©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
The Ligand Recognition Specificity of Integrins (*)

(Received for publication, November 28, 1995; and in revised form, February 12, 1996)

Kazuhisa Suehiro (1)(§) Jeffrey W. Smith (2) Edward F. Plow (1)(¶)

From the  (1)Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, The Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the (2)The Burnham Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alphabeta(3) (platelet membrane glycoprotein IIb-IIIa) and alpha(v)beta(3) are members of the beta(3) subfamily of integrin adhesion receptors. A cyclic peptide, KYGC(s-s)HarGDWPC(s-s) (cHarGD), originally described by Scarborough et al. (Scarborough, R. M., Naughton, M. A., Teng, W., Rose, J. W., Phillips, D. R., Nannizzi, L., Arsten, A., Campbell, A. M., and Charo, I. F.(1993) J. Biol. Chem. 268, 1066-1073) has been employed as a high affinity ligand for alphabeta(3) to examine the specificity of the beta(3) integrins. cHarGD interacted with high affinity with purified alphabeta(3) (K = 10 nM) or with platelets (K = 120 nM). While cHarGD was specific for alphabeta(3) in the presence of Ca, it bound to both beta(3) integrins in the presence of Mn. Barbourin, a snake venom disintegrin containing a reactive KGD sequence, remained alphabeta(3)-specific, even in the presence of Mn. cHarGD became cross-linked to a site in beta(3) of alphabeta(3), which is distinct from that of RGD peptides. These results allow identification of at least four classes of beta(3) ligands: Class I, represented by RGD peptides and vitronectin, react similarly with alphabeta(3) and alpha(v)beta(3); Class II, represented by cHarGD, -chain peptides and fibrinogen, react with both receptors in the presence of Mn but only with alphabeta(3) in the presence of Ca; Class III, represented by barbourin, are alphabeta(3)-specific under all cation conditions; Class IV, represented by osteopontin, bind primarily to alpha(v)beta(3).


INTRODUCTION

alphabeta(3) (platelet membrane glycoprotein IIb-IIIa) and alpha(v)beta(3) are members of the beta(3) subfamily of integrin heterodimeric adhesion receptors (1, 2, 3, 4) . alphabeta(3) is essential for platelet aggregation and controls platelet function in thrombosis and hemostasis (5, 6, 7) . alpha(v)beta(3) is expressed on many cells, where it influences cell migration and impacts on angiogenesis, restenosis, tumor cell invasion, and atherosclerosis(8, 9, 10) . These two integrins share the same beta subunit, beta(3), but have distinct alpha subunits; alpha and alpha(v) exhibit about 36% primary amino acid sequence identity(11) . Both integrins are oligospecific: their common macromolecular ligands include fibrinogen, fibronectin, thrombospondin, von Willebrand factor, and vitronectin(12, 13, 14, 15) . These common ligands contain Arg-Gly-Asp (RGD) sequences, and RGD-containing peptides inhibit binding of these ligands to both receptors(16) . When RGD peptides were bound and cross-linked to alphabeta(3) and alpha(v)beta(3), the cross-linking sites in both receptors are overlapping and confined to a stretch of about 100 amino acids in beta(3)(17, 18) . Moreover, single point mutations at several positions within beta(3) abrogated the binding of RGD peptides and macromolecular ligands to both receptors(19, 20, 21, 22) . These data are compatible with a role of the common beta(3) subunit of both receptors in establishing their RGD ligand recognition.

In addition to their common RGD recognition, several ligands with proported specificity for alphabeta(3) or alpha(v)beta(3) have been identified. The prototype of these are the fibrinogen -chain peptides. Peptides with sequences corresponding to the carboxyl terminus of the -chain of fibrinogen block ligand binding to alphabeta(3), but are poor inhibitors of ligand binding to alpha(v)beta(3)(16) . -Chain peptides cross-link to alpha, specifically to alpha 294-314, providing a basis for alphabeta(3) selective recognition(23) . Barbourin, a member of the disintegrin family of snake venom proteins, was reported to inhibit the binding of fibrinogen to alphabeta(3) but not to block vitronectin binding to alpha(v)beta(3)(24) . In contrast, several other disintegrins block ligand binding to both receptors. Distinguishing barbourin from other disintegrins is the presence of a KGD, rather than a RGD, sequence; and the Lys residue is critical for specificity(24) . In following this observation, Scarborough et al.(25) synthesized a series of peptides and ultimately identified small, conformationally constrained cyclic derivatives as highly potent agonists of alphabeta(3) function. A cyclic homoarginine (Har)(^1)-glycine-aspartic acid-containing peptide was one of the most potent representative of this series. This compound showed substantial specificity for alphabeta(3)versus alpha(v)beta(3), as compared to linear RGD-containing peptides, although it could block alpha(v)beta(3)-mediated cell adhesion(25) .

