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INTRODUCTION |
IIb
3 (GPIIb-IIIa) is a member of
integrin family of cell adhesion receptors (1-3) and is the most
abundant membrane protein on the platelet surface (4). On nonstimulated
platelets,
IIb
3 is incapable of binding
most of its soluble macromolecular ligands (5), but after exposure of
the cells to appropriate agonists, the receptor undergoes a
conformational change (6) as a consequence of signal transmission from
inside the cell to the extracellular domain of the receptor (inside-out
signaling (7, 8)) and becomes competent to interact with several plasma
protein ligands, including fibrinogen, fibronectin, and von Willebrand
factor (9-11). Fibrinogen inhibits the binding of other two ligands to
IIb
3 (10-13), and a common set of
monoclonal antibodies (mAbs)1
to the receptor blocks the interaction of these adhesive ligands with
IIb
3 (9). Two sets of ligand peptides,
chain peptides, which correspond to the sequence at the
carboxyl-terminal sequence of the fibrinogen
chain, and
RGD(X) peptides, which correspond to sequences present in
all three macromolecular ligands, define the recognition specificity of
IIb
3 for its macromolecular ligands (reviewed in Ref. 14). Both peptide sets inhibit the binding of protein
ligands to the receptor, and RGD and
chain peptides bind directly
to
IIb
3 and interfere with the
interaction of each other with the receptor (15-20). Naturally
occurring mutations within
IIb
3 block the
binding of both peptides to the receptor (21, 22). Furthermore, both
peptides can stimulate a transition of
IIb
3 from a resting to a ligand-competent
state, thereby acting as agonists as well as antagonists (23). The
chain and RGD peptides also can elicit ligand-induced binding sites, LIBS, epitopes that are expressed primarily by the occupied receptor but not by its resting or active conformers. Such observations suggest
that the binding sites recognized by these peptide ligands are
overlapping (17) or closely related (24, 25). Nevertheless, other
observations suggest that these two peptide ligands interact with
distinct sites within the macromolecular ligand binding pocket of
IIb
3 (20, 26-32). For example,
bifunctional reagents cross-link the two peptides to different subunits
of the receptor (26, 28, 29), and the peptides differentially inhibit
the binding of certain mAbs to
IIb
3 (33).
Furthermore, even though fibrinogen contains two sets of RGD sequences
within its dimeric structure, only its
chain sequences are
essential for a productive interaction of the ligand with the receptor
(34). Thus, the relationship between the peptide binding subsites
within
IIb
3 and the binding pocket for
macromolecular ligands remains uncertain.
A major limitation in the analyses of the interactions of the peptide
ligands with
IIb
3 is their low affinity
for the receptor. Fibrinogen itself has a relatively low affinity for
IIb
3 (Ka = 3 µM
1), and the peptides are estimated to
have 30-100-fold lower affinities for the receptor (24, 35). Certain
of the disintegrins derived from snake venoms have high affinities for
IIb
3 but are still relatively large and
complex ligands (36). The high affinity of the disintegrins can be
attributed to the placement of peptide ligand sequences, RGD or KGD,
within a constraining disulfide loop (37). Scarborough et
al. (38) synthesized a series of cyclized peptides and
demonstrated their high affinity and specificity for
IIb
3. One of these peptides,
cyclo(S,S)KYGCHarGDWPC (cHarGD), was found to be a high affinity
surrogate for fibrinogen and its
chain peptide based upon its
reactivity with
IIb
3 and its sister
receptor,
v
3, under different cation
conditions (30).
In this study, we have used cHarGD and a second, very closely
related cyclic peptide, cyclo(S,S)KYGCRGDWPC (cRGD), as probes of
IIb
3 recognition specificity. Both cyclic
peptides are shown to react with
IIb
3 and
V
3 with high affinities. By using
fluorescence energy transfer methodology, we demonstrate that these
ligands bind to different sites in the ligand binding domain of
IIb
3, and the distance between these
sites is estimated. A specific recombinant fragment of the
3 subunit is shown to contain one of these sites.
Furthermore, occupancy of these sites by the cyclic peptide ligands
results in distinct conformational alterations of the receptor and its
microenvironment as indicated by distinct patterns of exposure of LIBS
epitopes and differential effects on membrane fluidity.
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EXPERIMENTAL PROCEDURES |
Peptide Synthesis--
The cyclic peptides, cyclo(S,S)
KYGCRGDWPC (cRGD) and cyclo(S,S)KYGCHarGDWPC (cHarGD), were synthesized
as described previously (39) and cyclized with potassium ferricyanide
(38). Other peptides used in this study were KYGRGDS, GRGESP,
fibrinogen
-(400-411) (HHLGGAKQAGDV). All peptides were purified to
homogeneity by high performance liquid chromatography (C18
Vydac column with a linear gradient formed over 50 min from 0 to 60%,
0.1% trifluoroacetic acid in acetonitrile to 0.1% trifluoroacetic
acid in water). Peptides were characterized by amino acid composition
and mass spectroscopy, and their retention of cyclic structure was
confirmed periodically.
The following procedure was used to couple fluorochromes to the cyclic
peptides. To 5 mg of cRGD (4.3 mmol) or cHarGD (4.3 mmol) in 500 µl
of 25 mM sodium borate, pH 9.0, at 4 °C was added 100 µl of 45 mM fluorescein isothiocyanate or 45 mM tetramethylrhodamine 5-isothiocyanate in
N,N-dimethylformamide, respectively. The mixtures were stirred for 3 h at 4 °C, and then 100 µl of
N,N-dimethylformamide and 500 µl of 0.2 M acetic acid were added. Monofluorescein cRGD and
monorhodamine cHarGD were recovered by high performance liquid chromatography (same conditions as above) as the major peak in each of
the mixtures.
Preparation of
IIb
3 and
3-(95-373)--
IIb
3
was isolated from outdated platelets by affinity chromatography on RGD
columns (40). Briefly, platelets were lysed in buffer containing 10 mM HEPES, 150 mM NaCl, 1 mM
CaCl2, 1 mM MgCl2, 0.1 mM leupeptin, 10 mM
n-ethylmaleimide, 1 mM
phenylmethanesulfonyl fluoride, and 50 mM
octyl-glucoside, pH 7.3, centrifuged for 1 h at 30,000 × g, and applied onto a GRGDSPK-Sepharose column (12 × 2.5 cm) equilibrated with the lysis buffer. The detergent extracts of
the platelets were cycled over the affinity matrix at flow rate of 0.5 ml/min at 4 °C. Unbound proteins were eluted with 10 column volumes
of column buffer, identical to lysis buffer except that the octyl
glucoside concentration was lowered to 25 mM. Proteins
remaining bound onto RGD affinity matrix were eluted with buffer
containing RGDF (1 mg/ml). Fractions were analyzed by electrophoresis
on 7% polyacrylamide gels in SDS under nonreducing conditions and
pooled according to purity and concentration of
IIb
3. Prior to ligand binding
experiments, samples of
IIb
3 were
dialyzed against 0.02 M HEPES buffer, pH 7.3, containing 0.15 M NaCl, 25 mM octyl-glucoside, and 1 mM CaCl2 or MnCl2.
