Introduction

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The _IIb3 integrin (glycoprotein IIb-IIIa) is a cell-surface membrane receptor present in high density on platelets. Its activation through inside-out signaling mechanisms by shear stress or by external agents such as ADP or thrombin results in a ligand-receptive state capable of binding fibrinogen and von Willebrand factor. As a final endpoint, fibrinogen makes cross-links between platelets, causing their aggregation and leading to the formation of arterial thrombi (Vermylen et al., 1986; Phillips et al., 1988).

Thrombus formation is a sequela of several occlusive cardiovascular diseases, including atherosclerosis, myocardial infarction, and cardiac ischemia. To prevent or reduce the formation of platelet clots, drugs such as aspirin and heparin have been used. More recently, attempts have been made to directly inhibit _IIb3 integrin and thus block platelet aggregation (Verstraete, 2000). Several ligands of integrins, including fibrinogen and von Willebrand factor, can bind them through an arginine-glycine-aspartic acid (RGD) motif (Hynes, 1992). The affinity of these proteins to RGD-dependent integrins is in the order of 10⁷ M. Shorter RGD peptides, 5 to 10 amino acids long, have about a 1000-fold lower affinity for RGD-dependent integrins and are therefore not suitable as antagonists. Snake venom disintegrins are exceptions: these toxins, such as echistatin, flavostatin, and others, are 5- to 8-kDa-long peptides in which the central RGD motif is framed out by several disulfide bridges (McLane et al., 1998). They have been reported to bind with 10⁹ M affinity to RGD-dependent integrins. It is thus evident that the RGD motif by itself is not sufficient to confer high affinity binding and that a three-dimensional structure is required. Based on these observations, drugs have been designed to interact with increased affinity to _IIb3 integrin. Several of these compounds are now available, or will be available soon, on the clinical market, and they are able to interact with an affinity of 10⁹ to 10⁸ M with _IIb3 integrin (Topol et al., 1999; Verstraete, 2000). Their chemical structures, based on the RGD motif, correspond either to cyclic RGD peptides or to RGD peptidomimetics. Although reports from the literature suggest that they may be specific for _IIb3 integrin, some results indicate that cyclic RGD peptides can also interact with other RGD-dependent integrins, in particular _v3 integrin (Brooks et al., 1994; Matsuno et al., 1994; Mogford et al., 1996). We thus wonder whether or not RGD peptidomimetics can also recognize RGD-dependent integrins.

We recently developed a novel pharmacological assay that can easily detect the presence and functional state of RGD-dependent integrins on cells and tissues (Thibault, 2000). This assay is based on the interaction of ¹²⁵I-echistatin with RGD-dependent integrins: once bound, ¹²⁵I-echistatin forms SDS-stable complexes that can be visualized by autoradiography after nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this approach, we demonstrated that rat cardiac fibroblasts harbor three different radioactive bands corresponding to ₈₁, _v3, and a heterogeneous mixture of ₃₁, ₅₁, and _v1. We therefore used this method to investigate the specificity of RGD compounds on rat cardiac fibroblast integrins as well as on human skin fibroblasts that possess _v1 and _v3 integrins. By radioligand binding filtration experiments, we have also assessed and compared the ligand properties of these compounds on basal and thrombin-activated rat and human platelet _IIb3 integrin.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

RGD Compounds. The RGD compounds used in the present studies (Table 1) were generously provided by different pharmaceutical companies. Eptifibatide was obtained from Dr. D. R. Phillips of COR Therapeutics, Inc. (South San Francisco, CA), XV459 (the active metabolite of roxifiban), DMP728, and XJ735 were from Dr. S. A. Mousa of DuPont Pharmaceuticals Co. (Wilmington, DE), SC-54701A (the active metabolite of xemilofiban) was from Dr. L. G. Frederick of Searle (Skokie, IL), Ro 44-3888 (the active metabolite of sibrafiban) and lamifiban were from Dr. P. Weber of F. Hoffmann-La Roche Ltd. (Basel, Switzerland), BIBU-52 (the active metabolite of lefradafiban) was from Dr. J. Krause of Boehringer Ingelheim Pharma KG (Biberach, Germany), SR 121566A (the active metabolite of SR 121787) was from Dr. J.-M. Herbert of Sanofi Recherche (Toulouse, France) and L-734,217 was from Dr. G. D. Hartman of Merck Research Laboratories (Rahway, NJ). ReoPro, the humanized monoclonal antibody against human _IIb3 integrin, was a gift from Dr. M. A. Mascelli of Centocor, Inc. (Malvern, PA). All drugs were dissolved according to the manufacturers' recommendations.