Divalent cations are required to support the ligand binding functions of the beta(3) integrins described above as well as for the recognition of most ligands by integrins(26, 27) . This requirement depends on the direct binding of cations to the integrin subunits, and it is generally accepted that the divalent cation binding and ligand binding sites reside in close proximity within integrins, including the beta(3) integrins(23, 28, 29, 30) . Ligand binding to many integrins is differentially regulated by divalent cations. Numerous examples have been described in which the ligand specificity of members of the beta(1) and beta(2) integrin subfamilies are divalent cation determined(31, 32, 33, 34, 35, 36) . Recently, differential regulation of ligand binding to beta(3) integrins also has been reported (37) as fibrinogen did not bind to alpha(v)beta(3) in the presence of Ca, but Mn did support this interaction. This observation has brought the entire issue of the relative specificity of beta(3) integrins into question.

In the present study, we have examined the interaction of a cyclic HarGD-containing peptide (cHarGD) with alphabeta(3) and alpha(v)beta(3). These studies were initiated to test the hypothesis that cHarGD would represent a high affinity mimetic of fibrinogen and its -chain peptides. The high affinity binding of this peptide to alphabeta(3) is demonstrated by direct binding studies with the isolated receptor and platelets. Overall, the results document the complexity of ligand recognition by beta(3) integrins and allow discrimination of multiple classes of beta(3) ligands.


MATERIALS AND METHODS

Purification of Proteins

Fibrinogen was purified from human fresh-frozen plasma by differential ethanol precipitation(38) . Vitronectin was purified from human plasma by heparin-Sepharose chromatography in 8 M urea(39) . alphabeta(3) was purified from octyl glucoside extracts of human platelets by affinity chromatography using GRGDSPK-Sepharose columns(40) . alpha(v)beta(3) was purified from human placenta by affinity chromatography with alpha(v)beta(3)-specific monoclonal antibody (mAb) LM609(18) . This material did not contain detectable levels of alphabeta(3) by enzyme-linked immunosorbent assay. Barbourin was purified from lyophilized snake venom, Sistrurus m. barbouri, purchased from Miami Serpentarium Labs (Punta Gorda, FL) as described (24, 41) with minor modifications. Briefly, venom was dissolved in 0.5 M acetic acid and was applied to a Sephadex G-50 column (1.0 times 100 cm) equilibrated in 0.5 M acetic acid. Fractions inhibiting fibrinogen binding to alphabeta(3) were lyophilized, and barbourin was purified to homogeneity by high performance liquid chromatography using a C18 Vydac column.

Peptides and Antibodies

RGD-containing peptides (KYGRGDS and GRGDSP), GRGESP peptide and fibrinogen -chain peptides, KYGGHHLGGAKQAGDV (K16) and HHLGGAKQAGDV (H12), were prepared as described(42) . Cyclic HarGD peptide (cHarGD), KYGC(s-s)HarGDWPC(s-s), was synthesized by the same method and cyclized with potassium ferricyanide as described(25) . The peptides were purified to homogeneity by high performance liquid chromatography using a C18 Vydac column and characterized by amino acid composition. Monoclonal antibody 7E3 (43) was kindly provided by Dr. Barry S. Coller, Mt. Sinai School of Medicine, NY.

Radioiodination

NaI (specific activity = 15-17 mCi I/µg of iodine) from Amersham Life Science Inc. was used for radioiodination. Fibrinogen and vitronectin were radiolabeled using a modified chloramine-T method(38) , and synthetic peptides and barbourin were radiolabeled using IODO-GEN (Pierce). The iodinated peptides were separated from free NaI by gel filtration on a Bio-Gel P-2 column (Bio-Rad). For selected experiments, cHarGD was ``mock-labeled''; nonradioactive iodine was substituted for the I, but all other aspects of the procedure were the same.

