A New Model of Dual Interacting Ligand Binding Sites on Integrin alpha IIbbeta 3*

Dana D. HuDagger §, Carol A. WhiteDagger , Susan Panzer-Knodle, James D. Page, Nancy Nicholson, and Jeffrey W. SmithDagger parallel

From the Dagger  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

    ABSTRACT
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Abstract
Introduction
References

The platelet integrin alpha IIbbeta 3 mediates platelet aggregation and platelet adhesion. This integrin is the key to hemostasis and also to pathologic vascular occlusion. A key domain on alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 even when it is already occupied by fibrinogen. Once bound, RGD-type ligands induce the dissociation of fibrinogen from alpha IIbbeta 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

Integrins are noncovalently associated alpha beta 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.

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 beta 3 integrins, alpha IIbbeta 3 and alpha vbeta 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.

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 alpha IIbbeta 3, an integrin that binds to a number of different ligands. The ligands for alpha IIbbeta 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 alpha IIbbeta 3 through a non-RGD sequence present in its gamma -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).

There is a considerable discrepancy regarding the nature of the ligand binding pocket on alpha IIbbeta 3. Some studies imply that the two types of ligands could bind to distinct sites on alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 contains distinct and interacting ligand binding sites.

    MATERIALS AND METHODS

Purified Ligand Receptor Binding Measurements-- Competition binding experiments were performed with purified alpha IIbbeta 3 according to methods we have previously published (33). Briefly, alpha IIbbeta 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 alpha IIbbeta 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 gamma  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.

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 gamma  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.

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 alpha IIbbeta 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).

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.

    RESULTS AND DISCUSSION

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 alpha IIbbeta 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 alpha IIbbeta 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.

RGD Ligands Competitively Inhibit Fibrinogen Binding to alpha IIbbeta 3-- A series of competition binding experiments were conducted to determine whether alpha IIbbeta 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 alpha IIbbeta 3 (the association rate) is comparable with that of fibrinogen (39, 41). Second, both ligands are monovalent and bind to alpha IIbbeta 3 in a reversible manner, allowing for meaningful kinetic measurements and comparisons.

As a first step, we measured the ability of the RGD mimetic SC 52012 to block the binding between purified alpha IIbbeta 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 alpha IIbbeta 3 and Fab-9 and also a competitive inhibitor of the binding between alpha IIbbeta 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 alpha IIbbeta 3. Competition binding studies were performed using purified alpha IIbbeta 3 as described previously (39). 125I-Fibrinogen (Fg) (A, B) or 125I-Fab-9 (C, D) were used as ligands for alpha IIbbeta 3. The ability of 0 (bullet ), 3 × 10-11 M (black-square), 6 × 10-11 M (black-triangle), and 1 × 10-10 M (black-down-triangle ) 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 gamma  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%.

Fibrinogen Fails to Compete for the Binding of RGD Ligands to alpha IIbbeta 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 alpha IIbbeta 3. These studies were performed on gel-filtered human platelets, although virtually identical results were obtained with purified alpha IIbbeta 3 (not shown). An extensive series of preliminary binding experiments showed that all of the alpha IIbbeta 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 alpha IIbbeta 3 (Fig. 3A). In several experiments of this type, we observed no significant effect of fibrinogen on the affinity of alpha IIbbeta 3 for [3H]SC 52012. In contrast, Fab-9 (an RGD ligand) did block binding of [3H]SC 52012 (black-square).


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Fig. 3.   Fibrinogen fails to block binding of RGD-type ligands to alpha IIbbeta 3. A, the binding of [3H]SC 52012 to alpha IIbbeta 3 on platelets was measured in the absence of competitor (black-triangle) and in the presence of 2.3 µM Fab-9 (black-square) or 2.3 µM fibrinogen (bullet ). B, the binding of 125I-Fab-9 to alpha IIbbeta 3 was measured in the absence of competitor (black-triangle), in the presence of 1 µM SC 52012 (black-square), or in the presence of 2.3 µM fibrinogen (bullet ). 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.

A similar binding study showed that fibrinogen had only a minimal effect on the binding of 125I-Fab-9 to alpha IIbbeta 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 alpha IIbbeta 3 on platelets (black-square).

