From the Program on Cell Adhesion, Cancer Research
Center, The Burnham Institute, La Jolla, California 92037 and the
¶ Department of Cardiovascular Disease Research, Searle, Skokie,
Illinois 60077
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ABSTRACT |
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The platelet integrin
Integrins are noncovalently associated First, many integrins exhibit a very broad ligand binding specificity
(4, 5). Some integrins, even those that bind to the RGD tripeptide
adhesion motif, can bind to ligands that lack an RGD sequence. For
example, the two Second, ligand binding to integrins can depend on activation. Signals
transmitted from the cytoplasm can activate the integrins ligand
binding function in the ectodomain, resulting in a proadhesive phenotype (2, 10-12). However, integrins can bind to some ligands in
the absence of cellular stimulation. Such binding is often referred to
as "activation-independent." Clearly then, there are two separable
mechanisms of ligand binding.
Third, when integrins bind their ligands, signals are transmitted in
the opposite direction, or "outside-in." These signals can
ultimately alter cellular physiology and gene expression (13-16). Recent work suggests that outside-in signaling can depend upon the type
of ligand bound to integrin (17). The biochemical basis for these
ligand-dependent differences in signaling is not understood.
One hypothesis that would have a major bearing on each of these issues
suggests that integrins contain multiple ligand binding pockets (18).
In fact, there is considerable circumstantial evidence to support this
hypothesis. However, the existence of two ligand binding sites remains
uncertain, particularly because there is little information to explain
mechanistically how two such sites would interact, if at all.
To test the two-site hypothesis, we examined the ligand binding
function of the platelet integrin There is a considerable discrepancy regarding the nature of the ligand
binding pocket on Purified Ligand Receptor Binding Measurements--
Competition
binding experiments were performed with purified
Platelet Ligand Binding Studies--
Equilibrium binding studies
were performed on freshly isolated human platelets (35). Platelets were
purified from platelet-rich plasma by gel filtration on Sepharose CL-4B
equilibrated in Tyrode's buffer containing 2 mM
MgCl2, 0.1% bovine serum albumin, and 0.1% glucose, pH
7.2. For binding studies, platelets were diluted in Tyrode's buffer
containing 0.2 mM CaCl2, 1.8 mM
MgCl2, 0.1% bovine serum albumin, and 0.1% glucose to a
concentration of 1 × 108 platelets/ml. The binding of
radiolabeled ligands was measured on platelets stimulated with 20 µM ADP at ambient temperature and under equilibrium
binding conditions (30 min). Bound and free ligand were separated by
centrifugation of the platelets through a cushion of 20% sucrose. The
platelet pellet was collected into tubes for Surface Plasmon Resonance--
SPR was performed using the
BIAcoreTM amine coupling kit according to previously
published methods (33). Importantly, the results reported here were
obtained when either the integrin or the ligand was immobilized to the
SPR sensor chip via the amine coupling kit. Only minor differences in
dissociation rates were observed when the configuration of the assay
was switched. Each sensorgram represents at least five similar
repetitions, encompassing at least four batches of purified
Platelet Aggregation Experiments--
Turbidimetric aggregometry
was performed as described (37) with slight modifications. ADP was used
as an agonist at 5 µM. Three minutes after the addition
of agonist to the aggregometer tube, SC 52012 or buffer control was
added to the reaction. The disaggregation of the platelets was
monitored for an additional 6 min. The ability of Fab-9 to induce
disaggregation was measured by adding fibrinogen-coated blue
polystyrene beads (6 µm in diameter) to platelet-rich plasma (38).
Following stimulation of a suspension of beads and platelets with 20 µM ADP, platelets adhered to the beads and simultaneously
aggregated. The coupled process was monitored in a microtiter plate
reader at 562 nm, which detects the light scattering properties of the
blue polystyrene beads.
Modes of Competition between Ligands for a Common
Receptor--
Two distinct classes of competitive inhibition between
ligands for a common receptor are illustrated in Fig.