The
3-(95-373) fragment was expressed as a fusion
protein with thioredoxin in Escherichia coli GI724, using
the expression vector pTrxFus (Invitrogen, San Diego, CA). The cDNA
encoding this fragment was amplified using the primers
5'GGCGGGTCTAAGATGATTCGAAGAATTTC3' (forward) and
5'AAAGTAGCTAGCGGTGGCATTGAAGGATAG3' (reverse) and inserted into pTrxFus
using XbaI and NheI restriction enzymes. To
facilitate detection of the expressed fragment, an ID4 tag (ASETSQVAPA)
was attached to the carboxyl-terminal end of the
3-(95-373) fragment. Also, the enterokinase cleavage
site between thioredoxin and the
3-(95-373) fragment
was replaced by a thrombin cleavage site (LVPRG). Expression of the
3 fusion was induced with tryptophan for 3 h at
37 °C. The cell pellet was collected, and inclusion bodies were
prepared after sonication. The fusion protein was then dissolved in 6 M urea and separated by ion-exchange chromatography on
Q-Sephadex using a gradient of NaCl from 0 to 1.0 M.
Refolding of the fusion protein was conducted by dialysis against 0.1 M bicarbonate buffer, pH 8.5, containing 100 mM
NaCl. After thrombin cleavage, the
3-(95-373) fragment
was obtained by rechromatography on Q-Sephadex using a linear gradient
of NaCl from 0 to 1.0 M.
Solid-phase Ligand Binding Assays--
The binding of fibrinogen
to immobilized
IIb
3 and
3-(95-373) was performed as described (41, 42).
Microtiter wells (Corning Costar Corp., Cambridge, MA) were coated by
incubation overnight at 4 °C with 200 µl of purified
IIb
3 or
3-(95-373) at the
concentration of 5 µg/ml in 10 mM Tris buffer, pH 7.4, containing 0.15 M NaCl. The plates were then washed and
postcoated with 4% bovine serum albumin overnight at 4 °C. In
binding experiments, 20 µl of radioiodinated fibrinogen (10) with a
specific radioactivity of about 0.2 µCi/µg was added with 100-µl
aliquots of the cyclic peptides or other inhibitors to each well and
incubated for 3 h at 37 °C. After extensive washing with the
Tris buffer, binding of fibrinogen was quantitated by direct gamma
counting of the plastic wells. Nonspecific binding, defined as the
residual binding observed in the presence of 5 mM EDTA, was
subtracted from the total binding to obtain specific binding values.
Radiolabeled mAb CRC64, specific for
IIb
3
(43), was used to quantitate the amount of immobilized receptor per
well. At saturation, 5.6 × 1010 molecules of CRC64
bound to the
IIb
3-coated wells; and at
saturation, 4 × 1010 125I-fibrinogen molecules bound
per well.
V
3 was purified from placenta
with slight modifications of previously described protocols (40, 44).
The immobilization of
V
3 and its binding
of radiolabeled vitronectin were conducted as reported previously
(30).
Fluorescence Measurements--
Binding of
N
-Flu-cRGD and
N
-Rho-cHarGD to
IIb
3 was analyzed using the purified
receptor in a concentration range of 0.1 to 1 µM
dissolved in 0.02 M HEPES buffer, pH 7.3, containing 0.15 M NaCl, 25 mM octyl glucoside, and 1 mM CaCl2 or 1 mM MnCl2
(the binding buffer). Prior to experiments, the
IIb
3 was dialyzed for 1 h at
22 °C against the binding buffer. The binding reaction contained
450-µl aliquots of
IIb
3 and 25-50 µl
of the cyclic peptides individually for fluorescence binding
experiments or their combination for fluorescence energy transfer
experiments. Complexes of
IIb
3 with the
peptide ligands then were separated from free peptides by rapid (within
3 min) gel filtration on Sephadex G-25 minicolumns (Amersham Pharmacia
Biotech, prepacked PD-10 columns) equilibrated with 0.02 M
HEPES buffer, pH 7.3, containing 0.15 M NaCl, 25 mM octyl glucoside, and 1 mM CaCl2
or MnCl2. To evaluate stability of the complexes of
IIb
3 with the peptide ligands, 15 min
after the first gel filtration, they were subjected to a second gel
filtration performed under the same conditions. Emission spectra,
excitation spectra, and luminescence intensity were recorded
immediately after either the first or second gel filtration and were
recorded within 2 min using 5-nm band passes for both the excitation
and emission monochrometers in a Perkin-Elmer LS-50 spectrofluorometer.
A cut-off filter in the emission beam was used to eliminate second
order wavelength interference. The excitation wavelengths used were 494 and 541 nm for N
-Flu-cRGD and
N
-Rho-cHarGD, respectively. Emission spectra
were corrected for the blank contribution and for the instrument
response and normalized to the same protein concentration, as estimated
from the intrinsic fluorescence emission of tryptophan residues
(excitation at 285 nm and emission at 340 nm) in a quartz cell with a
1-cm path length. Excitation and emission spectra were automatically
corrected for lamp intensity variations. All the fluorescence
measurements were performed at the temperature of 23 ± 1 °C.
Buffers were degassed by bubbling nitrogen to prevent quenching of
fluorescence by dissolved oxygen. The fluorescence emission signals
were stable to photobleaching under the experimental conditions of
measurement. The luminescence maxima of
N
-Flu-cRGD and
N
-Rho-cHarGD were at 520 and 572 nm,
respectively, whether the peptides were free or bound to
IIb
3. The amount of each cyclic peptide
bound to receptor was quantitated at these maxima using the free
fluorescent peptides to construct calibration curves. The apparent
interchromophore separation, R, the distance separating the
receptor-bound N
-Flu-cRGD and
N
-Rho-cHarGD, was calculated by the Forster
equation: r = R0(1/E
1)1/6,
where E is the efficiency of energy transfer from donor to
acceptor. E = 1
FDA/
FD, and R0 is the distance for 50%
transfer efficiency E. FDA and FD
are the fluorescence intensities of the donor in the presence and
absence of the acceptor, respectively (45, 46). FDA
and FD were measured at 520 nm, the emission
maximum for the fluorescein within
N
-Flu-cRGD bound to
IIb
3.