View this table: [in this window] [in a new window]   TABLE 1 Platelet IIb3 integrin antagonists

Fibroblast Cultures. Primary cultures of rat cardiac fibroblasts were obtained by trypsin digestion of cardiac ventricles as already described (Fareh et al., 1997). Cells were grown in Dulbecco's modified Eagle's medium in the presence of 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) until they reached confluency. Only primary cultures were used.

Isolation of Platelets. Sprague-Dawley rats, under pentobarbital anesthesia (60 mg/100 g of body weight), were exsanguinated via the abdominal aorta. Blood collected under 3.8% trisodium citrate was centrifuged at room temperature at 1400g for 3 min to obtain platelet-rich plasma. The supernatant was then centrifuged at 2250g for 15 min to sediment the platelets, which were gently resuspended once in washing buffer (0.113 M NaCl, 4.3 mM K₂HPO₄, 4.3 mM Na₂HPO₄, 24.4 mM NaH₂PO₄, and 6.5 mM glucose), centrifuged, and diluted in a resuspension buffer [0.14 M NaCl, 15 mM Tris (Tris(hydroxymethyl)aminomethane)-HCl, pH 7.4, and 5.5 mM glucose] (Baezinger and Majerus, 1974). Platelets were counted in a microhematocrit tube assuming that a reading of 1% corresponds to a concentration of 1 × 10⁹ platelets/ml.

Analysis of Binding by Filtration Experiments. Basal-state platelets were diluted in binding buffer (consisting of 0.05 M HEPES, pH 7.4, and 5 mM MnCl₂) at a concentration of 1.5 × 10⁶ human platelets or 5 × 10⁶ rat platelets/100 µl. To get an equivalent radioactivity signal, a 3-fold higher concentration was used for thrombin-activated platelets. Platelets (100 µl) were incubated with 50 µl of ¹²⁵I-echistatin (250,000 cpm) in the presence of increasing concentrations of RGD compounds in a total volume of 250 µl of binding buffer. After 90-min incubation at room temperature, the samples were filtered on 34 glass fiber paper (Schleicher & Schuell, Keene, NH), then washed three times with 3 ml of 0.05 M Tris-HCl, pH 7.4, and 0.154 M NaCl on a 30-well Brandel cell harvester (Gaithersburg, MD). The filters were presoaked for 1 h in washing buffer containing 5% dry skim milk (Carnation, Nestlé, Don Mills, Ontario, Canada) to reduce nonspecific adsorption. Radioactivity was counted in a gamma counter with an efficiency of 80%. Competition curves were analyzed by the Hill equation. Under these conditions, a signal of 5,000 to 15,000 cpm for specific binding was consistently obtained. Nonspecific binding was measured in the presence of 10 mM EDTA and was less than 0.4% of total radioactivity.

Analysis of Binding by Nondenaturing SDS-PAGE and Autoradiography. NP-40-solubilized proteins (10-15 µg) were incubated in the presence of 250,000 cpm ¹²⁵I-echistatin and increasing concentrations of RGD compounds in a total volume of 20 µl of binding buffer plus 0.1% NP-40. After a 90-min incubation, SDS sample buffer (containing 0.188 M Tris-HCl, pH 6.8, 30% glycerol, 6% SDS, and 0.15% bromphenol blue, without -mercaptoethanol) was added to reach a SDS concentration of 0.8 to 1.2%. The samples were not heated. Proteins were then loaded on 6% polyacrylamide gel and separated according to Laemmli (1970) in a Mini-Protean II cell system (Bio-Rad Laboratories). Gels were stained with Coomassie Blue R-250, dried, and exposed for about 1 h to X-OMAT AR5 film (Eastman Kodak, Rochester, NY). For the quantification of radioactivity in individual bands, the gels were submitted to a phosphor screen and analyzed in a Storm 860 system (Molecular Dynamics, Sunnyvale, CA).