1. Simple competitive inhibition, or
same-site competitive inhibition (Scheme 1), is a
competition between ligands A and B for the same binding pocket. In
such a case, one ligand alters the affinity of a receptor for the other
ligand. A separate type of competitive inhibition is allosteric, and it
is more difficult to identify (Scheme 2). Allosteric inhibitors bind at physically separate sites. Like same-site
inhibition, an allosteric inhibitor will change the overall binding
affinity of a receptor for its ligand. However, allosteric inhibition
differs because the two ligands bind at separate sites, and it may be possible for one ligand to interact with the receptor when the other
ligand is already bound. Hence, ligand B could still interact with its
binding pocket, even when ligand A is bound at a separate site. Here we
show that RGD Ligands Competitively Inhibit Fibrinogen Binding to
As a first step, we measured the ability of the RGD mimetic SC 52012 to
block the binding between purified Fibrinogen Fails to Compete for the Binding of RGD Ligands to
A similar binding study showed that fibrinogen had only a minimal
effect on the binding of 125I-Fab-9 to
In other studies, we attempted to favor the binding of fibrinogen over
both RGD ligands by allowing a 20-min pre-binding step with competing
fibrinogen. However, even under these conditions, fibrinogen did not
interfere with the binding of [3H]SC 52012 or
125I-Fab-9. It is important to emphasize that we have found
that Fab-9 and fibrinogen associate with
The Allosteric Nature of the Two Ligand Binding Pockets on
SC 52012 prevented the association of Fab-9 with
SC 52012 had markedly different effects on the interaction between
fibrinogen and RGD Ligands Dissociate Platelet Aggregates--
An important
prediction of the two-site model is that integrins that are already in
contact with the extracellular matrix could still bind ligands at the
second ligand binding pocket and be redirected to other functions.
Indeed, the two-site model also has important implications for
anti-integrin therapy because it suggests a novel approach toward
reversing integrin-mediated matrix contact. To test these predictions,
we used platelet aggregates as a physiologic model of the interaction
between an integrin and its matrix. We measured the ability of the two
RGD ligands to dissociate an existing platelet aggregate, a structure
that is formed by the binding of fibrinogen and
The Two Ligand Binding Pockets on
To explore this possibility further, we compared the binding of Fab-9
and fibrinogen on resting versus activated platelets. The
binding studies were done with 250 nM
125I-Fab-9, a concentration we found to just saturate the
number of
Because Fab-9 and fibrinogen bind to separate sites on
Do Some Glanzmann's Thrombasthenics Have a Defect at Only One of
the Ligand Binding Pockets on
The Impact of a Two-site Model on Anti-integrin Therapy--
The
knowledge that IIb
3 mediates platelet aggregation and
platelet adhesion. This integrin is the key to hemostasis and also to
pathologic vascular occlusion. A key domain on
IIb
3 is the ligand binding site, which
can bind to plasma fibrinogen and to a number of Arg-Gly-Asp (RGD)-type
ligands. However, the nature and function of the ligand binding pocket
on
IIb
3 remains controversial. Some
studies suggest the presence of two ligand binding pockets, whereas
other reports indicate a single binding pocket. Here we use surface
plasmon resonance to show that
IIb
3 contains two distinct ligand binding pockets. One site binds to fibrinogen, and a separate site binds to RGD-type ligands. More importantly, however, the two ligand binding pockets are interactive. RGD-type ligands are capable of binding to
IIb
3 even when it is already occupied by
fibrinogen. Once bound, RGD-type ligands induce the dissociation of
fibrinogen from
IIb
3. This allosteric cross-talk has important implications for anti-platelet therapy because
it suggests a novel approach for the dissolution of existing platelet thrombi.
INTRODUCTION
Top
Abstract
Introduction
References
heterodimers that
serve as a primary link between the extracellular matrix and the cytoplasm (1-4). Integrins contribute to the structure of cells and
tissues by providing the physical contact between a cell and the
matrix. However, integrins are also involved in bidirectional signaling
events that greatly influence development, angiogenesis, wound repair,
and a variety of pathological conditions. Although considerable
progress has been made toward identifying the members in the integrin
protein family and toward assigning their physiologic ligands, there
are several biochemical properties of integrins for which the
underlying principles are not well understood.
