(Received for publication, September 30, 1994; and in revised form, January 13, 1995)
From the
Activated platelets expose a specific, reversible high affinity (K
10 nM)
binding site (n
1500 sites/platelet) for factor XI that
requires the presence of high molecular weight kininogen (HK) and
ZnCl
(Greengard, J. S., Heeb, M. J., Ersdal, E., Walsh, P.
N., and Griffin, J. H.(1986) Biochemistry 25,
3884-3890). Synthetic, conformationally constrained peptides from
four tandem repeat (Apple) domains were tested for their capacity to
inhibit
I-factor XI binding to platelets. A peptide from
the Apple 3 (A3) domain (Asn
-Arg
) inhibits
factor XI binding to platelets in the presence of HK (42 nM),
CaCl
(2 mM), and ZnCl
(25
µM), with a K
10
nM which is identical to the K
for factor XI binding to platelets. A peptide from the A1 domain
(Phe
-Ser
) partially inhibits factor XI
binding to platelets (K
6
µM) by inhibiting factor XI binding to HK, whereas
peptides from the A2 and A4 domains have no effect. Using computer
modeling for rational design, conformationally constrained peptides
were synthesized (Pro
-Gln
,
Thr
-Leu
, and
Ser
-Ser
) each of which acted alone and
synergistically when added together to inhibit factor XI binding to
platelets. Finally, the
I-labeled A3 domain peptide
(Asn
-Arg
) was found to bind to
thrombin-activated platelets in a specific, reversible, and saturable
manner. Thus, the sequence of amino acids Asn
-Arg
of the A3 domain of factor XI comprises a contact surface for
interaction with a platelet receptor.
Factor XI, an important component in the contact phase of
intrinsic coagulation, is a homodimer (160,000 daltons) consisting of
two identical disulfide-linked polypeptide chains each of which is
cleaved at a single peptide bond by factor XIIa to give rise to factor
XIa(1, 2) . This glycoprotein circulates in plasma as
a complex with its cofactor high molecular weight kininogen
(HK)()(3, 4) . The activation of factor XI
involves the proteins factor XII, prekallikrein, and HK. It has been
shown that these proteins are assembled on negatively charged surfaces
and form a contact factor complex capable of generating factor XIa
activity on the surface. Previous studies, from which this model was
derived, employed nonphysiological charged surfaces such as kaolin,
glass, or dextran sulfate(5) . Useful as these studies have
been for elucidating molecular events in contact activation, little is
known of physiological sites involved in intrinsic coagulation. It has
been demonstrated in experiments using highly purified contact factors
and platelets that activated platelets in the presence of HK promote
the proteolytic activation of factor XI by factor XIIa(6) . It
was demonstrated that the proteolytic cleavage products of activated
factor XI are associated with platelets(6) . In addition, it
was determined that both HK (7) and factor XI (8) bind
specifically and with high affinity to the surface of stimulated human
platelets in the presence of Zn
and Ca
ions(8) . The present study was undertaken to examine the
interactions of factor XI with the platelet surface and thereby
determine which domain(s) may serve as a contact site of interaction.
Four repeat sequences (designated A1, A2, A3, and A4 or Apple domains)
are present in the heavy chain of factor XI. Their primary structure
has been elucidated from the sequence of a cDNA insert coding for
factor XI(2) . We have previously reported evidence for the
presence of a HK-binding site in the A1 domain, a substrate binding
site for factor IX in the A2 domain, and recently a binding site for
factor XIIa in the A4
domain(9, 10, 11, 12) . Evidence for
a specific high affinity binding site in the A3 domain of the heavy
chain of factor XI that is an important contact site for the
interaction with platelets is reported in the present study.
Figure 1:
The effect of factor XI heavy
chain-derived synthetic peptides on clotting activity. The clotting
activity was determined, as described under ``Materials and
Methods,'' as a function of the concentration of the peptide given
in the abscissa. The symbols denote assays in which platelets
(4.2 10
platelets/ml) (
) were substituted for
phospholipids (
).