Immunoblotting and Immunoneutralization. SDS-stable complexes between RGD-dependent integrins and ¹²⁵I-echistatin on NP-40-solubilized human skin fibroblasts were identified by immunoblotting and immunoneutralization as described previously (Thibault, 2000). Specific antisera were obtained from Dr. R. O. Hynes (Howard Hughes Medical Institute, Cambridge, MA) (anti-₁, no. 130) and from Chemicon International Inc. (Temecula, CA) (anti-_v, MAB1953; anti-_v3, MAB1976).

Statistical Analysis. Differences between the potencies of _IIb3 integrin antagonists were compared by the unpaired t test with a p  0.05 level of significance.

	Results

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Specificity on Human and Rat Platelets. We first assessed, in radioligand binding and filtration experiments, the capacity of several RGD compounds to displace ¹²⁵I-echistatin on basal and thrombin-activated _IIb3 integrin of human and rat platelets. The results on activated platelets are shown in Fig. 1, and the IC₅₀ values are presented in Table 2. All the RGD compounds on human platelets, except XJ735, a cyclic RGD mimetic designed to interact with _v3 integrin, demonstrated high potency. Activation of platelets by thrombin increased potency by 2- to 60-fold, with L-734,217 and BIBU-52 showing the most significant difference. On basal rat platelets, only eptifibatide, XJ735, and DMP728 were able to demonstrate some activity. Surprisingly, activation of rat _IIb3 integrin by thrombin decreased potency of the ligands by a factor of 2- to 6-fold.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Displacement curves of RGD compounds on thrombin-activated human (A) and rat (B) platelets. Platelets were incubated with 125I-echistatin in the presence of increasing concentrations of RGD compounds for 90 min. Incubation was stopped by rapid filtration and washing on glass fiber filters. Each curve is the mean ± S.E.M. of three different filtration experiments. , echistatin; , eptifibatide; , L-734,217; , lamifiban; , Ro 44-3888; , SR 121566A; , XJ735; , DMP728; , BIBU-52; , SC-54701A; , XV459.

View this table: [in this window] [in a new window]   TABLE 2 Comparison of IIb3 integrin antagonists (IC50, nM) on rat and human platelets Results are the means ± S.E.M. of three different experiments.

Specificity on Other RGD-Dependent Integrins. We used two different cell lines to investigate the specificity of the RGD compounds on integrins. We have already identified, on cultured rat cardiac fibroblasts, five different RGD-dependent integrins by a novel pharmacological method (Thibault, 2000). As illustrated in Fig. 2D, three radioactivity bands were associated with ₈₁; a mixture of ₃₁, ₅₁, and _v1; and _v3 integrin; respectively. By using increasing concentrations of the test agents and by measuring the intensity of the bands on a phosphor screen, it was possible to construct displacement curves and simultaneously evaluate the potency of the corresponding agent on each integrin. Figure 2, A, B, and C, presents the displacement curves of XJ735, DMP728, and eptifibatide, respectively, and the IC₅₀ calculated from the Hill equation is shown in Table 3. The fact that the second band is heterogeneous, consisting of several integrins, prevents the adequate evaluation of potency on each integrin.