3 integrins,
IIb
3 and
v
3, can bind to as many as 13 different
ligands representing several protein families. Furthermore, some
integrins can bind to adhesive proteins and to proteases, two classes
of ligands with seemingly opposing functions (6-9). It is not known
how binding to ligands with such disparate functions is coordinated or regulated.
IIb
3,
an integrin that binds to a number of different ligands. The ligands
for
IIb
3 can be grouped into two general
categories. One class of ligands contains the well known RGD integrin
binding motif (19, 20). The other class of ligands is represented
solely by fibrinogen. Fibrinogen binds to
IIb
3 through a non-RGD sequence present in its
-chain (21-23). Both types of ligands have physiologic importance for regulating platelet adhesion to the subendothelial matrix (24, 25) and in mediating aggregation with other platelets to
form a thrombus (26).
IIb
3. Some studies
imply that the two types of ligands could bind to distinct sites on
IIb
3 (27, 28), whereas other reports
indicate the existence of a common, or overlapping, binding pocket (29,
30). A third hypothesis argues that the receptor contains a single
binding pocket that can have different depths (31). Most of the ligand
binding studies on
IIb
3 have been
performed under equilibrium conditions and show that the two types of
ligands competitively inhibit the binding of each other. However, these
prior measurements failed to distinguish between the two mechanisms of
competitive inhibition, same site competitive inhibition
versus allosteric competitive inhibition (32). Here we apply
real time kinetic analysis to show that
IIb
3 contains distinct and interacting
ligand binding sites.
MATERIALS AND METHODS
IIb
3 according to methods we have
previously published (33). Briefly,
IIb
3
purified from platelet lysates by RGD affinity chromatography (34) was
immobilized in the wells of microtiter plates. Radiolabeled ligand was
added to the immobilized
IIb
3 along with
competing ligand until the binding had reached equilibrium (empirically
determined to be 90 min). Free ligand was removed by extensive washing,
and bound ligand was solubilized using boiling 1 N NaOH.
Each sample was transferred to a glass 12 × 77-mm tube, and the
bound ligand was quantified by
counting. Each experiment was
performed at least three times, and all points are the average of
triplicate measurements. Data were analyzed and plotted using SigmaPlotTM. Double-reciprocal plots were generated using
the 95% confidence interval of a least squares analysis.
counting or
scintillation counting by excising the bottom of the centrifuge tube.
Nonspecific binding was typically less than 10% of the total binding
of each ligand and was measured by inclusion of a 500-fold molar excess
of Searle Compound (SC)1
52012 or Fab-9 with radiolabeled ligands. All points are the average of
triplicate data points, and each experiment was repeated at least three
times, yielding virtually identical results.
IIb
3 and three batches of human
fibrinogen (Enzyme Research Laboratories). The association (k1) and dissociation
(k
1) rate constants were calculated from
sensorgrams as described (36).
RESULTS AND DISCUSSION
IIb
3 has two separate binding
pockets that interact in an allosteric manner (Scheme 3).
The evidence in support of this conclusion is presented below.
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Fig. 1.
Model depicting two modes of competitive
inhibition. Competitive inhibitors change the affinity of a
receptor for its ligand. Competitive inhibition can occur when two
ligands compete for the same binding site (Scheme 1) or when
the two ligands allosterically influence the binding of one another by
binding to physically distinct sites (Scheme 2). A model of
the ligand binding sites of integrin IIb
3
is shown in Scheme 3. Binding Site I binds to
fibrinogen. Binding Site II interacts with RGD-type ligands,
including Fab-9 and SC 52012. The binding of ligands to Site
II has as profound an influence on ligand association and
dissociation rates for fibrinogen as Site I.
IIb
3--
A series of competition
binding experiments were conducted to determine whether
IIb
3 contains two ligand binding pockets. Such measurements cannot be performed properly with natural RGD-type ligands like vitronectin and fibronectin because, in comparison with
fibrinogen, they exhibit very slow association rates (39) and because
they are multivalent. Both properties invalidate many types of kinetic
analysis, a method that must be used to distinguish simple competitive
inhibition from allosteric competitive inhibition. Therefore, we
employed two model RGD ligands; Fab-9 is a human antibody engineered by
phage display to contain an RGD in the antigen binding site (40, 41),
and SC 52012 is a small molecule mimic of RGD (37). These two model
ligands have several advantages. First, the rate at which they bind
to
IIb
3 (the association rate) is
comparable with that of fibrinogen (39, 41). Second, both ligands
are monovalent and bind to
IIb
3 in a
reversible manner, allowing for meaningful kinetic measurements and comparisons.