Flow Cytometry--
Isolated platelets were suspended at 1 × 108/ml in modified Tyrode's buffer containing 0.1%
BSA, 1 mM CaCl2 or MnCl2, 10 µg/ml prostaglandin I2, and 2 µM
D-phenylalaninyl-L-prolyl-arginine chloromethylketone
and incubated with cRGD (10 µM), cHarGD (10 µM), or with no additions for 60 min at room temperature.
LIBS1 mAb (47), PMI-1 (48), or control mouse IgG was added at a final
concentration of 50 µg/ml. After 30 min, the platelets were washed by
centrifugation in the same buffer, incubated with fluorescein isothiocyanate/goat anti-mouse IgG on ice for 20 min, and then analyzed
by flow cytometry. Flow cytometry was performed using a FACScan
instrument (Becton Dickinson, San Jose, CA); 10,000 events were
recorded; and the data were analyzed using the CellQuest software
program (version 1.2).
Spin Labeling and Electron Spin Resonance (ESR)
Measurements--
Human blood (100 ml) was collected into one-sixth
volume of acid/citrate/dextrose from a forearm vein through an 18-gauge needle. Platelets were isolated by differential centrifugation (49),
and the resulting platelet pellet was resuspended in the modified
Tyrode's buffer (140 mmol/liter NaCl, 10 mmol/liter glucose, and 15 mmol/liter Tris-HCl, pH 7.4), supplemented with apyrase (0.1 g/liter)
and prostaglandin E1 (20 µg/liter). The platelets were
subsequently washed three times in this buffer. All steps in platelet
isolation were performed in plastic and at room temperature, and the
final platelet count was adjusted to 3 × 108 cells
per ml. For spin labeling experiments, the washed platelets were
incubated with a final concentration of 50 µM
5-doxylstearic acid for 30 min at room temperature. Specifically, 5 µl of a 100 mM solution of the spin labeled fatty acid in
dilute ethanol was added to 100 µl of platelet suspension (3 × 108 cells/ml), and the suspension was mixed gently. The
final ethanol concentration in the platelet suspension did not exceed
0.05% (v/v).
ESR measurements were performed at the ambient temperature (23 ± 1 °C) in a Brüker SX-300E spectrometer. For all the ESR spectra shown, the ordinate represents the amplitude of the ESR signal
and is expressed in arbitrary units. ESR scannings were routinely
recorded as the first derivatives of the absorption spectra. The
estimated ratios were calculated from the ESR plots by measuring the
amplitudes as the heights of the peaks. The ratio h+1/h0 was calculated,
where h+1 and h0
correspond to height of the low field and middle field lines of the
spectra, respectively.
Analytical Procedures--
The protein content of the platelet
membrane preparations was measured by the Lowry procedure.
Na125I (specific activity 15-17 mCi of 125I
per mg of iodine from Amersham Pharmacia Biotech) was used for radioiodination. Fibrinogen and synthetic peptides were labeled using
IODO-GEN (Pierce). The iodinated peptides were separated from free
Na125I by gel filtration on Bio-Gel P2 columns (Bio-Rad).
The specific radioactivity of the radioiodinated protein and peptides
ranged from 0.5 to 1.0 Ci/g. Aliquots of the radioiodinated protein and peptide were stored at
20 °C for no longer than 2 weeks before use.
Statistical Analysis--
The
h+1/h0 ratios derived
from the ESR spectra were expressed as the relative change compared
with controls, which were assigned values of 100% and were calculated
as the average of two replicates for each sample. All final data are
presented as the means of replicates ± S.D. The normal
distribution of data was confirmed using the Shapiro-Wilk's test. The
analysis of variance and Tukey's test for multiple comparisons (50)
were employed to assess the significance of differences among groups.
 |
RESULTS |
The Cyclic Peptides Are Potent Mimetics of the Linear
Chain and
RGD Peptides--
Previous studies have shown that cHarGD is a potent
surrogate of the fibrinogen
-chain peptide based upon its reactivity with
IIb
3 and
V
3 under different cation conditions
(30). Specifically, like the
-chain peptide, cHarGD reacts with
IIb
3 in the presence of Ca2+
and Mn2+, whereas it only binds well to
V
3 in the presence of Mn2+.
cRGD was synthesized as a high affinity mimetic of linear RGD peptides,
and the effects of these two cyclic peptides on fibrinogen binding to
purified
IIb
3 were compared. As shown in
Fig. 1, both cyclic peptides effectively
inhibited 125I-fibrinogen binding to immobilized
IIb
3 in a cation-dependent manner. In the presence of 1 mM Ca2+,
IC50 values for cRGD and cHarGD were 100 ± 10 and
50 ± 4.5 nM, respectively (Fig. 1). When
Ca2+ was replaced by 1 mM Mn2+,
these values were 100- and 500-fold lower at 1 ± 0.07 nM for cRGD and 0.1 ± 0.008 nM for
cHarGD. Under similar conditions, IC50 values for
representative linear
-chain and RGD peptides are 200- 1000-fold
higher (14). Studies also were conducted to determine the reactivity of
the cyclic peptides with
V
3 as the
receptor. While fibrinogen does not bind to
V
3 in the presence of 1 mM
Ca2+, vitronectin does (51). cRGD was a potent inhibitor of
125I-vitronectin binding to immobilized
V
3 (IC50 = 4.5 ± 2.6 nM; n = 3), whereas, consistent with
previous results (30), cHarGD produced minimal inhibition at a 50-fold
higher concentration. Thus, the cyclic peptides are potent inhibitors
of ligand binding to the
3 integrins under specified
cation conditions, are substantially more active than their linear
counterparts, and cHarGD and cRGD mimic the behavior of
-chain and
RGD peptides, respectively.

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Fig. 1.
Inhibition of fibrinogen binding to
IIb 3
by cRGD and cHarGD. 125I-Fibrinogen (25 nM) and various concentrations of cRGD ( ) or cHarGD
( ) were added to microtiter wells coated with
IIb 3 in the presence of 1 mM
CaCl2. In parallel, binding experiments were performed in
the presence of MnCl2 (cRGD ( ); cHarGD ( )). After
3 h at 37 °C, bound fibrinogen was quantitated by counting
radioactivity in a counter. The data represent the means from three
separate experiments, each with triplicate measurements.