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 alpha IIbbeta 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 alpha IIbbeta 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).

The Allosteric Nature of the Two Ligand Binding Pockets on alpha IIbbeta 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 alpha IIbbeta 3, we reasoned that the RGD binding site is likely to be accessible even when fibrinogen and alpha IIbbeta 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 alpha IIbbeta 3 and fibrinogen or Fab-9. Particular interest was paid to the effects of the compound on ligand dissociation.

SC 52012 prevented the association of Fab-9 with alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3. SPR was used to measure the effect of SC 52012 on the association (A, C) and dissociation (B, D) between alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 in the analyte solution. To measure the effect of SC 52012 on ligand dissociation, the alpha IIbbeta 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.

SC 52012 had markedly different effects on the interaction between fibrinogen and alpha IIbbeta 3. It blocked association between fibrinogen and alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 even when fibrinogen is bound. Consequently, SC 52012 and fibrinogen bind to separate sites on the integrin (Fig. 1, Scheme 3).

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 alpha IIbbeta 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/alpha IIbbeta 3 required to enact 50% disaggregation ranged from 0.5:1 to 1:1. Calculations are based on an estimate of 80,000 alpha IIbbeta 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 alpha IIbbeta 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.

The Two Ligand Binding Pockets on alpha IIbbeta 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 alpha IIbbeta 3 are regulated by different means. The fibrinogen binding function of alpha IIbbeta 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 alpha IIbbeta 3 on resting platelets is of such low affinity that it cannot be measured (35). Fibronectin, an RGD ligand for alpha IIbbeta 3, cannot bind to resting platelets or even to platelets stimulated by ADP. It will only bind alpha IIbbeta 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 alpha IIbbeta 3 are regulated independently by activation.

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 alpha IIbbeta 3 molecules on the platelet surface when platelets were stimulated with ADP (see Fig. 3B). 125I-Fab-9 bound the same number of alpha IIbbeta 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 alpha IIbbeta 3 molecules on the platelet as reported by the binding of 125I-abciximab, an antibody that binds to alpha IIbbeta 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 alpha IIbbeta 3 molecules.

Because Fab-9 and fibrinogen bind to separate sites on alpha IIbbeta 3, these observations are consistent with the conclusion that the two ligand binding pockets on alpha IIbbeta 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 alpha IIbbeta 3. Although Fab-9 and fibronectin are both RGD-type ligands, Fab-9 binds alpha IIbbeta 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 alpha IIbbeta 3 can be regulated independently by physiologic stimuli.

Do Some Glanzmann's Thrombasthenics Have a Defect at Only One of the Ligand Binding Pockets on alpha IIbbeta 3?-- Glanzmann's thrombasthenia is a series of genetic disorders in which patients either fail to express alpha IIbbeta 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 alpha IIbbeta 3. There are reports of defects in which alpha IIbbeta 3 fails to mediate platelet aggregation or bind to fibrinogen but retains the ability to bind RGD ligands. The two-site model of alpha IIbbeta 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 beta 3 subunit (45). Interestingly, Strasbourg alpha IIbbeta 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.

The Impact of a Two-site Model on Anti-integrin Therapy-- The knowledge that alpha IIbbeta 3 contains two interacting ligand binding pockets also has bearing on the application of small molecule antagonists of alpha IIbbeta 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 alpha IIbbeta 3 interacted with two classes of ligands in a "mutually exclusive" manner (27, 30), then antagonists of alpha IIbbeta 3 could function only by preventing the association of fibrinogen with the integrin. In such cases, drugs directed toward alpha IIbbeta 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 alpha IIbbeta 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 alpha IIbbeta 3 (Fig. 1, Scheme 3) may provide an additional benefit to the patient by enacting the dissolution of existing thrombi.

    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.

parallel 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.

    ABBREVIATIONS

The abbreviations used are: SC, Searle Compound; SPR, surface plasmon resonance.