IIb
3
and the two macromolecular ligands Fab-9 and fibrinogen. The binding
isotherms are shown in Fig. 2 along with
double-reciprocal transformations of the data. The shape of these plots
enables one to distinguish noncompetitive inhibition from competitive
inhibition. Increasing concentrations of competing SC 52012 shift the
binding isotherms for both 125I-fibrinogen (Fig.
2A) and 125I-Fab-9 (Fig. 2C) to the
right, indicating a change in their overall binding affinity. The
double-reciprocal transformations of both sets of binding data
intersect on the y axis (Fig. 2, B and
D), a hallmark of competitive inhibition (32). Based on
these findings, we conclude that the small molecule RGD mimetic, SC
52012, is a competitive inhibitor of the binding between
IIb
3 and Fab-9 and also a competitive
inhibitor of the binding between
IIb
3 and
fibrinogen. Although these findings are consistent with previously published reports indicating competitive inhibition between RGD ligands
and fibrinogen, they do not distinguish between same site competitive
inhibition (Fig. 1, Scheme 1) and allosteric competitive inhibition (Fig. 1, Scheme 2).
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Fig. 2.
The RGD mimetic SC 52012 is a competitive
inhibitor of the binding of Fab-9 and fibrinogen to
IIb
3.
Competition binding studies were performed using purified
IIb
3 as described previously (39).
125I-Fibrinogen (Fg) (A, B) or
125I-Fab-9 (C, D) were used as ligands for
IIb
3. The ability of 0 (
), 3 × 10
11 M (
), 6 × 10
11
M (
), and 1 × 10
10 M
(
) SC 52012 to interfere with the binding of each ligand was
assessed under equilibrium conditions. At the end of the binding
reactions, microtiter plates were washed three times and the bound
ligand was harvested and quantified by
counting. Binding isotherms
are shown in A and C. The data were transformed
to double-reciprocal plots (32) by replotting the inverse of each
value. The double-reciprocal plots were generated with SigmaPlot using
a 95% confidence interval for the construction of lines for each data
series. The character of the double-reciprocal plots indicates the type
of inhibition. A plot in which the series of fitted lines
intersect on the y axis indicates competitive inhibition
(see plots B and D). Each plot represents an
experiment that was repeated at least three times. All
points are the average of triplicate points in which the
S.E. was less than 12%.
IIb
3--
As a second step, we performed
the converse experiment and measured the ability of fibrinogen to block
the binding of each of the RGD-type ligands to
IIb
3. These studies were performed on
gel-filtered human platelets, although virtually identical results were
obtained with purified
IIb
3 (not shown).
An extensive series of preliminary binding experiments showed that all
of the
IIb
3 molecules on the platelet
surface could be saturated at a fibrinogen concentration of 500 nM. Hence, fibrinogen was used in excess of this
concentration (2.3 µM) in attempts to compete for the
binding of [3H]SC 52012 and 125I-Fab-9.
Fibrinogen failed to block the binding of [3H]SC 52012 to
IIb
3 (Fig.
3A). In several experiments of
this type, we observed no significant effect of fibrinogen on the
affinity of
IIb
3 for [3H]SC
52012. In contrast, Fab-9 (an RGD ligand) did block binding of
[3H]SC 52012 (
).
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Fig. 3.
Fibrinogen fails to block binding of RGD-type
ligands to
IIb
3.
A, the binding of [3H]SC 52012 to
IIb
3 on platelets was measured in the
absence of competitor (
) and in the presence of 2.3 µM
Fab-9 (
) or 2.3 µM fibrinogen (
). B, the
binding of 125I-Fab-9 to
IIb
3
was measured in the absence of competitor (
), in the presence of 1 µM SC 52012 (
), or in the presence of 2.3 µM fibrinogen (
). All experiments were performed with
gel-filtered human platelets stimulated with 20 µM ADP.
