(Received for publication, November 28, 1995; and in revised form, February 12, 1996)
From the
(platelet membrane
glycoprotein IIb-IIIa) and
are
members of the
subfamily of integrin adhesion
receptors. A cyclic peptide, KYGC(s-s)HarGDWPC(s-s) (cHarGD),
originally described by Scarborough et al. (Scarborough, R.
M., Naughton, M. A., Teng, W., Rose, J. W., Phillips, D. R., Nannizzi,
L., Arsten, A., Campbell, A. M., and Charo, I. F.(1993) J. Biol.
Chem. 268, 1066-1073) has been employed as a high affinity
ligand for
to examine the
specificity of the
integrins. cHarGD interacted with
high affinity with purified
(K
= 10 nM) or with
platelets (K
= 120 nM).
While cHarGD was specific for
in
the presence of Ca
, it bound to both
integrins in the presence of Mn
. Barbourin, a
snake venom disintegrin containing a reactive KGD sequence, remained
-specific, even in the presence of
Mn
. cHarGD became cross-linked to a site in
of
, which is
distinct from that of RGD peptides. These results allow identification
of at least four classes of
ligands: Class I,
represented by RGD peptides and vitronectin, react similarly with
and
; Class II, represented by cHarGD,
-chain peptides and fibrinogen, react with both receptors in the
presence of Mn
but only with
in the presence of
Ca
; Class III, represented by barbourin, are
-specific under all cation
conditions; Class IV, represented by osteopontin, bind primarily to
.
(platelet membrane
glycoprotein IIb-IIIa) and
are
members of the
subfamily of integrin heterodimeric
adhesion receptors (1, 2, 3, 4) .
is essential for platelet
aggregation and controls platelet function in thrombosis and hemostasis (5, 6, 7) .
is expressed on many cells, where it influences cell migration
and impacts on angiogenesis, restenosis, tumor cell invasion, and
atherosclerosis(8, 9, 10) . These two
integrins share the same
subunit,
, but have
distinct
subunits;
and
exhibit about 36% primary amino acid sequence
identity(11) . Both integrins are oligospecific: their common
macromolecular ligands include fibrinogen, fibronectin, thrombospondin,
von Willebrand factor, and
vitronectin(12, 13, 14, 15) . These
common ligands contain Arg-Gly-Asp (RGD) sequences, and RGD-containing
peptides inhibit binding of these ligands to both
receptors(16) . When RGD peptides were bound and cross-linked
to
and
, the cross-linking sites in both
receptors are overlapping and confined to a stretch of about 100 amino
acids in
(17, 18) . Moreover, single
point mutations at several positions within
abrogated
the binding of RGD peptides and macromolecular ligands to both
receptors(19, 20, 21, 22) . These
data are compatible with a role of the common
subunit
of both receptors in establishing their RGD ligand recognition.
In
addition to their common RGD recognition, several ligands with
proported specificity for or
have been identified. The prototype
of these are the fibrinogen
-chain peptides. Peptides with
sequences corresponding to the carboxyl terminus of the
-chain of
fibrinogen block ligand binding to
, but are poor inhibitors of ligand
binding to
(16) .
-Chain peptides cross-link to
, specifically to
294-314, providing a basis for
selective
recognition(23) . Barbourin, a member of the disintegrin family
of snake venom proteins, was reported to inhibit the binding of
fibrinogen to
but not to block
vitronectin binding to
(24) .
In contrast, several other disintegrins block ligand binding to both
receptors. Distinguishing barbourin from other disintegrins is the
presence of a KGD, rather than a RGD, sequence; and the Lys residue is
critical for specificity(24) . In following this observation,
Scarborough et al.(25) synthesized a series of
peptides and ultimately identified small, conformationally constrained
cyclic derivatives as highly potent agonists of
function. A cyclic homoarginine
(Har)(
)-glycine-aspartic acid-containing peptide was one of
the most potent representative of this series. This compound showed
substantial specificity for
versus
, as compared
to linear RGD-containing peptides, although it could block
-mediated cell adhesion(25) .
Divalent cations are required to support the ligand binding
functions of the integrins described above as well as
for the recognition of most ligands by
integrins(26, 27) . This requirement depends on the
direct binding of cations to the integrin subunits, and it is generally
accepted that the divalent cation binding and ligand binding sites
reside in close proximity within integrins, including the
integrins(23, 28, 29, 30) .
Ligand binding to many integrins is differentially regulated by
divalent cations. Numerous examples have been described in which the
ligand specificity of members of the
and
integrin subfamilies are divalent cation
determined(31, 32, 33, 34, 35, 36) .