View larger version (36K): [in this window] [in a new window]   Fig. 2.   Displacement curves of RGD compounds on rat cardiac fibroblast extracts. NP-40-solubilized rat fibroblasts were incubated with 125I-echistatin in the presence of increasing concentrations of RGD compounds for 90 min. Displacement was assessed by separation of proteins by nondenaturing SDS-PAGE and quantification of radioactive bands on the dried gel by a phosphor screen. Each curve is the mean ± S.E.M. of three different binding experiments. Displacement curves are only shown for XJ735 (A), DMP728 (B), and eptifibatide (C). Typical autoradiograms are also shown (D).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Identification of RGD-dependent integrins on human skin fibroblasts by immunoblotting (A) and immunoneutralization (B) experiments. For immunoblotting, NP-40-solubilized fibroblasts were incubated with 125I-echistatin, proteins were separated by nondenaturing SDS-PAGE and transferred to nitrocellulose, and autoradiograms were obtained (auto.). The nitrocellulose sheet was then incubated with anti-integrin antisera, as specified in the figure, and developed (blot). In some cases, three different fibroblast extracts were used (1, 2, and 3). For immunoneutralization, NP-40-solubilized fibroblasts were first incubated with anti-integrin antisera, as specified in the figure, then with 125I-echistatin. Proteins were separated by nondenaturing SDS-PAGE, gels were stained and dried, and autoradiograms were obtained. cont., control.

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View larger version (29K): [in this window] [in a new window]   Fig. 4.   Displacement curves of RGD compounds on human skin fibroblast extracts. NP-40-solubilized human fibroblasts were incubated with 125I-echistatin in the presence of increasing concentrations of RGD compounds for 90 min. Displacement was assessed by separation of proteins by nondenaturing SDS-PAGE and quantification of radioactive bands on the dried gel by a phosphor screen. Each curve is the mean ± S.E.M. of three different binding experiments. Displacement curves are only shown for XJ735 (A), DMP728 (B), and eptifibatide (C). Typical autoradiograms are also shown (D).

View this table: [in this window] [in a new window]   TABLE 3 Comparison of IIb3 integrin inhibitors (IC50, µM) on rat and human RGD-dependent integrins Results are the means ± S.E.M. of three different experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Integrins are subdivided into different families based on the structural composition of their  and  subunits, their expression in specific cell types, or their affinity toward certain groups of extracellular matrix proteins. Among them, several integrins, namely, _IIb3, ₅₁, ₈₁, _v1, _v3, _v5, _v6, and _v8 and, under special conditions, _{31 41}, ₂₁, and ₁₁, have been documented to bind through an RGD motif. This motif is present in proteins such as fibronectin, fibrinogen, von Willebrand factor, vitronectin, osteopontin, tenascin, and others (Ruoslahti, 1997). With the exception of _IIb3 integrin that has an expression restricted to platelets and megakaryocytes, all other RGD-dependent integrins are widely distributed. Attribution of a functional, cellular role for each integrin has been impaired by the fact that multiple integrins with similar specificity can be expressed on one cell type. However, some information indicates that _v3 integrin may be implicated in migration of vascular smooth muscle cells (Byzova et al., 1998), whereas other reports have observed an association between the expression of ₈₁ integrin in myofibroblasts and deposition of fibrotic material in the lungs, the kidneys, and the liver (Hartner et al., 1999; Levine et al., 2000).

Antagonists of _IIb3 integrin were basically designed on the RGD motif of the matrix proteins. Accordingly, the first group of substances to be synthesized was short, linear, and later cyclic peptides that contain an RGD sequence. These peptides presented moderate specificity and affinity, as exemplified by eptifibatide, XJ735, and DMP728. More recently, RGD peptidomimetics were chemically designed and selected for their potential to interact exclusively with _IIb3 integrin. Our results confirmed that these drugs have interaction limited to _IIb3 integrin. This specificity is particularly important if interactions with other RGD-binding integrins prove to have deleterious effects. On the other hand, _v3 integrin has been proposed as an interesting target to inhibit cellular migration as observed in vascular restenosis or in tumor propagation (Brooks et al., 1994; Matsuno et al., 1994). In that case, an integrin antagonist should solely interact with _v3 integrin and not disturb blood homeostasis. Therefore, a method that can test rapidly the specificity of these RGD-based molecules on several integrins is of great interest.

So far, investigation into the specificity of either RGD peptides or peptidomimetics on other RGD-dependent integrins was limited by the type of method used. Inhibition of adhesion of integrin-transfected cells is one example: the results on inhibition of adhesion reflect not only the strength of interaction between integrin and adhesion protein but also the extent of cell spreading on the matrix (McClay and Hertzler, 1999). Estimation of the displacement of adhesion proteins on purified integrins in solid phase assay is another example: adsorption of a specific purified integrin to a plastic matrix may change its conformation and alter its binding properties; in addition, purification of different integrins from several species is a considerable task. For these reasons, results on the specificity of RGD compounds are rather scarce.