Each point is the average of triplicate data points in which
the S.E. was typically less than 12%. Each plot is representative of
at least three experiments that yielded nearly identical results.
IIb
3 (Fig. 3B). The presence
of saturating levels of fibrinogen (2.3 µM) caused only a
2-3-fold shift in the Kd of whole platelets for
Fab-9 (1.9 ± 0.9 nM in the absence of fibrinogen to
5.9 ± 2.9 nM in the presence of fibrinogen
(n = 3)). In contrast, SC 52012 was able to block
virtually all of the specific binding between 125I-Fab-9
and
IIb
3 on platelets (
).
IIb
3 at similar rates, so fibrinogen's
inability to block the binding of Fab-9 is not a kinetic artifact.
Collectively, the results show that even though RGD-type ligands are
competitive inhibitors of fibrinogen binding to
IIb
3, fibrinogen fails to interfere with
the binding of either RGD ligand. Such findings are inconsistent with
same site competitive inhibition (Fig. 1, Scheme 1) and
strongly hint that the RGD ligands are allosteric inhibitors of
fibrinogen binding (Fig. 1, Scheme 2).
IIb
3 Is Revealed by Plasmon
Resonance--
Because our measurements indicated that RGD ligands are
competitive inhibitors of fibrinogen binding, we sought to perform a
definitive test that would distinguish between same site competitive inhibition (Fig. 1, Scheme 1) and allosteric competitive
inhibition (Fig. 1, Scheme 2). Measuring
Kd under equilibrium binding conditions cannot
provide such a distinction because both modes of inhibition alter the
overall binding affinity between receptor and ligand. Therefore, we
used SPR because it allows one to measure the two components of overall
affinity, ligand association and ligand dissociation, independently. In
essence, SPR allows one to observe the binding reaction in real time,
i.e. as it happens. Because fibrinogen failed to block the
binding of RGD ligands to
IIb
3, we
reasoned that the RGD binding site is likely to be accessible even when
fibrinogen and
IIb
3 are in a complex. We
further suspected that the binding of RGD might induce the dissociation
of fibrinogen from the integrin. Such an observation would prove the
two-site model. To test this idea, we measured the effects of SC 52012 on the association and dissociation rates between
IIb
3 and fibrinogen or Fab-9. Particular
interest was paid to the effects of the compound on ligand dissociation.
IIb
3 (Fig.
4A) but had no effect on the
dissociation rate for this ligand (Fig. 4B). This behavior
is consistent with the conclusion that SC 52012 and Fab-9 bind the same
binding pocket on
IIb
3. SC 52012 cannot
gain access to the RGD binding pocket and induce dissociation of Fab-9
because that binding site is already occupied by Fab-9 (same site
competitive inhibition, Fig. 1, Scheme 1).
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Fig. 4.
RGD-type ligands induce the dissociation of
fibrinogen from
IIb
3.
SPR was used to measure the effect of SC 52012 on the association
(A, C) and dissociation (B, D) between
IIb
3 and Fab-9 (A, B) or
fibrinogen (C, D). In these experiments, Fab-9 or fibrinogen
was immobilized to the sensor chip using the amine coupling kit
(BIAcore). Binding was measured by passing purified
IIb
3 over the surface of the sensor chip.
The ability of different concentrations (noted) of SC 52012 to block
ligand association was measured by adding the compound along with
IIb
3 in the analyte solution. To measure
the effect of SC 52012 on ligand dissociation, the
IIb
3 integrin was first allowed to bind
to ligand on the sensor chip, and then the analyte was changed to
contain either a buffer control or the noted concentration of SC 52012 (arrow, panels B and D). This
experiment represents at least seven similar repetitions. Nearly
identical results were obtained when the assay was done in the converse
format with the integrin immobilized on the surface of the sensor
chip.
IIb
3. It blocked
association between fibrinogen and
IIb
3
(Fig. 4A), but more importantly, SC 52012 induced the dissociation of prebound fibrinogen from the integrin (Fig.
4C). Saturating levels of SC 52012 increase the off-rate
between fibrinogen and
IIb
3 by 160-fold
(from 6.2 × 10
5 s
1 to 1 × 10
2 s
1). Because SPR reports ligand binding
in real time (as the binding reaction is occurring), the latter
observation unequivocally demonstrates that SC 52012 can bind to
IIb
3 even when fibrinogen is bound. Consequently, SC 52012 and fibrinogen bind to separate sites on the
integrin (Fig. 1, Scheme 3).