Figure 2:
Effects of factor XI heavy chain synthetic
peptides on the binding of I-factor XI binding to
platelets. The effects of factor XI and various synthetic peptides on
the binding of
I-factor XI to platelets were examined
including: factor XI (
), Phe
-Ser
(
), Ala
-Ala
(
),
Asn
-Arg
(
),
Ala
-Gly
(
).
I-factor XI
(0.2 µg/ml), gel-filtered platelets (4.2
10
platelets/ml), ZnCl
(25 µM), CaCl
(2 mM), thrombin peptide (25 µM), and HK
(42 nM) were incubated for 30 min at 37 °C either with the
designated peptide at the concentration designated or with buffer
solution. Aliquots were removed and centrifuged as described under
``Materials and Methods.'' Each point is an average of
triplicate determinations and the maximum variation of counts/min bound
for each observation was <2% of total counts/min bound. One-hundred
% binding of factor XI represents an average of 100,190 cpm bound
whereas 0% binding of factor XI represents 0% bound after subtracting
an average of 140 cpm representing a control in which labeled factor XI
was incubated with platelets at 0 time.
Figure 3: Molecular model of the A3 domain of the heavy chain of factor XI. The molecular model was constructed utilizing the Sybyl Software Package (Triobos and Ass., St. Louis, MO) and a Silicon Graphics Onyx Parallel Processing Supercomputer (see ``Materials and Methods''). Using the primary structure of the A3 domain (2) and its known disulfide linkages (21) and energy minimization calculations, a plausible three-dimensional structure was calculated. The figure depicts the protein backbone with the positions of amino acids numbered according to their sequence in the mature protein.
Figure 4:
Effect of factor XI heavy chain synthetic
peptides on the binding of I-factor XI to platelets. This
experiment examines the effects of the following peptides on the
binding of
I-factor XI to platelets:
Pro
-Gln
(c) (
);
Thr
-Leu
(c) (
);
Ser
-Ser
(c) (
);
Ala
-Ser
(c) (
);
Gln
-Asn
(c) (
);
Ser
-Lys
(c) (
); the following peptides
were added in combination at equimolar concentrations:
Pro
-Gln
(c) plus
Thr
-Leu
(c) plus
Ser
-Ser
(c) (
).
I-factor
XI (0.2 µg/ml), gel filtered platelets (4.2
10
platelets/ml), ZnCl
(25 µM), CaCl
(2 mM), thrombin receptor peptide, SFLLRN-amide (25
µM), and HK (42 nM) were incubated for 30 min at
37 °C either with the designated peptide(s) at the concentration
designated or with buffer solution. Aliquots were removed, and
separation of bound from free ligand was accomplished as described
under ``Materials and Methods.'' Each point is an average of
triplicate determinations. When
I-factor XI was incubated
with platelets at 0 time, the amount of
I-factor XI bound
was <1% of the control value (incubated for 30 min), and the maximum
variation of counts/min bound for each experimental observation was
<2% of total counts/min bound. One-hundred % binding of factor XI
represents an average of 100,580 cpm bound whereas 0% binding of factor
XI represents 0 cpm bound after subtracting an average of 120 cpm
representing the negative control in which
I-factor XI
was incubated with platelets at 0 time.
Figure 5:
Binding of I-Asn
-Arg
to platelets.
Platelets were incubated without stirring at 37 °C with
I-Asn
-Arg
(1.85 µM or 6.34 µg/ml), HK (5 µg/ml), ZnCl
(25
µM), CaCl
(2 mM), and thrombin
receptor peptide, SFLLRN-amide (25 µM) (
), all the
above components in the absence of ZnCl
(
), in the
absence of CaCl
(
), or in the absence of HK
(
). At the times indicated, aliquots were removed and centrifuged
as described under ``Materials and Methods.'' Each point is
an average of triplicate determinations and the maximum variation of
counts/min bound for each experimental observation was <2% of total
cpm bound.