After adequate identification of the integrins present on a specific cell type, the assay that we described represents a relatively rapid method of evaluating the binding properties and comparing the specificity of RGD compounds on different RGD-dependent integrins from the same species and also from other species. Using this approach, we evaluated the specificity of several platelet _IIb3 integrin antagonists on human _v1 and _v3 integrins and on rat ₈₁ and _v3 integrins.

On thrombin-activated human platelets, all _IIb3 integrin antagonists displayed potency between 0.3 and 30 nM, with XV459 showing the greatest potency. The potency was 2- to 60-fold higher than on basal platelets. The results on basal platelets correspond well to those reported in the literature, as assessed by direct binding or by the displacement of ¹²⁵I-fibrinogen from activated platelets, with values ranging from 0.1 to 150 nM (Mousa et al., 1993, 1994, 1998; Scarborough et al., 1993; Nicholson et al., 1995; Zablocki et al., 1995; Weller et al., 1996; Askew et al., 1997; Badorc et al., 1997; Muller et al., 1997; Bednar et al., 1998; Bernat et al., 1999). One important issue of _IIb3 integrin antagonists, as discussed recently by Scarborough et al. (1999), is their potency on unstimulated platelets. Ideally, _IIb3 integrin antagonists should only bind to activated platelets to achieve inhibition of platelet aggregation and not to resting platelets to not affect normal blood homeostasis and cause bleeding. Our results indicate that only L-734,217 and BIBU-52 showed interesting potency differences (60- and 20-fold, respectively) between basal and stimulated platelets.

Cyclic RGD peptides demonstrated weak interactions (0.1-0.2 µM) on rat platelets. Surprisingly, their potencies were lower on thrombin-activated platelets than on resting platelets. Although echistatin bound in a comparable manner on rat and human platelets, none of the RGD mimetics were able to interact with rat basal and stimulated platelets. These results explain previous observations showing that the activity of _IIb3 integrin antagonists, particularly the peptidomimetics, was very weak in inhibiting rodent platelet aggregation (Mousa et al., 1994; Cook et al., 1996; Phillips and Scarborough, 1997; Bernat et al., 1999). The reasons underlying the differences of binding properties of the RGD mimetics on rat and human platelets are presently unclear. Comparative studies of the three-dimensional structure of the rat and human _IIb3 integrins will certainly help to resolve these issues.

We also explored the potency of _IIb3 integrin antagonists on other RGD-dependent integrins. With the exception of cyclic RGD mimetics, none of these antagonists showed any significant interaction with rat ₈₁ and _v3 integrins, and human _v1 and _v3 integrins. The interaction (micromolar range) of eptifibatide, XJ735, and DMP728 with integrins of the _v subunit family probably reflects the presence of an RGD motif. XJ735 and DMP728 showed equivalent potency on these RGD-dependent integrins, although it has been reported that XJ735 interacts with an affinity of 40 nM on purified human _v3 integrin (Srivasta et al., 1997). Interestingly, the potency of XJ735 was similar on human platelets and _v3. Although no such results are available for DMP728, this compound has a 1000-fold preference for human _IIb3 over _v3. ReoPro demonstrated a good interaction only with human _v3 integrin, confirming previous results (Tam et al., 1998).

In summary, we have investigated the specificity of _IIb3 integrin inhibitors not only on rat and human platelets but also on other RGD-dependent integrins present on rat and human fibroblasts. For that purpose, we have used a novel pharmacological approach based on the fact that ¹²⁵I-echistatin forms SDS-resistant complexes with RGD-dependent integrins. This allows direct access to visualize and determine the specificity of RGD compounds. On the RGD-dependent integrins that we have tested, all RGD mimetics demonstrated a specificity restricted to human _IIb3 integrin. Cyclic RGD peptides have a larger specificity, being able to interact with 100-fold lower potency on rat _IIb3 integrin and with 1000-fold weaker potency on rat and human _v3 integrins.