Recently, differential regulation of ligand binding to
integrins also has been reported (37) as fibrinogen did
not bind to
in the presence of
Ca
, but Mn
did support this
interaction. This observation has brought the entire issue of the
relative specificity of
integrins into question.
In the present study, we have examined the interaction of a cyclic
HarGD-containing peptide (cHarGD) with and
. These studies were
initiated to test the hypothesis that cHarGD would represent a high
affinity mimetic of fibrinogen and its
-chain peptides. The high
affinity binding of this peptide to
is demonstrated by direct binding studies with the isolated
receptor and platelets. Overall, the results document the complexity of
ligand recognition by
integrins and allow
discrimination of multiple classes of
ligands.
Figure 1:
Inhibition of fibrinogen binding to
by cHarGD, RGD, and
-chain
peptides.
I-Fibrinogen (20 nM) and various
concentrations of cHarGD (
), GRGDSP (
), and
-chain
peptide HHLGGAKQAGDV (
) were simultaneously added to microtiter
wells coated with
in the presence
of 1 mM Ca
, Mg
, and
Mn
. After 3 h at 37 °C, bound fibrinogen was
quantitated by counting the bound radioactivity in a
-counter.
Binding in the absence of the peptides was assigned a value of 100%.
The data shown are means and standard deviation from three separate
experiments each with quadruplicate
measurements.
Figure 2:
Specificity of the interaction of cHarGD
with .
I-cHarGD (20
nM) was added to microtiter wells coated with
in the presence or absence of
excess unlabeled cHarGD (2 µM),
-chain peptide
HHLGGAKQAGDV (100 µM), GRGDSP (100 µM),
GRGESP (100 µM), fibrinogen (5 µM),
transferrin (5 µM), and monoclonal antibody 7E3 (20
µg/ml) in the presence of 1 mM Ca
,
Mg
, and Mn
, and incubated for 60
min at 37 °C. Values represent the means and standard deviation of
3-5 determinations and are expressed as percent of a control
lacking inhibitors.
The specific binding of I-cHarGD
to immobilized
was saturable (Fig. 3A). Addition of a 100-fold excess unlabeled
cHarGD displaced more than 95% of the bound radiolabeled cHarGD. The
data from the binding isotherm in Fig. 3A were graphed
as a Scatchard plot (Fig. 3B) which suggested that the
interaction could be described by a single class of high affinity
binding sites with an apparent dissociation constant (K
) of 9.7 ± 0.9 nM (n = 3).
I-cHarGD binding to immobilized
was observed to approach 1 to 1
molar stoichiometry of ligand to receptor. Using a radiolabeled
monoclonal anti-
, PMI-1, to quantitate the amount of
immobilized receptor, as described by Du et al.(47) ,
the ratio of B
for radiolabeled cHarGD/PMI-1 was
0.92.
Figure 3:
Saturation isotherm and Scatchard analysis
of I-cHarGD binding to
. A, binding isotherms of
cHarGD to immobilized
were
constructed by incubating increasing concentrations of
I-cHarGD with immobilized
. Specific binding was calculated
by subtracting nonspecific binding, measured as the residual binding in
the presence of a 100-fold excess of nonlabeled cHarGD, from the total
binding. The data shown are representative of three separate
experiments. B, Scatchard plots of cHarGD binding to
. The apparent dissociation
constant (K
) estimated from the slope of
the line was 9.7 ± 0.9 nM (n =
3).
In a second set of studies, I-cHarGD binding to
platelets was evaluated.
I-cHarGD did not bind to
non-stimulated platelets in the presence of Ca
or
EDTA, but the binding was supported in the presence of Mn
(Fig. 4).
I-cHarGD bound to
thrombin-stimulated platelets in the presence of Ca
as well as Mn
, and the latter cation supported
higher binding.
I-cHarGD binding to thrombin-stimulated
platelets was inhibited by
-chain peptide, RGD-containing
peptides, fibrinogen, and mAb 7E3 (data not shown), using these
reagents at the conditions specified in Fig. 2. Analysis of
saturable cHarGD binding to thrombin-stimulated platelets by Scatchard
plots indicated a single class of binding sites with an estimated K
of 120 ± 16 nM (n = 3) and a maximum of 28,700 ± 900 molecules bound
per platelet (data not shown). Taken together, these observations
indicate that cHarGD fulfills the specification of a low molecular
weight peptide ligand which binds with high affinity to
, either in purified form or on
platelets.
Figure 4:
Effect
of divalent cations on I-cHarGD binding to platelets.