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Interaction of Fluoresceinated cHarGD and cRGD with
IIb
3--
The cyclic peptides were
labeled with either fluoresceinyl (Flu) or rhodaminyl (Rho)
fluorophores. Exclusive modification of the amino-terminal group by the
fluorophores was accomplished by selection of the pH of the reaction
and the electrophilic reagent used (52). Thus, cRGD was derivatized
with an equimolar amount of fluorescein isothiocyanate at pH 9.0 to
yield the mono-substituted compound,
N
-Flu-cRGD. Reaction of cHarGD with an
equimolar quantity of tetramethylrhodamine 5-isothiocynate gave the
amino-terminally labeled N
-Rho-cHarGD.
Labeling of these peptides did not affect their functional properties,
based upon their IC50 values and the slopes of their inhibition curves in inhibiting fibrinogen binding to
IIb
3 under different cations conditions
(not shown).
N
-Flu-cRGD and
N
-Rho-cHarGD were incubated with purified
IIb
3 for 1 h at 22 °C, and the
complexes were separated from free peptide by rapid gel filtration.
Within 3 min of beginning the gel filtration, the fluorescence spectra
of
IIb
3 complexed with either
N
-Flu-cRGD or
N
-Rho-cHarGD were initiated. The spectra of
either bound N
-Flu-cRGD or
N
-Rho-cHarGD showed the same characteristics
as those of free fluorescein and rhodamine, respectively. Upon
excitation at 494 nm, N
-Flu-cRGD bound to
IIb
3 could be detected by its emission at 520 nm, whereas bound N
-Rho-cHarGD yielded an
emission peak at 572 nm when excited at 541 nm. As shown in Fig.
2, the specific binding of each cyclic peptide to
IIb
3 was saturable. The
concentrations of the cyclic peptides required for half-maximal binding
were in the 0.5-1.0 µM range, approximately 10-fold
higher than their IC50 values for inhibiting fibrinogen
binding to immobilized receptor. A 10-fold difference also has been
observed in the apparent affinity under the following circumstances:
(a) cHarGD binding to immobilized
IIb
3 as compared with its binding to
IIb
3 on intact platelets (30), and
(b) fibrinogen binding to immobilized
IIb
3 (53) versus its binding
to
IIb
3 on platelets (10) or in liposomes (54).

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Fig. 2.
Binding of
N -Flu-cRGD and
N -Rho-cHarGD
to
IIb 3.
Purified IIb 3 (0.1 µM) was
incubated with various concentrations of
N -Flu-cRGD (A) or
N -Rho-cHarGD (B) for 60 min at
room temperature. Total volume of the mixture was 500 µl, and all
components were dissolved in 0.02 M HEPES buffer, pH 7.3, containing 0.15 M NaCl, 1 mM CaCl2,
and 25 mM octyl glucoside. Then,
IIb 3 complexed with the fluorescent probe
was separated from free ligand by rapid gel filtration on Sephadex G-25
mini-columns equilibrated and eluted with the same buffer. Binding of
fluorescent probes was evaluated by fluorescence measurements at 520 and 572 nm upon excitation at 494 and 541 nm for
N -Flu-cRGD and
N -Rho-cHarGD, respectively. Data obtained in
the presence of 1 mM EDTA ( ), the nonspecific binding,
were subtracted from total binding ( ) to obtain specific binding
( ). Data represent the means from three separate binding
experiments.
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Simultaneous Binding of cHarGD and cRGD to
IIb
3--
IIb
3
was incubated with a mixture of the two cyclic peptides under the same
conditions and was separated from the free peptides by gel filtration.
When excited at 494 nm, the fluorescence emission spectrum of the
complexes had two peaks, one at 520 nm and a second at 572 nm (Fig.
3). This pattern was accompanied by a
decrease in the N
-Flu-cRGD fluorescence at
520 nm. This spectrum is indicative of considerable energy transfer
from the excited fluorescein to the rhodamine, providing evidence that
N
-Flu-cRGD and
N
-Rho-cHarGD are bound in close proximity
within the receptor. Two experiments were performed to verify that the
observed energy resonance transfer involves donors and acceptors
associated with the same receptor molecule rather than arising from
their binding to different receptor molecules. First, we analyzed the
efficiency of energy transfer as a function of
IIb
3 concentration. The efficiency of
energy transfer from donor to acceptor, E, was determined to
be 0.24 ± 0.05 and 0.27 ± 0.04 when
IIb
3 was used at 0.1 and 1.0 µM, respectively. Since our previous studies showed that
IIb
3 itself, or in complex with peptide
ligands, does not form stable aggregates (55), the independence of the
efficiency of energy transfer on
IIb
3
concentration indicates that the fluorochromes must be bound to the
same receptor. Second, complexes of
IIb
3 with N
-Flu-cRGD or
N
-Rho-cHarGD were separated by gel filtration
as indicated; these samples were immediately mixed together, and
spectral analyses were performed. Under this condition, no energy
transfer was observed. This result provides independent evidence that
the two ligands must bind to the same receptor for efficient energy
transfer. Based upon an efficiency of energy transfer of 0.27 and
assuming a Forster critical distance (R0) range
of 5.0-5.2 nm for a donor rhodamine (56), the distance between donor
(fluorescein linked to cRGD) and acceptor (rhodamine linked to cHarGD)
was calculated to be 6.13 ± 0.5 nm.

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Fig. 3.
Fluorescence resonance energy transfer
between
N -Flu-cRGD and
N -Rho-cHarGD
bound to
IIb 3.
Emission spectra of IIb 3 (0.5 µM) complexed with N -Flu-cRGD
(---), N -Rho-cHarGD (···), or both
peptides (- - -). A, the fluorescein was excited at 494 nm, and in B, the rhodamine was excited at 541 nm. The
decrease in the emission spectrum at 520 nm and appearance of the
second peak at 572 nm with both cyclic peptides present in A
reflects energy transfer from the bound
N -Flu-cRGD to the bound
N -Rho-cHarGD.
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The next series of experiments was designed to test stability and
stoichiometry of complexes of
IIb
3 with
the cyclic peptide ligands.