IIb
3. As shown in Fig.
5A, the RGD mimic, SC 52012, enacted the complete dissolution of the aggregate within a period of
minutes. When platelet aggregates were allowed to incubate for extended
periods of time before the addition of the RGD-type ligand, less
dissolution of the platelet aggregate occurred. Yet, even when
aggregation was allowed to proceed for 15 min in the presence of
maximal platelet stimulation (20 µM ADP), before
challenge with SC 52012, 40-60% of the aggregate was consistently
dissociated by the compound. Using a slightly different assay of
platelet aggregation (38), we found that Fab-9 also dissociated
platelets that were aggregated with fibrinogen-coated polystyrene beads
(Fig. 5B). In five separate experiments of this type, the
molar ratio of Fab-9/
IIb
3 required to
enact 50% disaggregation ranged from 0.5:1 to 1:1. Calculations are
based on an estimate of 80,000
IIb
3
molecules/platelet. A platelet aggregate is a complex structure, and we
cannot exclude the possibility that there are other parameters that
influence the dissolution of aggregates when challenged with an RGD
compound. However, the simplest interpretation of these findings is
that dissolution of the aggregate is enacted by the same mechanism that
induces dissociation of complexes between purified
IIb
3 and fibrinogen.
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Fig. 5.
RGD-type ligands promote the disaggregation
of platelets. A, the effect of SC 52012 on platelet
aggregates was measured using turbidimetric aggregometry. Platelet-rich
plasma was stimulated with 5 µM ADP, and an aggregate was
allowed to form for 3 min. Then, SC 52012 or control buffer was added
to the reaction at the noted concentration. The status of the aggregate
was monitored for an additional 6 min. This particular plot is
representative of six similar repetitions. B, the effect of
Fab-9 on platelet aggregates was examined using an assay that measures
the combined agglutination of fibrinogen-coated blue polystyrene beads
with platelets as they aggregate. Once aggregates between platelets and
beads were formed, Fab-9 or the control buffer was added to the
aggregate. Disaggregation was monitored in a microtiter plate reader
using the absorbance of the sample at 562 nm. The absorbance of the
sample increases as the beads and platelets dissociate. Substantial
disaggregation occurred within 3 min (40-60%) and was virtually
complete within 20 min. The data represent triplicate points in which
the S.E. was typically less than 10%. This experiment represents four
similar repetitions. Virtually identical results were obtained with SC
52012 when this bead-based agglutination assay was used.
IIb
3 Can Be Regulated Independently by
Activation of the Integrin--
Consideration must also be given to
the idea that the two ligand binding sites on
IIb
3 are regulated by different means. The fibrinogen binding function of
IIb
3
is tightly controlled by platelet stimulation, a process that can be
brought about by a host of physiologic stimuli such as ADP, thrombin,
or collagen (2). In fact, the binding of fibrinogen to
IIb
3 on resting platelets is of such low
affinity that it cannot be measured (35). Fibronectin, an RGD ligand
for
IIb
3, cannot bind to resting platelets or even to platelets stimulated by ADP. It will only bind
IIb
3 on the platelet surface when the
platelets have been stimulated by thrombin (42). Thus, the activation
requirement for ligand binding appears to depend on the type of ligand
being examined. In light of the findings in the current report, another interpretation of the observation of Plow and Ginsberg (42) is that the
two ligand binding pockets on
IIb
3 are
regulated independently by activation.
IIb
3 molecules on the platelet
surface when platelets were stimulated with ADP (see Fig.
3B). 125I-Fab-9 bound the same number of
IIb
3 molecules on resting and stimulated
platelets. In binding studies performed on blood from four separate
donors, 125I-Fab-9 bound to between 30,000 and 100,000 sites/platelet, depending on the donor. The number of molecules of
Fab-9 bound was equivalent in each case on resting versus
ADP-stimulated platelets. The number of binding sites for Fab-9 was
also equivalent to the number of
IIb
3
molecules on the platelet as reported by the binding of 125I-abciximab, an antibody that binds to
IIb
3 in an activation-independent manner
(43). As expected, parallel binding studies performed on the same
platelets showed that 125I-fibrinogen is unable to bind
specifically to resting platelets. However, upon activation with ADP,
125I-fibrinogen bound to the full complement of
IIb
3 molecules.