Washed platelets (1
10
/ml), non-stimulated or
stimulated with
-thrombin (0.1 unit/ml), were incubated with
I-cHarGD at a final concentration of 50 nM for
15 min at 22 °C in the presence of 5 mM EDTA, 1 mM Ca
, or 1 mM Mn
as
described under ``Materials and Methods.'' The values shown
are means and standard deviation from three separate experiments
performed with triplicate measurements.
Figure 5:
Differential effects of divalent cations
on the binding of I-fibrinogen and
I-cHarGD
to
and
. The effects of divalent cations on
I-fibrinogen (A) and
I-cHarGD (B) binding to immobilized
and
were examined. Binding was
measured in the presence of 1 mM Ca
or 1
mM Mn
, and the binding of each ligand to
in the presence of 1 mM Ca
was assigned a value of 100%. The data shown
are means and standard deviation from three
experiments.
Further evidence for the
cation-dependent regulation of cHarGD with integrins
was obtained in competition experiments using fibrinogen as a
prototypic
macromolecular ligand
and vitronectin as a prototypic
ligand. While cHarGD was an effective inhibitor of fibrinogen
binding to
in the presence of
Ca
, it was a poor inhibitor of vitronectin binding to
(Fig. 6A). In the
presence of Mn
, however, cHarGD possessed a similar
inhibitory activity against vitronectin binding to
and fibrinogen binding to
(Fig. 6B). These
results clearly show that cHarGD interacts with
in the presence of Mn
although its interaction with
is minimal in the presence of Ca
.
Figure 6:
Differential effects of cHarGD on
fibrinogen binding to and
vitronectin to
. The ability of
cHarGD to block the interaction of fibrinogen (Fgn) with
(
) or vitronectin (Vn) with
(
) is
compared in the presence of 1 mM Ca
(A) or 1 mM Mn
(B).
Radiolabeled fibrinogen or vitronectin (20 nM) was added in
quadruplicate to microtiter wells coated with
or
, respectively, and incubated in the
presence of various concentrations of cHarGD for 3 h at 37 °C.
Binding was expressed as a percent of the control without cHarGD. The
data shown are representative of three separate
experiments.
In
interacting with in the presence of
Mn
, cHarGD possessed a similar inhibitory activity as
a RGD peptide; its IC
was 40 nM compared to 25
nM for GRGDSP (Fig. 7). We also found that a
-chain peptide, H12, was inhibitory (IC
= 40
µM) in the presence of Mn
although it
was substantially less potent than cHarGD. The relationship between
Mn
concentration and
I-cHarGD binding
to
was examined. Little cHarGD bound
to the receptor at Mn
concentrations below 1
µM (Fig. 8); maximal binding was supported by
concentrations above 100 µM; and 50% maximal binding was
obtained at 8 ± 1 µM (n = 3). This
latter value is similar to that determined for fibrinogen binding to
(37) .
Figure 7:
Inhibition of fibrinogen binding to
by cHarGD, RGD, and
-chain
peptides. Quadruplicate samples of
I-fibrinogen and
various concentrations of cHarGD (
), GRGDSP (
), and the
-chain peptide HHLGGAKQAGDV (
), were incubated with
immobilized
in the presence of 1
mM Mn
for 3 h at 37 °C. Binding was
expressed as percent of the control without inhibitor. The results
shown are representative of two
experiments.
Figure 8:
Effect of Mn concentration on
I-cHarGD binding to
. Quadruplicate samples of
I-cHarGD (20 nM) were incubated with immobilized
in the presence of various
concentrations of Mn
(
). Binding was measured
after 60 min incubation at 37 °C. Binding in the presence of 1
mM Ca
(
) is shown for comparison. The
experiment shown is a representative of three
determinations.
Figure 9:
Effects of divalent cations on the binding
of barbourin to and
.
I-Barbourin (20
nM) binding to immobilized
and
was examined in the
presence of 1 mM Ca
or 1 mM Mn
.
I-Barbourin binding to
in the presence of 1 mM Ca
was assigned a value of 100%. The values
shown are means and standard deviation derived from three
experiments.
Figure 10:
A, chemical cross-linking of cHarGD, RGD,
and -chain peptides to
on
platelets.
I-cHarGD (lane 1) at 100 nM was bound to thrombin-stimulated platelets for 15 min at 22 °C
and cross-linked with BS
(0.2 mM) for 10 min at 22
°C.
I-KYGRGDS (lane 2) and
I-KYGGHHLGGAKQAGDV (lane 3), each at 1
µM, were bound to thrombin-stimulated platelets for 45 min
at 22 °C and cross-linked with BS
(0.2 mM).