IIb
3 (0.5 µM) was incubated with
N
-Flu-cRGD and
N
-Rho-cHarGD, separated by gel filtration,
and used for spectroscopic measurements as described above. Then, after
15 min, the samples were refractionated by gel filtration, and the
emission spectra were again recorded. The concentration of each peptide
in the
IIb
3 fraction after the first and
second gel filtration was quantitated from their fluorescence
intensities using the free cyclic peptides to construct calibration
curves. In addition, the efficiency of energy transfer was estimated
for each sample. The results of these analyses are summarized in Table
I.
IIb
3 purified from the free peptides by the first rapid gel filtration contained 0.45 mol of cRGD and 0.85 mol of cHarGD per mol of receptor. Thus, under the conditions of the initial spectral analysis,
substantial amounts of each peptide were bound to the receptor, and the
distance between the two bound peptides allowed for energy transfer
with an efficiency of 0.24. After 15 min, the emission spectrum
(excitation at 494 nm) was again recorded. The emission peak at 572 nm
had almost completely disappeared indicating that the peptides had dissociated from the receptor, and energy transfer no longer occurred. This sample then was subjected to a second gel filtration, which reduced the concentrations of both cyclic peptides significantly (cRGD,
0.06 mol/mol; cHarGD, 0.18 mol/mol). These data indicate that both
cyclic peptides were bound with high stoichiometry and reversibility to
IIb
3. Corroborating data were obtained by
a second experimental approach. A spectrum was taken of the
peptide-
IIb
3 complex after the first gel
filtration; then EDTA was added at a final concentration of 1 mM, and the spectrum was analyzed again. EDTA affected the
fluorescent characteristics of N
-Flu-cRGD and
N
-Rho-cHarGD only slightly but completely
abolished energy transfer (Table I). Thus, EDTA dissociated the bound
peptides from
IIb
3, and the free
peptides, although present in the mixture at the same concentration as
when bound to
IIb
3, could not transfer energy.
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Table I
Stoichiometry and stability of N -Flu-cRGD and
N -Rho-cHarGD complexed with IIb 3
IIb 3 (0.5 µM) was incubated with
both cyclic peptides, and the free peptide ligands were rapidly removed
by gel filtration (I). The concentrations of both cyclic peptides,
quantitated from their fluorescence intensities as described under
"Experimental Procedures," are expressed in moles per mol of
IIb 3. Emission spectra were taken immediately
after the first gel filtration, 15 min later, or immediately after a
second gel filtration (II). The efficiency of energy transfer was
estimated as described. EDTA (1 mM) was added to the
IIb 3 complexed with N -Flu-cRGD
and N -Rho-cHarGD after the first gel filtration
and analyzed for energy transfer.
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Cation Dependence and Specificity of Sites for
N
-Flu-cRGD and N
-Rho-cHarGD--
The
binding of both peptides required the presence of divalent cations,
Ca2+ or Mn2+. When
IIb
3 was depleted from cations by
incubation with EDTA, interaction with
N
-Flu-cRGD and
N
-Rho-cHarGD was almost completely abolished
(Fig. 4). Substitution of
Mn2+ for Ca2+ resulted in a significant
increase in fluorescence emission at 572 nm as indicated by higher
ratio of luminescence at 572 nm to that at 520 nm (0.588 and 0.823 in
the presence of Ca2+ and Mn2+, respectively).
This increase in fluorescence energy transfer was not associated with a
change in the efficiency of energy transfer (E); in the
presence of Mn2+, E was calculated to be
0.25 ± 0.04. Direct excitation of rhodamine at 541 nm also showed
increased fluorescence emission at 572 nm, suggesting that
N
-Rho-cHarGD binds more efficiently to the
receptor in Mn2+, probably arising from the enhanced
affinity of the receptor for ligands in the presence of this cation
(51).

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Fig. 4.
Effects of cations on fluorescence emission
spectra of
IIb 3
complex with
N -Flu-cRGD and
N -Rho-cHarGD.
Binding of fluorescent probes to IIb 3
(0.5 µM) was performed in the presence of
Ca2+ (---), Mn2+ (- - -), or EDTA (-·-).
Emission spectra were recorded upon excitation at 494 nm (A)
or 541 nm (B).
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|
The effects of the nonlabeled cyclic peptides on the signals given by
the fluorescent peptides bound to the receptor were evaluated (Fig.
5). In these experiments, binding of both
N
-Flu-cRGD (0.8 µM) and
N
-Rho-cHarGD (0.8 µM) to
IIb
3 (0.1 µM) was performed
in the presence of various concentrations of nonlabeled peptides.
Emission spectra were recorded after excitation at 494 and 541 nm. The
relative change in fluorescence emission was quantified at 520 and 572 nm for N
-Flu-cRGD and
N
-Rho-cHarGD bound to the receptor,
respectively. Addition of a 100-fold excess of nonlabeled cRGD
decreased the fluorescence intensity of fluorescein and rhodamine by
~80 and 20%, respectively. In contrast, cHarGD added at 100-fold
molar excess caused a 25 and 85% reduction of
N
-Flu-cRGD and
N
-Rho-cHarGD luminescence, respectively. The
differential effects of cRGD and cHarGD observed in these experiments
suggest that these peptides preferentially bind to distinct sites in
the receptor but that they can weakly and mutually inhibit the binding
of each other.

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Fig. 5.
Displacement of
N -Flu-cRGD and
N -Rho-cHarGD
bound to
IIb 3
by nonlabeled peptides. Binding of both fluorescent probes to
IIb 3 (0.2 µM) was performed
in the presence of either nonlabeled cRGD ( ) or cHarGD ( ). The
extent of binding of N -Flu-cRGD and
Na-Rho-cHarGD was evaluated by measurement of
fluorescence intensity at 520 and 572 nm upon excitation at 494 (A) and 541 nm (B), respectively. Each point
represents a mean of three separate experiments.
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We also evaluated the capacity of a linear RGD and
-chain peptide to
inhibit the binding of N
-Flu-cRGD and
N
-Rho-cHarGD to
IIb
3. The data shown in Table
II indicate that the linear counterparts
of the cyclic peptides yielded similar patterns of inhibition. GRGDSP
was more effective in inhibiting N
-Flu-cRGD
than N
-Rho-cHarGD binding, whereas the
-chain peptide corresponding to fibrinogen
-(400-411) was more
effective in inhibiting N
-Rho-cHarGD than
N
-Flu-cRGD binding to
IIb
3.