Solid-phase Ligand Binding Assay

The binding of fibrinogen, cHarGD, and barbourin to immobilized alphabeta(3) and alpha(v)beta(3) was performed as described by Charo et al.(15, 44) with minor modifications. alphabeta(3) (250 µg/ml) was diluted 1:50 with a buffer containing 20 mM Tris, 150 mM NaCl, pH 7.4 (Buffer A), immobilized in 96-well microtiter plates at 500 ng/well, and incubated overnight at 4 °C. alpha(v)beta(3) (400 µg/ml) was diluted 1:80 with Buffer A, and immobilized at 500 ng/well. After the plates were blocked with 20 mg/ml bovine serum albumin, I-labeled ligands were added in Buffer A containing 1 mg/ml bovine serum albumin and selected divalent ions at 1 mM and incubated for 60-180 min at 37 °C. Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled ligand. K(d) and B(max) values were derived by a curve fitting analyses of specific binding isotherms using the program DeltaGraph for Macintosh (DeltaPoint Inc.). Data were determined as the mean of quadruplicate measurements at each experimental point.

Binding of cHarGD to Washed Platelets

Platelets, isolated as described (12) and suspended at 1 times 10^8/ml in the modified Tyrode's buffer, were stimulated with alpha-thrombin at 0.1 unit/ml, and 3 µMD-phenylalanyl-L-prolyl-arginine chloromethyl ketone was added 10 min after thrombin stimulation. I-cHarGD was then added at a final concentration of 50 nM in the presence of appropriate divalent ions. At selected times, platelet-bound ligand was separated by centrifugation through 20% sucrose for 2.5 min at 22 °C in Beckman Microfuge, and the cell-bound radioactivity was measured in a -counter.

Cross-linking Studies

Platelets were suspended at 5 times 10^8/ml in Tyrode's buffer, pH 7.2, containing 1 mg/ml bovine serum albumin, and activated by 0.5 unit/ml alpha-thrombin. Radiolabeled cHarGD was then added at a concentration of 100 nM and incubated for 15 min at 22 °C in the presence of 1 mM each CaCl(2), MgCl(2), and MnCl(2). I-KYGRGDS and I-K16, at concentrations of 1 µM, were also tested. Bis(sulfosuccinimidyl) suberate (BS^3), 3,3`-dithiobis(sulfosuccinimidyl propionate) and dithiobis(succinimidyl propionate) were used as cross-linking reagents. All three cross-linking reagents, purchased from Pierce, were used at concentrations 0.05-0.2 mM. The cross-linking reaction was terminated after 10 min at 22 °C by addition of 10 mM Tris, pH 7.0. The platelets were harvested by centrifugation through 20% sucrose and were extracted in phosphate-buffered saline (0.15 M NaCl, 0.01 M sodium phosphate buffer, pH 7.3), containing 1% Nonidet P-40 and 10 mMN-ethylmaleimide (Sigma). Extracted proteins were analyzed by SDS-PAGE on 7.5% polyacrylamide gel and subjected to autoradiography. For cross-linking of cHarGD to purified alphabeta(3), I-cHarGD, at a final concentration of 2 µM, was incubated with 1 µg of purified alphabeta(3) in 150 mM NaCl, 10 mM HEPES, pH 7.5, in the presence of 1 mM cation (CaCl(2) or MnCl(2)) for 60 min at 22 °C. The cross-linking reaction was terminated after 10 min at 22 °C and analyzed by SDS-PAGE. Cross-linking of I-KYGRGDS to alphabeta(3) was performed by the same method, but the I-KYGRGDS concentration was 100 µM, and the incubation time was 3 h. Cross-linked samples were immunoprecipitated as described (42) using mAbs: PMI-1 to the heavy chain of alpha(45, 46) , PMI-2 to beta(3)(45) , and control TSPI-1 to thrombospondin. Enzymatic fragmentation of cross-linked I-cHarGD- or I-KYGRGDS-alphabeta(3) complexes was performed in the presence of alpha-chymotrypsin (Sigma) for 18 h at 37 °C using the 1:1 (w/w) enzyme-to-substrate ratio in 50 mM ammonium bicarbonate, pH 8.0, containing 5 mM EDTA and 0.1% SDS. Samples were boiled for 5 min to inactivate the enzyme and analyzed by SDS-PAGE.