The cross-linked samples were extracted, analyzed on 7.5%
polyacrylamide gel under nonreducing conditions, and subjected to
autoradiography. B, cross-linking of cHarGD to
on platelets at different cation
conditions.
I-cHarGD at 100 nM was bound to
thrombin-stimulated platelets in the presence of either 1 mM Ca
(lane 1) or 1 mM Mn
(lane 2) and cross-linked with
BS
(0.2 mM).
To explore the
inter-relationship between the cHarGD and RGD cross-linking sites in
, enzymatic digestion of cross-linked samples was
performed. When
I-KYGRGDS or
I-cHarGD was
cross-linked to
, digested with
chymotrypsin and subjected to SDS-PAGE, the patterns of digestion were
exactly the same by the silver staining of the gels (Fig. 11).
However, the autoradiograms of the gels were not identical. As shown in Fig. 11, when
I-KYGRGDS-
complex
was digested with chymotrypsin, a 60-kDa chymotryptic fragment was the
major band. Chymotryptic digestion of
I-cHarGD-
complex, however, gave a
predominant 46/40 kDa doublet, and the 60-kDa band was not present.
Under reducing conditions, the
I-KYGRGDS-
digest showed no radioactive band above 20 kDa, although
I-cHarGD-
digest still contained a
doublet at a slightly increased molecular mass. These results indicate
that the site of cHarGD cross-linking differs from that of the RGD
peptide.
Figure 11:
Chymotryptic digestion of
after cross-linking to cHarGD and
RGD peptide.
I-KYGRGDS at 100 µM was bound
to purified
for 3 h at 22 °C
and cross-linked with BS
(0.1 mM) (lanes 1, 3, and 5).
I-cHarGD at 2 µM was
bound to purified
for 60 min at 22
°C and cross-linked with BS
(0.1 mM) (lanes 2, 4, and 6). Cross-linked samples were
digested with
-chymotrypsin for 18 h at 37 °C using the 1:1
(w/w) enzyme-to-substrate ratio and analyzed on 12.5% polyacrylamide
gel. Lanes 1 and 2 are silver staining of the gel
under nonreducing conditions. Lanes 3 and 4 and lanes 5 and 6 are autoradiograms of the gels under
nonreducing and reducing conditions,
respectively.
In this study, we have characterized the direct interaction
of cHarGD with and have compared
the recognition specificity of the
integrins for this
ligand and other peptide and macromolecular ligands. The following
conclusions are drawn from these analyses. First, cHarGD interacts with
, either in purified form or on
platelets, with high affinity. Second, the capacity of cHarGD to
interact with
is divalent cation
determined. Third, although cHarGD and fibrinogen are not specific for
, as they react with
in the presence of
Mn
, other ligands may still be specific for
: barbourin is a notable example.
Fourth, cHarGD becomes cross-linked to a site in
of
, which is distinct from the
previously identified RGD and
-chain cross-linking sites. Fifth,
based upon differential interactive properties, at least four
categories of
ligands can be distinguished.
cHarGD
was a potent inhibitor of fibrinogen binding to isolated
with an IC
of 10
nM. This value is identical to its estimated K
for the receptor and is very similar to the K
of barbourin and fibrinogen binding to immobilized
(24, 44) .
I-cHarGD also bound to platelets with high affinity; its K
of 120 nM for thrombin-stimulated
platelets is similar to that of fibrinogen(49) . Binding of
cHarGD to purified
or to platelets
was divalent cation dependent and was inhibited by fibrinogen, mAb 7E3,
-chain peptide, and RGD-containing peptides. Taken together, these
characteristics, including the K
values, closely
recapitulate the binding properties of fibrinogen to
and indicates that cHarGD may be
used as a low molecular weight, high affinity surrogate of fibrinogen.
cHarGD binding to the integrins was regulated by
divalent cations: cHarGD binding to
was supported by Mn
but not by
Ca
. Cation regulation of ligand binding has been
noted for other integrins as well (31, 33, 35, 36, 37, 50, 51) and
may be explained by two possible mechanisms. First, specific cations
may selectively modulate integrin conformation; e.g. Mn
may induce a conformation which is favorable
for ligand binding. Recent studies using mAb 9EG7, which not only
recognizes an epitope induced by Mn
but also
stimulates
integrin adhesive functions(52) ,
support this mechanism. Second, integrin, cation, and ligand initially
may form a ternary coordination complex, and the cation may eventually
be displaced as the complex of ligand and integrin
stabilizes(30) . Mn
may be able to form the
ternary complex when cHarGD binds to
but Ca
cannot.
