stretch of
amino acids is 65% identical to the comparable sequence in FXI (Fig. 6). Moreover, a peptide with the amino acid sequence
Pro
-Lys
of PK had no effect on
thrombin-catalyzed FXI activation, unlike
Ala
-Arg
of FXI, which was the most
effective inhibitor ( Fig. 3and Table 2). These two
regions of FXI and PK have only 18% identity (Fig. 6), and it
seems reasonable that PK does not contain a binding site for thrombin
since thrombin does not activate PK(40) . We have also
attempted to fine map the important contact sites of peptide
Ala
-Arg
(C) that interacts with
thrombin. Apparently, Asp
is important in this
interaction. However, amino acid residues in the first half of the
peptide Ala
-Pro
also appear to be
important in its interaction with thrombin (Fig. 3).
Figure 6: Comparison of amino acid sequences of portions of the Apple 1 domains of FXI and PK. The positions that have identical residues are boxed. The primary structure of the A1 domain in FXI and PK (7) was utilized.
The data
presented in this paper support the conclusion that the thrombin and HK
binding sites in the A1 domain, while contiguous, are separate and
distinct. However, these two binding sites appear to overlap since they
share a common sequence of amino acids
(Val-Arg
). The relationship between the
putative thrombin and HK binding sites is depicted in the molecular
model shown in Fig. 5. The model and our results predict that if
HK is bound to the A1 domain, thrombin-mediated activation of FXI would
be blocked and FXIIa-mediated activation of FXI would be favored. The
reverse may also occur, i.e. the binding of thrombin to the A1
domain should prevent HK binding and FXIIa-mediated activation of FXI.
Therefore, these two contiguous partially overlapping sites could
constitute a point of regulation to determine by which pathway (contact
activation versus feedback activation) and by which protease
(FXIIa or thrombin) FXI might be activated.