RESULTS

cHarGD as a Potent Inhibitor of Fibrinogen Binding to alphabeta(3)

To explore the binding specificity of the beta(3) integrins, a cyclic peptide, cHarGD, was synthesized as a predicted high affinity ligand for alphabeta(3)(25) . Initially, the effect of cHarGD on I-fibrinogen binding to purified and immobilized alphabeta(3) was examined. cHarGD was a potent inhibitor of fibrinogen binding to alphabeta(3) as compared to RGD and -chain ligand peptides. As shown in Fig. 1, its IC for inhibiting fibrinogen binding to isolated alphabeta(3) was 10 ± 1.7 nM (n = 3). This value is considerably lower than that of the -chain peptide, H12, at 10 ± 2.0 µM (n = 3) and RGD-containing peptide, GRGDSP, at 2.0 ± 0.6 µM (n = 3), and is consistent with the observations of Scarborough et al.(25) .


Figure 1: Inhibition of fibrinogen binding to alphabeta(3) by cHarGD, RGD, and -chain peptides. I-Fibrinogen (20 nM) and various concentrations of cHarGD (), GRGDSP (bullet), and -chain peptide HHLGGAKQAGDV () were simultaneously added to microtiter wells coated with alphabeta(3) in the presence of 1 mM Ca, Mg, and Mn. After 3 h at 37 °C, bound fibrinogen was quantitated by counting the bound radioactivity in a -counter. Binding in the absence of the peptides was assigned a value of 100%. The data shown are means and standard deviation from three separate experiments each with quadruplicate measurements.



Direct Binding of cHarGD to alphabeta(3)

To assess direct binding of cHarGD to alphabeta(3), the peptide was radiolabeled to a specific activity of 20-40 µCi/µg. We verified that iodination did not alter its ability to inhibit fibrinogen binding to alphabeta(3): the IC values for nonlabeled and mocked labeled cHarGD were 14 and 16 nM, respectively. Interaction of I-cHarGD with immobilized alphabeta(3) was then assessed. The time course of I-cHarGD (20 nM) binding to immobilized alphabeta(3) was analyzed and found to reach a constant level within 60 min at 37 °C (data not shown). This interaction was divalent cation dependent and was inhibited by EDTA. With the cations at 1 mM, at the 60 min time point, higher binding was obtained in the presence of Mn (relative value = 1.3) and the combination of Ca, Mg, and Mn (relative value = 1.4) than in the presence of Ca (assigned relative value = 1.0). To further characterize I-cHarGD binding to alphabeta(3), several competitors were tested. Nonlabeled cHarGD, -chain peptide, RGD-containing peptides, fibrinogen, and mAb 7E3 inhibited binding, but RGE-containing peptides and unrelated proteins, such as transferrin and ovalbumin, did not (Fig. 2). These characteristics were the same as reported for fibrinogen binding to alphabeta(3)(44) .


Figure 2: Specificity of the interaction of cHarGD with alphabeta(3). I-cHarGD (20 nM) was added to microtiter wells coated with alphabeta(3) in the presence or absence of excess unlabeled cHarGD (2 µM), -chain peptide HHLGGAKQAGDV (100 µM), GRGDSP (100 µM), GRGESP (100 µM), fibrinogen (5 µM), transferrin (5 µM), and monoclonal antibody 7E3 (20 µg/ml) in the presence of 1 mM Ca, Mg, and Mn, and incubated for 60 min at 37 °C. Values represent the means and standard deviation of 3-5 determinations and are expressed as percent of a control lacking inhibitors.



The specific binding of I-cHarGD to immobilized alphabeta(3) was saturable (Fig. 3A). Addition of a 100-fold excess unlabeled cHarGD displaced more than 95% of the bound radiolabeled cHarGD. The data from the binding isotherm in Fig. 3A were graphed as a Scatchard plot (Fig. 3B) which suggested that the interaction could be described by a single class of high affinity binding sites with an apparent dissociation constant (K(d)) of 9.7 ± 0.9 nM (n = 3). I-cHarGD binding to immobilized alphabeta(3) was observed to approach 1 to 1 molar stoichiometry of ligand to receptor. Using a radiolabeled monoclonal anti-alpha, PMI-1, to quantitate the amount of immobilized receptor, as described by Du et al.(47) , the ratio of B(max) for radiolabeled cHarGD/PMI-1 was 0.92.