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Table II
Inhibition of N -Flu-cRGD and N -Rho-cHarGD binding
to IIb 3 by linear RGD and fibrinogen chain
peptides
Data represent percent of inhibition ± S.D. of binding of the
fluoresceinated cyclic peptides by the indicated linear peptides as
estimated by the spectroscopic measurements described under
"Experimental Procedures" and in the legend to Fig. 2.
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N
-Flu-cRGD but Not N
-Rho-cHarGD Binds
to
3-(95-373)--
To characterize the binding sites
for the peptide ligands, a recombinant fragment corresponding to
3-(95-373) was expressed, renatured, and purified. The
fragment preparation used in subsequent ligand binding studies was
homogeneous as assessed by polyacrylamide gel electrophoresis in sodium
dodecyl sulfate under reducing and nonreducing conditions. This
fragment would contain the putative metal ion-dependent
adhesion site (MIDAS motif) in the
3 subunit (57, 58).
As shown in Fig. 6,
3-(95-373) bound fibrinogen in a
cation-dependent manner. Similar to
IIb
3, this binding was inhibited by EDTA
and supported by Ca2+ and Mn2+. However, in
contrast to the intact
IIb
3, higher
concentrations of Ca2+ inhibited the binding of fibrinogen
to the recombinant fragment; 125I-fibrinogen binding to
3-(95-373) was significantly lower in 1 mM
Ca2+ than in 0.1 mM Ca2+. A
suppressive effect of Ca2+ on fibrinogen binding is
observed with
V
3 (51). Binding of 125I-fibrinogen to
3-(95-363) was blocked
effectively of cRGD (72% inhibition of specific binding at 1 µM), whereas a high concentration (100 µM)
of cHarGD only partially inhibited the interaction.

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Fig. 6.
Specificity of the interaction of fibrinogen
with 3-(95-373) fragment.
125I-Fibrinogen (25 nM) was added to microtiter
wells coated with 3-(95-373) in the presence of
Ca2+ (1 and 0.1 mM), Mn2+ (0.1 mM), and EDTA (1 mM). The effect of peptides,
cRGD (1 µM), cHarGD (1 µM), GRGESP (100 µM), and fibrinogen (3 µM) was tested in
the presence of 0.1 mM Ca2+. Values represent
the means ± S.D. of three determinations and are expressed as a
percent of a control with 0.1 mM Ca2+ lacking
inhibitors.
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|
The direct binding of the cyclic peptides to the recombinant fragment
was tested in spectroscopic analyses (Fig.
7). The
3-(95-373) fragment was incubated with N
-Flu-cRGD and
N
-Rho-cHarGD as described for
IIb
3. As shown in Fig. 7A,
when excited at 494 nm, a single emission peak was observed at 520 nm,
reflecting the binding of N
-Flu-cRGD to the
recombinant fragment. No emission peak was observed at 572 nm,
indicating that N
-Rho-cHarGD could not be
excited by energy transfer from N
-Flu-cRGD
bound to
3-(95-373). The lack of
N
-Rho-cHarGD binding to the
3
fragment was further demonstrated by the absence of an emission
spectrum upon direct excitation of rhodamine at 541 nm (Fig.
7B).

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Fig. 7.
Binding of
N -Flu-cRGD
to 3-(95-373). The purified
3-(95-373) (10 µM) was incubated with
N -Flu-cRGD or
N -Rho-cHarGD in the presence of 0.1 mM Ca2+ (---) or 0.1 mM
Mn2+ (- - -). Complexes of 3 fragment with
peptide ligands were purified as described in the legend to Fig. 2 and
under "Experimental Procedures." The extent of binding of
N -Flu-cRGD was evaluated by measurement of
fluorescence intensity at 520 nm upon excitation at 494 nm
(A). The extent of N -Rho-cHarGD
binding was evaluated by measurement of the fluorescence intensity at
572 nm upon excitation at 541 nm (B).
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Binding of cRGD or cHarGD to Platelets Induces Different Functional
Responses--
If cRGD and cHarGD bind to different sites in
IIb
3, the two cyclic peptides might
initiate distinct functional responses. This possibility was explored
initially by assessing the differential induction of LIBS in
IIb
3. LIBS are the target epitopes of
mAbs that react selectively with the occupied receptor (47). Induction of two prototypic LIBS epitopes, one expressed by the
3
subunit and recognized by mAb LIBS-1 (59) and a second expressed by the
IIb subunit and recognized by mAb PMI-1 (60), was
analyzed upon binding of the cyclic peptides to platelets by
fluorescence-activated cell sorter. As summarized in Table
III, binding of cRGD and cHarGD to
IIb
3 on platelets resulted in a
differential induction of the two LIBS epitopes. cRGD increased
expression of both the LIBS-1 and PMI-1 epitopes as indicated by the
increase in the mean fluorescence intensity of the platelets upon
incubation with the two antibodies. The capacity of cRGD to induce both
LIBS epitopes was observed in the presence of either Ca2+
or Mn2+. In contrast, whereas cHarGD elicited exposure of
LIBS-1, the PMI-1 epitope was minimally induced by this peptide. This
differential induction of LIBS epitopes by cHarGD was observed in the
presence of either Ca2+ or Mn2+.
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Table III
Differential effect of the cyclic peptides on expression of PMI-1 and
LIBS1 epitopes by IIb 3
Data represent mean fluorescence measured by flow cytometry of
platelets incubated with cyclic peptides in the presence of
Ca2+ or Mn2+ as described under "Experimental
Procedures."
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|
As a second functional parameter, the capacity of cHarGD and cRGD to
affect differentially the fluidity of the membrane lipid bilayer of
platelets was assessed. For this analysis, platelets were labeled with
5-doxylstearic acid, a spin probe that reports fluidity changes
proximal to the outer surface of the platelet membrane bilayer (61).
ESR spectra were taken in the presence of various concentrations of the
two cyclic peptides, and representative spectra are shown in Fig.