Figure 6:
Saturable, specific binding of I-Asn
-Arg
to platelets. Platelets
were incubated at 37 °C with ZnCl
(25 µM),
CaCl
(2 mM), thrombin receptor peptide,
SFLLRN-amide (25 µM), HK (42 nM), and mixtures of
I-factor XI Asn
-Arg
and
unlabeled factor XI at various concentrations. Binding was determined
at 30 min. A, amount of Asn
-Arg
bound at different input concentrations. Specific binding (
)
is shown after subtracting the amount bound in the presence of a large
molar excess of unlabeled factor XI. B, Scatchard analysis of
the data shown in A. The line is best fit of data from
triplicate samples. The apparent dissociation constant and number of
binding sites were calculated as
parameters.
Stimulated platelets have been demonstrated to promote the
assembly of contact factors leading to factor XI activation (6) in part by exposing binding sites for factor
XI(8) . Specific, high affinity factor XI binding occurs at
physiological levels of the metal ions ZnCl and CaCl
when HK is present(8) . It was demonstrated that binding
required platelet stimulation and was specific, reversible, and
saturable(8) . Scatchard analysis of the binding yielded
approximately 1,500 binding sites/platelet with an apparent
dissociation constant of approximately 10 nM(8) . The
similarity between the concentrations of the metal ions optimal for
factor XI binding and those optimal for HK binding (7) suggests
the possibility that factor XI and HK may form a complex on the
platelet surface as they do in solution and on negatively charged
surfaces(5) . The purpose of the present study was to
investigate the role of the Apple domains in the binding of factor XI
to platelets and to delineate a sequence of amino acids which may
indirectly or directly bind the platelet surface.
One important
conclusion derived from our study is that platelets make a unique and
an important contribution to the in vitro clotting process
since the A3 peptide (Asn-Arg
) was a potent
inhibitor of clotting activity in the presence of platelets but not in
the presence of phospholipids (Fig. 1). This key experiment also
gave us a clue as to the role of the A3 domain in the interaction of
factor XI with the platelet surface. The results of this and other
experiments reported here support the hypothesis that a sequence of
amino acids (Pro
-Ser
) in the A3 domain of
the factor XI heavy chain region contains three antiparallel
-strands connected by
-turns, which comprise a continuous
surface that interacts with a platelet receptor. The evidence
supporting this possibility is listed as follows: 1) an A3-derived
peptide (Asn
-Arg
) inhibits
I-factor XI binding to platelets with a K
(
10 nM) equivalent to that observed with unlabeled
factor XI (Fig. 2), suggesting that the peptide as well as
factor XI may bind directly to the platelet surface. 2) A model of the
A3 domain derived from energy minimization computations (Fig. 3)
predicts the presence of three
-stranded loop structures
(Pro
-Gln
,
Thr
-Leu
, and
Ser
-Ser
) that may fold together to form a
solvent-accessible surface comprising a binding site for platelets. 3)
Based on this model three conformationally constrained peptides were
synthesized all of which were found to inhibit the binding of
I-factor XI to platelets (Fig. 4). 4) Using
equimolar mixtures of the three folded peptides, it was found that the
inhibitory effects were mildly synergistic (Fig. 4). 5) A
tyrosinated and radiolabeled A3 domain peptide
(Asn
-Arg
) encompassing this putative
solvent-accessible surface is able to bind directly to the activated
platelet surface (Fig. 5), in a specific and saturable manner (Fig. 6) in the absence of HK and the metal ions CaCl
and ZnCl
which are required for factor XI binding to
platelets (Fig. 5).
We have used computer modeling to predict
the secondary and tertiary structures of the A3 domain of factor XI
that appears to form a contact surface with platelets. In the absence
of defined structural information from x-ray crystallography or nuclear
magnetic resonance studies, this model was derived from molecular
dynamic, energy minimization calculations which suggested that this
domain may have a structural motif consisting of three antiparallel
-strands connected by
-turns. This construct was used to
design synthetic peptides conformationally constrained with disulfide
bonds. Thus, the model has been used as a tool to generate an
hypothesis about the possible secondary and tertiary structure of the
A3 domain that was then tested in functional studies of factor XI
binding to platelets. Our results are consistent with the possibility
that the predicted structural motif (Fig. 3) has some validity.