Figure 3: Saturation isotherm and Scatchard analysis of I-cHarGD binding to alphabeta(3). A, binding isotherms of cHarGD to immobilized alphabeta(3) were constructed by incubating increasing concentrations of I-cHarGD with immobilized alphabeta(3). Specific binding was calculated by subtracting nonspecific binding, measured as the residual binding in the presence of a 100-fold excess of nonlabeled cHarGD, from the total binding. The data shown are representative of three separate experiments. B, Scatchard plots of cHarGD binding to alphabeta(3). The apparent dissociation constant (K) estimated from the slope of the line was 9.7 ± 0.9 nM (n = 3).



In a second set of studies, I-cHarGD binding to platelets was evaluated. I-cHarGD did not bind to non-stimulated platelets in the presence of Ca or EDTA, but the binding was supported in the presence of Mn (Fig. 4). I-cHarGD bound to thrombin-stimulated platelets in the presence of Ca as well as Mn, and the latter cation supported higher binding. I-cHarGD binding to thrombin-stimulated platelets was inhibited by -chain peptide, RGD-containing peptides, fibrinogen, and mAb 7E3 (data not shown), using these reagents at the conditions specified in Fig. 2. Analysis of saturable cHarGD binding to thrombin-stimulated platelets by Scatchard plots indicated a single class of binding sites with an estimated K(d) of 120 ± 16 nM (n = 3) and a maximum of 28,700 ± 900 molecules bound per platelet (data not shown). Taken together, these observations indicate that cHarGD fulfills the specification of a low molecular weight peptide ligand which binds with high affinity to alphabeta(3), either in purified form or on platelets.


Figure 4: Effect of divalent cations on I-cHarGD binding to platelets. Washed platelets (1 times 10^8/ml), non-stimulated or stimulated with alpha-thrombin (0.1 unit/ml), were incubated with I-cHarGD at a final concentration of 50 nM for 15 min at 22 °C in the presence of 5 mM EDTA, 1 mM Ca, or 1 mM Mn as described under ``Materials and Methods.'' The values shown are means and standard deviation from three separate experiments performed with triplicate measurements.



Receptor Specificity of cHarGD Is Regulated by Divalent Ions

The specificity of cHarGD for the two beta(3) integrins was explored using solid-phase assays. In initial experiments, we verified that I-fibrinogen did not bind to alpha(v)beta(3) in the presence of Ca but did bind in the presence of Mn (Fig. 5A)(37) . Next, cHarGD binding to alphabeta(3) and alpha(v)beta(3) was compared. While I-cHarGD bound selectively to alphabeta(3) in the presence of Ca, cHarGD lost its selectivity and bound to both alphabeta(3) and alpha(v)beta(3) in the presence of Mn (Fig. 5B).


Figure 5: Differential effects of divalent cations on the binding of I-fibrinogen and I-cHarGD to alphabeta(3) and alpha(v)beta(3). The effects of divalent cations on I-fibrinogen (A) and I-cHarGD (B) binding to immobilized alphabeta(3) and alpha(v)beta(3) were examined. Binding was measured in the presence of 1 mM Ca or 1 mM Mn, and the binding of each ligand to alphabeta(3) in the presence of 1 mM Ca was assigned a value of 100%. The data shown are means and standard deviation from three experiments.



Further evidence for the cation-dependent regulation of cHarGD with beta(3) integrins was obtained in competition experiments using fibrinogen as a prototypic alphabeta(3) macromolecular ligand and vitronectin as a prototypic alpha(v)beta(3) ligand. While cHarGD was an effective inhibitor of fibrinogen binding to alphabeta(3) in the presence of Ca, it was a poor inhibitor of vitronectin binding to alpha(v)beta(3) (Fig. 6A). In the presence of Mn, however, cHarGD possessed a similar inhibitory activity against vitronectin binding to alpha(v)beta(3) and fibrinogen binding to alphabeta(3) (Fig. 6B). These results clearly show that cHarGD interacts with alpha(v)beta(3) in the presence of Mn although its interaction with alpha(v)beta(3) is minimal in the presence of Ca.


Figure 6: Differential effects of cHarGD on fibrinogen binding to alphabeta(3) and vitronectin to alpha(v)beta(3). The ability of cHarGD to block the interaction of fibrinogen (Fgn) with alphabeta(3) () or vitronectin (Vn) with alpha(v)beta(3) (bullet) is compared in the presence of 1 mM Ca (A) or 1 mM Mn (B). Radiolabeled fibrinogen or vitronectin (20 nM) was added in quadruplicate to microtiter wells coated with alphabeta(3) or alpha(v)beta(3), respectively, and incubated in the presence of various concentrations of cHarGD for 3 h at 37 °C. Binding was expressed as a percent of the control without cHarGD. The data shown are representative of three separate experiments.