8A. The low and middle field
maxima are described by their amplitudes, h+1
and h0, respectively, and these values were
quantitated. The h+1/h0
ratios derived in the presence of the two cyclic peptides and 0.1 mM Ca2+ are displayed in Fig. 8B. A
substantial reduction of the
h+1/h0 ratio was observed
upon binding of cRGD to platelets, indicating a significant decrease in
the fluidity of lipid bilayer. The effect of cRGD approached that
induced by increasing the Ca2+ concentration from 0.1 to 1 mM. In sharp contrast, cHarGD elevated the
h+1/h0 ratios, indicating
an increase in membrane lipid fluidity upon binding of this peptide to
platelets. This increase was greater than that observed when 0.1 mM Mn2+ was used instead of 0.1 mM
Ca2+. Even at the lowest concentration of the peptide
ligands tested, 2.5 nM, the differences in membrane
fluidity evoked by the two cyclic peptides were significantly different
(p < 0.001). The effects of each cyclic peptide on
membrane fluidity was concentration-dependent. However,
the changes were induced by very low concentrations of the cyclic
peptides, substantially lower than their Kd value
for
IIb
3. This was also observed with the
linear ligand peptides (31) and indicates that only limited occupancy
of
IIb
3 is sufficient to cause changes in
membrane fluidity as detectable by ESR.

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Fig. 8.
Effect of cRGD and cHarGD binding to
IIb 3
on fluidity of lipid bilayer in platelet membranes.
Microenvironmental changes in the vicinity of
IIb 3 were analyzed by ESR spectra of
5-doxylstearic acid incorporated into the membrane lipid bilayer of
platelets. Platelets labeled with the spin probe were incubated with
various concentrations of cyclic peptides in the presence of 0.1 mM CaCl2. Typical spectra obtained in the case
of control platelets (curve a), platelets with bound c-HarGD
(curve b), or cRGD (curve c) are shown in
A. B, the
h+1/h0 ratios calculated
from ESR spectra, where h+1 and
h0 correspond to heights of the low field and
middle field lines of the spectra, respectively, are presented.
Reduction in the h+1/h0
ratio indicates a lower fluidity of the lipid bilayer relative to the
0.1 mM CaCl2. The effects of EDTA, 1 mM CaCl2, and 0.1 mM
MnCl2 are shown as controls. Data represent means ± S.D. calculated from five separate experiments each with triplicate
measurements.
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|
 |
DISCUSSION |
In this report, we have utilized two structurally similar but
nonidentical cyclic peptides to examine the ligand binding properties of
IIb
3. Evidence is developed to
indicate that cHarGD and cRGD interact with two distinguishable binding
sites within
IIb
3. Moreover, occupancy of
these sites by ligand initiates different conformational changes in the
receptor which may ultimately lead to distinct functional responses in
platelets. These observations shed light on a long-standing question in
the
IIb
3 area regarding the relationship
between its peptide ligand-binding sites, may provide insights into how
macromolecular ligands may bind to
IIb
3, and may even be relevant to the biological activities of the new class
of antithrombotic drugs, the GPIIb-IIIa blockers (62).
Fluorescent derivatives of cRGD and cHarGD were developed to aid in our
analyses of the interaction of these cyclic peptides with
IIb
3.
N
-Flu-cRGD and
N
-Rho-cHarGD were found to have properties
functionally analogous to their respective parent cyclic peptides. They
bound to
IIb
3 with high affinity in a
cation-dependent manner, could be displaced by nonlabeled
peptides, and efficiently inhibited fibrinogen binding to
IIb
3. Utilization of these labeled
peptide ligands in fluorescence resonance energy transfer experiments
provided evidence that both probes could bind to the same
IIb
3 molecules. Since the energy transfer
between fluorochromes was largely independent of receptor concentration, and only occurred if the peptides had the opportunity to
bind simultaneously to the same receptor, a primary conclusion of this
study is that
IIb
3 expresses two distinct
binding sites for these peptide ligands. Based upon the assumptions of
the Forster equations, the distance between the two bound fluorophores
was estimated to be 6.13 ± 0.5 nm. Molecular models of the two
cyclic peptides have been constructed (Insight II, Molecular
Simulations, San Diego, CA), and the disulfide bond is predicted to
substantially restrict the flexibility of each peptide. The maximum
length of the two peptides in these models, from the center of the
fluorochrome to the aspartic acid residue is 1.5 ± 0.1 and
0.96 ± 0.017 nm for cHarGD and cRGD, respectively. If the
fluorochromes are placed as the most distant residues in the
receptor-bound orientation of the two cyclic peptides, overlap between
the contact sites would still not be possible.
For the purpose of discussion, these two binding sites for the cyclic
peptides are designated as A and B. Site A reacts preferentially with
cHarGD, and its affinity for ligand is highly modulated by cations.
This site does bind its ligand in the presence of Ca2+, but
its affinity for cHarGD was significantly increased by
Mn2+. Recombinant
3-(95-373) did not bind
cHarGD, suggesting that site A is not renatured or not fully located
with this recombinant fragment. Site B preferentially interacts with
cRGD in the presence of either Ca2+ or Mn2+ and
is located in recombinant
3-(95-373). Although sites A
and B are spatially and functionally distinct, they are not fully independent. Displacement experiments indicate that the two cyclic peptides can mutually inhibit binding of each other to intact receptor.
Whether this cross-talk between sites A and B arises from lack of
complete specificity or is allosterically induced cannot be
distinguished by the present data. A previous study (30) had suggested
that cHarGD is a high affinity surrogate for linear
-chain peptides
based upon its differential reactivity with
IIb
3 and
v
3
under specific cation conditions. The correspondence between cHarGD and
cRGD to linear
-chain and RGD peptides, respectively, is supported
by several additional observations in the present study. First, the two
cyclic peptides show the same differential regulation by divalent
cations in the reactivity with
IIb
3 and
v
3 as observed with the linear peptides,
i.e. cHarGD and the
-chain peptide interact with
IIb
3 in the presence of Ca2+
but Ca2+ inhibits their binding to
v
3. This inhibitory effect of
Ca2+ is not as marked with cRGD or linear RGD peptides.
Second, the binding of each cyclic peptide to
IIb
3 was preferentially inhibited by the
corresponding linear peptide. Third, the cyclic peptide and its linear
counterpart exerted the same effects on membrane fluidity: cHarGD and
the
-chain peptide increased membrane fluidity, whereas cRGD and the
RGD peptide decreased it. Based upon this correspondence,
-chain
peptides would react preferentially with site A and RGD peptides
preferentially with site B. The preferential inhibition of cRGD binding
by a linear RGD peptide and cHarGD binding by the fibrinogen
-chain
peptide is consistent with this model. A two-site model has been
proposed for integrin
5
1, which recognizes the RGD and a synergistic sequence in fibronectin (63).