    REFERENCES
Top
Abstract
Introduction
References
  1. Albelda, S. M., and Buck, C. A. (1990) FASEB J. 4, 2868-2880[Abstract]
  2. Phillips, D. R., Charo, I. F., and Scarborough, R. M. (1991) Cell 65, 359-362[Medline] [Order article via Infotrieve]
  3. Ruoslahti, E. (1991) J. Clin. Invest. 87, 1-5[Medline] [Order article via Infotrieve]
  4. Hynes, R. O. (1992) Cell 69, 11-25[Medline] [Order article via Infotrieve]
  5. Humphries, M. J. (1990) J. Cell Sci. 97, 585-592[Medline] [Order article via Infotrieve]
  6. Brooks, P. C., Silletti, S., von Schalscha, T. L., Friedlander, M., and Cheresh, D. A. (1998) Cell 92, 391-400[Medline] [Order article via Infotrieve]
  7. Mesri, M., Plescia, J., and Altieri, D. C. (1998) J. Biol. Chem. 273, 744-748[Abstract/Free Full Text]
  8. Brooks, P. C., Stromblad, S., Sanders, L. C., von Schalscha, T. L., Aimes, R. T., Stetler-Stevenson, W. G., Quigley, J. P., and Cheresh, D. A. (1996) Cell 85, 683-693[Medline] [Order article via Infotrieve]
  9. Byzova, T. V., and Plow, E. F. (1997) J. Biol. Chem. 272, 27183-27188[Abstract/Free Full Text]
  10. Williams, M. J., Hughes, P. E., O'Toole, T. E., and Ginsberg, M. H. (1994) Trends Biochem. Sci. 4, 109-112
  11. O'Toole, T. E., Katagiri, Y., Faull, R. J., Peter, K., Tamura, R., Quaranta, V., Loftus, J. C., Shattil, S. J., and Ginsberg, M. H. (1994) J. Cell Biol. 124, 1047-1059[Abstract]
  12. Hughes, P. E., Diaz-Gonzalez, F., Leong, L., Wu, C., McDonald, J. A., Shattil, S. J., and Ginsberg, M. H. (1996) J. Biol. Chem. 271, 6571-6574[Abstract/Free Full Text]
  13. Pelletier, A. J., Bodary, S. C., and Levinson, A. D. (1992) Mol. Biol. Cell 3, 989-998[Abstract]
  14. Juliano, R. L., and Haskill, S. (1993) J. Cell Biol. 120, 577-585[Medline] [Order article via Infotrieve]
  15. Kornberg, L. J., Earp, H. S., Turner, C. E., Prockop, C., and Juliano, R. L. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 8392-8396[Abstract]
  16. Rosales, C., O'Brien, V., Kornberg, L., and Juliano, R. (1995) Biochim. Biophys. Acta 1242, 77-98[CrossRef][Medline] [Order article via Infotrieve]
  17. Hocking, D. C., Sottile, J., and McKeown-Longo, P. J. (1998) J. Cell Biol. 141, 241-253[Abstract/Free Full Text]
  18. Loftus, J. C., and Liddington, R. C. (1997) J. Clin. Invest. 99, 2302-2306[Free Full Text]
  19. Plow, E. F., Pierschbacher, M. D., Ruoslahti, E., Marguerie, G. A., and Ginsberg, M. H. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 8057-8061[Abstract]
  20. Plow, E. F., Pierschbacher, M. D., Ruoslahti, E., Marguerie, G., and Ginsberg, M. H. (1987) Blood 70, 110-115[Abstract]
  21. Farrell, D. H., and Thiagarajan, P. (1994) J. Biol. Chem. 269, 226-231[Abstract/Free Full Text]
  22. Kloczewiak, M., Timmons, S., and Hawiger, J. (1983) Thromb. Res. 29, 249-255[Medline] [Order article via Infotrieve]
  23. Ruggeri, Z. M., Houghten, R. A., Russell, S. R., and Zimmerman, T. S. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5708-5712[Abstract]
  24. Ruggeri, Z. M., De Marco, L., Gatti, L., Bader, R., and Montgomery, R. R. (1983) J. Clin. Invest. 72, 1-12[Medline] [Order article via Infotrieve]