IIb
3, these observations are consistent
with the conclusion that the two ligand binding pockets on
IIb
3 are regulated independently by
activation of the integrin. However, our findings do not resolve all of
the discrepancies in binding data relating to activation of
IIb
3. Although Fab-9 and fibronectin are
both RGD-type ligands, Fab-9 binds
IIb
3
in the absence of platelet stimulation, whereas fibronectin will bind
only when platelets are stimulated with thrombin. Nevertheless, in
conjunction with prior reports, the results presented here suggest that
the distinct ligand binding pockets on
IIb
3 can be regulated independently by
physiologic stimuli.
IIb
3?--
Glanzmann's thrombasthenia
is a series of genetic disorders in which patients either fail to
express
IIb
3 on the platelet surface or
express a dysfunctional form of the integrin (44). Glanzmann's
patients suffer from chronic bleeding problems. Interestingly, however,
not all Glanzmann's defects are associated with the complete dysfunction of
IIb
3. There are reports of
defects in which
IIb
3 fails to mediate
platelet aggregation or bind to fibrinogen but retains the ability to
bind RGD ligands. The two-site model of
IIb
3 proposed in Fig. 1, Scheme
3,provides a basis for such observations. The Strasbourg
variant of Glanzmann's thrombasthenia contains a point mutation of
arginine to tyrosine at residue 214 in the integrin
3
subunit (45). Interestingly, Strasbourg
IIb
3 bound to small RGD peptides but not
to fibrinogen. It is reasonable to suggest that the Strasbourg variant
results from a defect specific to the fibrinogen binding site even
though the RGD ligand binding pocket remains functional.
IIb
3 contains two
interacting ligand binding pockets also has bearing on the application
of small molecule antagonists of
IIb
3 in
anti-platelet therapy. Several such drugs are currently being tested as
antithrombotic agents in large clinical trials (46-48). These trials
are aimed at eliminating the ischemic complications that often
accompany cardiac interventions like balloon angioplasty. During
coronary intervention, complications are presumed to arise when
platelets aggregate to form a thrombus, occluding a vessel and reducing
blood supply. Anti-platelet therapy has been proposed as a solution to
this problem. If
IIb
3 interacted with two
classes of ligands in a "mutually exclusive" manner (27, 30), then
antagonists of
IIb
3 could function only
by preventing the association of fibrinogen with the integrin. In such
cases, drugs directed toward
IIb
3 would
be effective only when applied before the formation of thrombi or in a
prophylactic manner. The results presented here indicate that the
binding of RGD-type antagonists to
IIb
3
will occur even when fibrinogen is already bound and when platelets
have already aggregated. Thus, such ligands could enact the dissolution
of an existing thrombus. Therefore, drugs that bind to site II on
IIb
3 (Fig. 1, Scheme 3) may
provide an additional benefit to the patient by enacting the
dissolution of existing thrombi.
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FOOTNOTES |
---|
* The study was supported by National Institutes of Health Grants HL58925 and AR42750 (to J. W. S.) and AR 45054 (to D. D. H.) and by Searle.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported in part by a fellowship from the California Affiliate of the American Heart Association and by a fellowship from the U. S. Army Breast Cancer Program. Present address: Monsanto Co., Discovery Pharmacology, Mail Code AA3C, 700 Chesterfield Village Parkway N., St. Louis, MO 63198.
Established investigator of the American Heart Association and
Genentech. To whom correspondence should be addressed: Program on Cell
Adhesion, Cancer Research Center, The Burnham Inst., 10901 N. Torrey
Pines Rd., La Jolla, CA 92037. Tel.: 619-646-3121; Fax: 619-646-3192;
E-mail: jsmith{at}ljcrf.edu.
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ABBREVIATIONS |
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The abbreviations used are: SC, Searle Compound; SPR, surface plasmon resonance.
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REFERENCES |
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