In interacting with alpha(v)beta(3) in the presence of Mn, cHarGD possessed a similar inhibitory activity as a RGD peptide; its IC was 40 nM compared to 25 nM for GRGDSP (Fig. 7). We also found that a -chain peptide, H12, was inhibitory (IC = 40 µM) in the presence of Mn although it was substantially less potent than cHarGD. The relationship between Mn concentration and I-cHarGD binding to alpha(v)beta(3) was examined. Little cHarGD bound to the receptor at Mn concentrations below 1 µM (Fig. 8); maximal binding was supported by concentrations above 100 µM; and 50% maximal binding was obtained at 8 ± 1 µM (n = 3). This latter value is similar to that determined for fibrinogen binding to alpha(v)beta(3)(37) .


Figure 7: Inhibition of fibrinogen binding to alpha(v)beta(3) by cHarGD, RGD, and -chain peptides. Quadruplicate samples of I-fibrinogen and various concentrations of cHarGD (), GRGDSP (bullet), and the -chain peptide HHLGGAKQAGDV (), were incubated with immobilized alpha(v)beta(3) in the presence of 1 mM Mn for 3 h at 37 °C. Binding was expressed as percent of the control without inhibitor. The results shown are representative of two experiments.




Figure 8: Effect of Mn concentration on I-cHarGD binding to alpha(v)beta(3). Quadruplicate samples of I-cHarGD (20 nM) were incubated with immobilized alpha(v)beta(3) in the presence of various concentrations of Mn (). Binding was measured after 60 min incubation at 37 °C. Binding in the presence of 1 mM Ca (bullet) is shown for comparison. The experiment shown is a representative of three determinations.



Specificity of Barbourin for alphabeta(3)

The specificity of the disintegrin, barbourin, for alphabeta(3) was originally defined in the presence of Ca. As cHarGD was designed from the barbourin structure(24) , we questioned whether the specificity of barbourin also was cation determined. As shown in Fig. 9, in contrast to fibrinogen and cHarGD, I-barbourin failed to bind to alpha(v)beta(3) in the presence of Mn as well as Ca. Binding of barbourin to alphabeta(3) in the presence of Ca, as reported by Scarborough et al.(24) , was demonstrable, verifying that the ligand was functional. These characteristics distinguish barbourin from cHarGD and fibrinogen.


Figure 9: Effects of divalent cations on the binding of barbourin to alphabeta(3) and alpha(v)beta(3). I-Barbourin (20 nM) binding to immobilized alphabeta(3) and alpha(v)beta(3) was examined in the presence of 1 mM Ca or 1 mM Mn. I-Barbourin binding to alphabeta(3) in the presence of 1 mM Ca was assigned a value of 100%. The values shown are means and standard deviation derived from three experiments.



Chemical Cross-linking of cHarGD to alphabeta(3)

With the change in specificity of cHarGD in the presence of Mnversus Ca, we sought to determine whether cations might affect the cross-linking site of cHarGD in alphabeta(3). When radiolabeled RGD peptide and -chain peptide were cross-linked to thrombin-stimulated platelets using BS^3 as a cross-linker in the presence of Ca, Mg, and Mn, RGD peptide cross-linked primarily to beta(3) and the -chain peptide to alpha (Fig. 10A) as reported(17, 23, 48) . In parallel analyses, I-cHarGD was cross-linked only to the beta(3) subunit. No cross-linking to alpha was detected even with prolonged exposure. Immunoprecipitation analyses using monoclonal antibodies specific for alpha (PMI-1) and beta(3) (PMI-2) verified that cHarGD cross-linking was to beta(3); i.e. the radioactive band was immunoprecipitated by PMI-2 but not by PMI-1 (data not shown). When the cross-linking was compared at Ca alone or Mn alone, cHarGD still only cross-linked to beta(3) (Fig. 10B). Similar results were obtained when I-cHarGD was cross-linked to purified alphabeta(3) (data not shown). When two additional cross-linking reagents, 3,3`-dithiobis(sulfosuccinimidyl propionate) and dithiobis(succinimidyl propionate), were tested, cHarGD again was cross-linked only to beta(3). The cross-linking of cHarGD to beta(3) was blocked by non-labeled cHarGD, RGD peptide, and -chain peptide (data not shown). Thus, cHarGD was specifically cross-linked only to the beta(3) subunit of alphabeta(3) under all divalent cation conditions tested. When I-barbourin was bound to alphabeta(3), it also cross-linked only to beta(3) in the presence of either Ca or Mn (data not shown).