As evidenced by the induction of the LIBS epitopes, LIBS1 and PMI-1,
occupancy of either site A or site B induced conformational changes in
IIb
3. However, the pattern of epitope
expression was different with the two ligands; binding of cRGD resulted
in induction of both LIBS epitopes, whereas binding of cHarGD caused only minimal LIBS1 epitope expression. These results indicate that the
occupied receptor can exist in at least two distinct conformational
states. Both LIBS epitopes have been at least partially localized:
PMI-1 to the carboxyl terminus of the
IIb heavy chain (60) and LIBS1 to the
3 subunit (47). Thus, occupancy of site B within
3-(95-373) by cRGD appears to transduce
conformational changes in both subunits of the receptor. In view of the
long range transmission of conformational changes in
IIb
3 induced by ligand occupancy (59), it
is not surprising that membrane fluidity might also be influenced by
occupancy of sites A and B. However, the two cyclic peptides exerted
opposing effects on membrane fluidity as evidenced by their
differential alteration of the
h+1/h0 ratio obtained
with 5-doxylstearic acid as a probe. Interaction of cRGD with platelets
reduced the h+1/h0 ratio,
indicating a significant rigidification of membrane lipid bilayer,
whereas cHarGD increased the
h+1/h0 ratio indicating an increase in membrane fluidity. This differential effect is consistent with previous studies in which linear RGD and
-chain peptides were used in ESR studies (31) and supports the hypothesis that
cRGD and cHarGD are the respective high affinity analogs of the linear
peptides. With the differential transmission of signals from sites A
and B, which are likely to reside in the amino-terminal aspects of
IIb
3, to the membrane proximal LIBS epitopes and then further into the membrane bilayer, it is not unreasonable to suggest that these peptides also would induce different
outside-in signaling events. Consistent with this possibility, the
linear RGD and
-chain peptides have been reported to induce different effects on intracellular Ca2+ mobilization
(64).
Binding of protein and peptide ligands to integrin receptors is
regulated by divalent cations (65-67). Two classes of functionally distinct Ca2+-binding sites have been identified in
3 integrins; the higher affinity sites can be occupied
by Ca2+ and other cations and support ligand binding,
whereas the lower affinity sites are Ca2+-selective and are
inhibitory (68). Recombinant
3-(95-373) bound
fibrinogen in the presence of Mn2+ and low concentrations
of Ca2+, whereas high concentrations of Ca2+
abolish this interaction. Based on these cation effects, it appears that this domain must contain an inhibitory Ca2+-binding
site if not both classes of sites. It should be noted that Gulino
et al. (69) concluded that a similar segment of
3 bound fibrinogen in a cation-independent manner. These
investigators only employed a high Ca2+ concentration which
may have suppressed ligand binding. Thus, our data showing that the
level of binding in 1 mM Ca2+ and EDTA is
similar are consistent with this previous report. At a structural
level, the extracellular domain of
IIb
3
is believed to contain two classes of cation-binding sites. The first
class is homologous to the EF-hand structures, such as found in
calmodulin, and four such sites are located in
IIb
subunit (55, 69). The second class is thought to be similar to a metal
ion-dependent adhesion site (MIDAS), originally identified
in the crystal structures of the I domain of the integrin
L and
M subunits (57, 70). It has been
proposed that integrin
subunits, including
3,
contain an I domain-like structure with a cation-binding site related (58) but not identical (71) to a MIDAS motif.
3-(95-373), which binds cRGD but not cHarGD, would
contain this I domain-like structure with its putative MIDAS motif.
Therefore, our data would suggest that this putative MIDAS domain could
function as a (not necessarily the only) Ca2+-inhibitory
site. Whether this region contains two Ca2+-binding sites
is the subject of ongoing investigations.
The involvement of sites A and B in fibrinogen binding to
IIb
3 can only be a subject for
speculation at this juncture. Macromolecular proteins such as
fibrinogen appear to function as polyvalent ligands and develop
multiple contacts with the receptor (72). Initial docking into the
activated integrin is reversible (72, 73) and requires the fibrinogen
-chain sequence (34). This initial step of ligand recognition was
found to be critically dependent upon the presence of cations such as
Ca2+ and Mn2+ (68, 74). In the context of our
current findings, site A must be occupied for a productive interaction
to occur, and divalent cations are required for engagement of site A. As additional contacts develop, including engagement of site B, the
receptor-ligand complex becomes stabilized so it becomes nondissociable
(65, 72). The reversible nature of the interaction of each cyclic
peptide with
IIb
3 is consistent with this
multiple contact model. The ligation of site B may be mediated by the
natural RGD sequences within the fibrinogen A(
) chain or other
sequences that can conform to fit site B. These two basic steps could
be distinguished experimentally when fibrinogen binding was evaluated
in the presence of RGD peptides (72) or using a recombinant Fab
fragment containing an RGD sequence (68). The engagement of each
contact in sequence may initiate a different wave of conformational
change in the receptor, which results in differential induction of LIBS
epitopes and the transmission of outside-in signals into the cell. In
association with this sequence of events, the platelet membrane would
undergo an initial increase in fluidity as the
-chain binds to site
A and then become more rigid as site B is occupied. The latter change
should lead to a greater exposure of membrane proteins, including
IIb
3 and other membrane receptors. This
model raises the possibility that binding of macromolecular ligands,
such as fibronectin or von Willebrand factor, to
IIb
3, which contain an RGD but not a
-chain motif and, therefore, may not engage site A, may induce
signaling pathways distinct from those initiated by fibrinogen. Also,
the occupancy of
V
3 by fibrinogen may
have very different consequences than its occupancy of
IIb
3. In this regard, the ligand contact site in
3-(95-373), site B, may be more crucial for
fibrinogen binding to
V
3, which depends
upon recognition of an RGD sequence (44), rather than for
IIb
3, which depends upon recognition of
the
-chain sequence (75). The fact that Ca2+ suppresses
fibrinogen binding to
3-(95-373) and
V
3, but not to
IIb
3, is consistent with this possibility.
A number of peptide and nonpeptide antagonists of
IIb
3 are now under development as
antithrombotic agents (reviewed in Ref. 62). The RGD peptide has often
been used as a starting structure for design of these drugs, and
preferential reactivity with
IIb
3 versus
V
3 has often been a
criteria for selection. Therefore, these drugs may very well be totally
selective or at least react differently with sites A and B within
IIb
3. Consequently, the mechanism of
antagonism of platelet aggregation by these drugs may be different as
may be their induction of LIBS epitopes, platelet microenvironmental
changes (membrane fluidity), and intracellular signaling. Indeed,
changes in membrane fluidity may occur even at very low levels of
IIb
3 occupancy. It will be interesting to
see if these differences influence the efficacy of these drugs.