  25. Plow, E. F., McEver, R. P., Coller, B. S., Woods, V. L., Jr., Marguerie, G. A., and Ginsberg, M. H. (1985) Blood 66, 724-727[Abstract]
  26. Farrell, D. H., Thiagarajan, P., Chung, D. W., and Davie, E. W. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10729-10732[Abstract]
  27. Bennett, J. S., Shattil, S. J., Power, J. W., and Gartner, T. K. (1988) J. Biol. Chem. 263, 12948-12953[Abstract/Free Full Text]
  28. Santoro, S. A., and Lawing, W. J., Jr. (1987) Cell 48, 867-873[Medline] [Order article via Infotrieve]
  29. Plow, E. F., Srouji, A. H., Meyer, D., Marguerie, G., and Ginsberg, M. H. (1984) J. Biol. Chem. 259, 5388-5391[Abstract/Free Full Text]
  30. Lam, S. C.-T., Plow, E. F., Smith, M. A., Andrieux, A., Ryckwaert, J.-J., Marguerie, G., and Ginsberg, M. H. (1987) J. Biol. Chem. 262, 947-950[Abstract/Free Full Text]
  31. Beer, J. H., Springer, K. T., and Coller, B. S. (1992) Blood 79, 117-128[Abstract]
  32. Segal, I. (1975) Enzyme Kinetics, John Wiley & Sons, New York
  33. Hu, D. D., Barbas, C. F., III, and Smith, J. W. (1996) J. Biol. Chem. 271, 21745-21751[Abstract/Free Full Text]
  34. Pytela, R., Pierschbacher, M. D., Argraves, S., Suziki, S., and Ruoslahti, E. (1987) Methods Enzymol. 144, 475-489[Medline] [Order article via Infotrieve]
  35. Marguerie, G. A., Edgington, T. S., and Plow, E. F. (1980) J. Biol. Chem. 255, 154-161[Free Full Text]
  36. Altschuh, D., Dubs, M.-C., Weiss, E., Zeder-Lutz, G., and Van Regenmortel, M. H. V. (1992) Biochemistry 31, 6298-6304[Medline] [Order article via Infotrieve]
  37. Panzer-Knodle, S. G., Jacqmin, P., Page, J. D., Nicholson, N. S., Zablocki, J. A., Engleman, V. W., and Feigen, L. P. (1995) Platelets 6, 288-295
  38. Coller, B. S., Lang, D., and Scudder, L. E. (1997) Circulation 95, 860-867[Abstract/Free Full Text]
  39. Smith, J. W., Piotrowicz, R. S., and Mathis, D. M. (1994) J. Biol. Chem. 269, 960-967[Abstract/Free Full Text]
  40. Barbas, C. F., Languino, L., and Smith, J. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10003-10007[Abstract]
  41. Smith, J. W., Hu, D., Satterthwait, A., Pinz-Sweeney, S., and Barbas, C. F. (1994) J. Biol. Chem. 269, 32788-32795[Abstract/Free Full Text]
  42. Plow, E. F., and Ginsberg, M. H. (1981) J. Biol. Chem. 256, 9477-9482[Free Full Text]
  43. Wagner, C. L., Mascelli, M. A., Neblock, D. S., Weisman, H. F., Coller, B. S., and Jordan, R. E. (1996) Blood 88, 907-914[Abstract/Free Full Text]
  44. Kato, A. (1997) Crit. Rev. Oncol. Hematol. 26, 1-23[CrossRef][Medline] [Order article via Infotrieve]
  45. Lanza, F., Stierle, A., Fournier, D., Morales, M., Andre, G., Nurden, A. T., and Cazenave, J. P. (1992) J. Clin. Invest. 89, 1995-2004[Medline] [Order article via Infotrieve]
  46. Coller, B. S. (1997) J. Clin. Invest. 99, 1467-1471[Free Full Text]
  47. Tcheng, J. E. (1997) Thromb. Haemostasis 78, 205-209[Medline] [Order article via Infotrieve]
  48. Moliterno, D. J., and Topol, E. J. (1997) Thromb. Haemostasis 78, 214-219[Medline] [Order article via Infotrieve]


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