Figure 10: A, chemical cross-linking of cHarGD, RGD, and -chain peptides to alphabeta(3) on platelets. I-cHarGD (lane 1) at 100 nM was bound to thrombin-stimulated platelets for 15 min at 22 °C and cross-linked with BS^3 (0.2 mM) for 10 min at 22 °C. I-KYGRGDS (lane 2) and I-KYGGHHLGGAKQAGDV (lane 3), each at 1 µM, were bound to thrombin-stimulated platelets for 45 min at 22 °C and cross-linked with BS^3 (0.2 mM). The cross-linked samples were extracted, analyzed on 7.5% polyacrylamide gel under nonreducing conditions, and subjected to autoradiography. B, cross-linking of cHarGD to alphabeta(3) on platelets at different cation conditions. I-cHarGD at 100 nM was bound to thrombin-stimulated platelets in the presence of either 1 mM Ca (lane 1) or 1 mM Mn (lane 2) and cross-linked with BS^3 (0.2 mM).



To explore the inter-relationship between the cHarGD and RGD cross-linking sites in beta(3), enzymatic digestion of cross-linked samples was performed. When I-KYGRGDS or I-cHarGD was cross-linked to alphabeta(3), digested with chymotrypsin and subjected to SDS-PAGE, the patterns of digestion were exactly the same by the silver staining of the gels (Fig. 11). However, the autoradiograms of the gels were not identical. As shown in Fig. 11, when I-KYGRGDS-beta(3) complex was digested with chymotrypsin, a 60-kDa chymotryptic fragment was the major band. Chymotryptic digestion of I-cHarGD-beta(3) complex, however, gave a predominant 46/40 kDa doublet, and the 60-kDa band was not present. Under reducing conditions, the I-KYGRGDS-beta(3) digest showed no radioactive band above 20 kDa, although I-cHarGD-beta(3) digest still contained a doublet at a slightly increased molecular mass. These results indicate that the site of cHarGD cross-linking differs from that of the RGD peptide.


Figure 11: Chymotryptic digestion of alphabeta(3) after cross-linking to cHarGD and RGD peptide. I-KYGRGDS at 100 µM was bound to purified alphabeta(3) for 3 h at 22 °C and cross-linked with BS^3 (0.1 mM) (lanes 1, 3, and 5). I-cHarGD at 2 µM was bound to purified alphabeta(3) for 60 min at 22 °C and cross-linked with BS^3 (0.1 mM) (lanes 2, 4, and 6). Cross-linked samples were digested with alpha-chymotrypsin for 18 h at 37 °C using the 1:1 (w/w) enzyme-to-substrate ratio and analyzed on 12.5% polyacrylamide gel. Lanes 1 and 2 are silver staining of the gel under nonreducing conditions. Lanes 3 and 4 and lanes 5 and 6 are autoradiograms of the gels under nonreducing and reducing conditions, respectively.




DISCUSSION

In this study, we have characterized the direct interaction of cHarGD with alphabeta(3) and have compared the recognition specificity of the beta(3) integrins for this ligand and other peptide and macromolecular ligands. The following conclusions are drawn from these analyses. First, cHarGD interacts with alphabeta(3), either in purified form or on platelets, with high affinity. Second, the capacity of cHarGD to interact with alpha(v)beta(3) is divalent cation determined. Third, although cHarGD and fibrinogen are not specific for alphabeta(3), as they react with alpha(v)beta(3) in the presence of Mn, other ligands may still be specific for alphabeta(3): barbourin is a notable example. Fourth, cHarGD becomes cross-linked to a site in beta(3) of alphabeta(3), which is distinct from the previously identified RGD and -chain cross-linking sites. Fifth, based upon differential interactive properties, at least four categories of beta(3) ligands can be distinguished.