Our observations with cHarGD
led us to re-evaluate whether barbourin is specific for
even in the presence of
Mn
. Barbourin did not bind to
either in the presence of
Ca
or Mn
. Thus, ligand
discrimination by
integrins can be demonstrated even
in the presence of Mn
. In the series of
XGD-containing cyclic peptides, Scarborough et al.(25) observed that guanidation of the lysine-containing
analogs to form homoarginine-containing inhibitors, led not only to a
significant increase in the affinity for
, but also increased the affinity
for
. Although the specificity of
such homoarginine-containing analogs for
may remain extremely high in the presence of
Ca
, their specificity is much lower in the presence
of Mn
. Thus, despite the subtlety of the Lys to Har
to Arg interchange, these alterations have significant bearing on
cation regulated recognition. Substitution at the amino acids flanking
the tripeptide ligand sequences also influence affinity for
and
(53) , emphasizing the fine specificity of ligand
recognition.
cHarGD became cross-linked only to when this peptide was bound to purified
or to platelets, regardless of the
divalent cation conditions. Thus, even in the presence of
Ca
, a condition where cHarGD only binds to
and not
, its cross-linking site still
resides in the
subunit. Although RGD peptide also
cross-linked to
, proteolytic digestion indicated that
the cHarGD and RGD cross-linking sites are distinct. A 60-kDa band in
the digest of cross-linked RGD peptide under nonreducing conditions is
predicted to contain two fragments held by the cysteine 5 and 435
disulfide bond in
(54) . The amino-terminal
aspect of this fragment is likely to correspond to the 19-kDa
chymotryptic fragment obtained by Ramsamooj et al.(55) and to the 23-kDa chymotryptic fragment obtained by
D'Souza et al.(17) which contains the RGD
cross-linking site
109-171. This composition
would explain the generation of a 60-kDa fragment under nonreducing
conditions which yields less than
20 kDa under reducing
conditions. The 46/40-kDa radioactive doublet in the digest of
cross-linked
I-cHarGD under nonreducing conditions with
only a slight decrease in mobility upon reduction, would correspond to
a distinct fragment. This fragment may contain the region to which
certain RGD disintegrins cross-link within
217-302(56) .
Based upon the behavior of the
various ligands described in this study and in the literature, at least
four classes of ligands can be distinguished (Table 1). RGD-containing peptides and vitronectin are
representative of Class I ligands which react well with both
integrins. Class II ligands, represented by cHarGD,
react with
in the presence of
Mn
but not Ca
and react with
with either cation present. Class
II are distinguished from Class I ligands on the basis of the
suppression of their binding to
by
Ca
. Moreover, these two categories also appear to be
distinguished in terms of their primary cross-linking sites. Barbourin,
which binds specifically to
(24) and does not react with
even in the presence of Mn
, represents a Class
III ligand. Osteopontin is the known representative of Class IV
ligands. Although it binds to
in the
presence of Mn
, it does not bind
(57) . Some caution must
be taken in assigning these classifications as cation determined
specificity may be somewhat relative; i.e. some binding of
nonreactive ligands to
integrins may occur over
extended time periods(37) .
Fibrinogen appears to be a
member of Class II, as cHarGD behaved like a low molecular weight
mimetic of fibrinogen in virtually all respects. The classification of
the fibrinogen -chain peptides presents an interesting challenge.
We found that
-chain peptides bound to
in the presence of 1 mM Mn
, indicating that
-chain peptides behave
like cHarGD and belong in Class II. This proposal raises the
possibility that soluble fibrinogen may bind to
in the presence of Mn
via its
-chain, whereas immobilized fibrinogen appears to be
recognized via its RGD sequences(58) . Although the
-chain
peptides cross-link to
(23) rather than
, such cross-linking may be influenced by peptide
length as well as by the greater molecular flexibility of
as compared to
(59) .
As a final
issue, the reactivity of cHarGD with in the presence of Mn
bear upon the development
of
antagonists for use as
antithrombotic drugs(60) . Binding of cHarGD to
was observed at Mn
concentrations above 1 µM. Tissue concentrations of
Mn
may be in the 1-14 µM range(37, 61) ; and liver concentrations may
reach 30 µM(62) . Therefore, physiological
Mn
concentrations could support cross-reactivity of
Class II ligands with
. The results
with barbourin suggest that the design of low molecular weight, high
affinity, and specific
agonists is
feasible. Antagonists which maintain a Lys or a faithful substitution
for it in XGD mimetics should uphold this specificity.