However, the model is not presented as evidence of the
three-dimensional structure of the A3 domain, which can only be
obtained using physical techniques.
The optimal binding of factor XI
to activated platelets requires the presence of Zn ions, calcium ions, and the cofactor HK (8) . The fact
that HK binding to platelets also requires Zn
and
Ca
ions, but occurs in the absence of factor
XI(7) , suggests the possibility that the factor XI-HK complex
that circulates in plasma (3, 4) may bind to activated
platelets via the platelet binding site for HK(7) . However,
the A3 domain-derived peptide Asn
-Arg
is
able to bind directly to platelets in the absence of HK,
CaCl
, and ZnCl
(Fig. 5). Moreover, not
only are the unlabeled peptide (Asn
-Arg
)
and factor XI identical in their capacity to inhibit
I-factor XI binding to platelets (Fig. 2), but the
iodinated peptide binds directly to a similar number of sites (n = 1,035) as factor XI with similar affinity (K
21 nM; Fig. 6). This
suggests that the A3 domain contains a solvent-accessible surface
(Asn
-Arg
) that contains all the structural
information required for binding of factor XI to the platelet surface.
We have also demonstrated that the A1 domain peptide
(Phe
-Ser
) is a less potent, partial inhibitor
of factor XI binding to platelets (Fig. 2). We have shown that
this peptide comprises the binding site for HK in the A1 domain of
factor XI(9, 11) . Thus, the binding of factor XI to
platelets requires an interaction of the A1 domain with HK in the
presence of ZnCl
. In contrast, the interaction of platelets
with the A3 domain peptide (Asn
-Arg
), and
thus with the A3 domain of factor XI, appears to be direct and does not
require ions or cofactors. It therefore seems reasonable to suggest
that the binding of HK to factor XI may regulate the binding of factor
XI directly to a platelet receptor, possibly by inducing a
conformational alteration in factor XI that exposes the A3
domain-binding site. Thus, the initial event in the interaction of
factor XI with platelets is the binding of factor XI to HK through the
A1 domain followed by conformational changes in the heavy chain which
permit the interaction of the A3 domain with platelets. The binding of
HK to its own receptor on the activated platelet surface (7) then may serve to further stabilize the factor XI
HK
complex on the platelet surface, or alternatively, the two proteins may
even dissociate from one another once individually bound to platelets
although there is no evidence to either confirm or refute this
possibility.
The plasma protein prekallikrein contains four tandem
repeat Apple domains which have 58% amino acid identity with the Apple
domains of factor XI(2) . Our studies show that neither
prekallikrein nor its A3 domain peptide,
Asn-Arg
, which is 53% identical to our
factor XI A3 domain peptide (Fig. 7), is capable of competing
with factor XI for platelet binding sites. This observation provides
strong evidence of the specificity of factor XI binding to platelets.
In addition, we reasoned that an examination of the A3 domain in
prekallikrein might give us important clues about which amino acids are
present on the surface of the factor XI A3 domain that are involved in
binding to platelets. As shown in Fig. 7, although a 53% amino
acid sequence identity exists between factor XI and prekallikrein in
the region between amino acids 235 and 266, there is one stretch of
nine amino acids(249-257) that are entirely different in factor
XI compared with prekallikrein. Interestingly, this sequence
corresponds closely with the sequence of the most active of the three
conformationally constrained A3 peptides, Ser
-Ser
( Table 1and Table 2and Fig. 4). Inspection
of the molecular model of the A3 domain (Fig. 3) suggests that a
surface loop structure consisting of residues KKSKAL(252-257)
might comprise the primary platelet binding site in factor XI and that
additional amino acid sequences contained within the region
Pro
-Arg
(i.e. Pro
-Gln
, and
Thr
-Leu
) might provide accessory
platelet-binding sites.
Figure 7: Comparison of amino acid sequences of portions of the Apple 3 domains of factor XI and prekallikrein (PK). The positions that have identical residues are boxed. The primary structure of the A3 domain in factor XI and prekallikrein (2) was utilized.