cHarGD was a potent inhibitor of fibrinogen binding to isolated alphabeta(3) with an IC of 10 nM. This value is identical to its estimated K(d) for the receptor and is very similar to the K(d) of barbourin and fibrinogen binding to immobilized alphabeta(3)(24, 44) . I-cHarGD also bound to platelets with high affinity; its K(d) of 120 nM for thrombin-stimulated platelets is similar to that of fibrinogen(49) . Binding of cHarGD to purified alphabeta(3) or to platelets was divalent cation dependent and was inhibited by fibrinogen, mAb 7E3, -chain peptide, and RGD-containing peptides. Taken together, these characteristics, including the K(d) values, closely recapitulate the binding properties of fibrinogen to alphabeta(3) and indicates that cHarGD may be used as a low molecular weight, high affinity surrogate of fibrinogen.

cHarGD binding to the beta(3) integrins was regulated by divalent cations: cHarGD binding to alpha(v)beta(3) was supported by Mn but not by Ca. Cation regulation of ligand binding has been noted for other integrins as well (31, 33, 35, 36, 37, 50, 51) and may be explained by two possible mechanisms. First, specific cations may selectively modulate integrin conformation; e.g. Mn may induce a conformation which is favorable for ligand binding. Recent studies using mAb 9EG7, which not only recognizes an epitope induced by Mn but also stimulates beta(1) integrin adhesive functions(52) , support this mechanism. Second, integrin, cation, and ligand initially may form a ternary coordination complex, and the cation may eventually be displaced as the complex of ligand and integrin stabilizes(30) . Mn may be able to form the ternary complex when cHarGD binds to alpha(v)beta(3) but Ca cannot.

Our observations with cHarGD led us to re-evaluate whether barbourin is specific for alphabeta(3) even in the presence of Mn. Barbourin did not bind to alpha(v)beta(3) either in the presence of Ca or Mn. Thus, ligand discrimination by beta(3) integrins can be demonstrated even in the presence of Mn. In the series of XGD-containing cyclic peptides, Scarborough et al.(25) observed that guanidation of the lysine-containing analogs to form homoarginine-containing inhibitors, led not only to a significant increase in the affinity for alphabeta(3), but also increased the affinity for alpha(v)beta(3). Although the specificity of such homoarginine-containing analogs for alphabeta(3) may remain extremely high in the presence of Ca, their specificity is much lower in the presence of Mn. Thus, despite the subtlety of the Lys to Har to Arg interchange, these alterations have significant bearing on cation regulated recognition. Substitution at the amino acids flanking the tripeptide ligand sequences also influence affinity for alphabeta(3) and alpha(v)beta(3)(53) , emphasizing the fine specificity of ligand recognition.

cHarGD became cross-linked only to beta(3) when this peptide was bound to purified alphabeta(3) or to platelets, regardless of the divalent cation conditions. Thus, even in the presence of Ca, a condition where cHarGD only binds to alphabeta(3) and not alpha(v)beta(3), its cross-linking site still resides in the beta(3) subunit. Although RGD peptide also cross-linked to beta(3), proteolytic digestion indicated that the cHarGD and RGD cross-linking sites are distinct. A 60-kDa band in the digest of cross-linked RGD peptide under nonreducing conditions is predicted to contain two fragments held by the cysteine 5 and 435 disulfide bond in beta(3)(54) . The amino-terminal aspect of this fragment is likely to correspond to the 19-kDa chymotryptic fragment obtained by Ramsamooj et al.(55) and to the 23-kDa chymotryptic fragment obtained by D'Souza et al.(17) which contains the RGD cross-linking site beta(3) 109-171. This composition would explain the generation of a 60-kDa fragment under nonreducing conditions which yields less than 20 kDa under reducing conditions. The 46/40-kDa radioactive doublet in the digest of cross-linked I-cHarGD under nonreducing conditions with only a slight decrease in mobility upon reduction, would correspond to a distinct fragment. This fragment may contain the region to which certain RGD disintegrins cross-link within beta(3) 217-302(56) .

Based upon the behavior of the various ligands described in this study and in the literature, at least four classes of beta(3) ligands can be distinguished (Table 1). RGD-containing peptides and vitronectin are representative of Class I ligands which react well with both beta(3) integrins. Class II ligands, represented by cHarGD, react with alpha(v)beta(3) in the presence of Mn but not Ca and react with alphabeta(3) with either cation present. Class II are distinguished from Class I ligands on the basis of the suppression of their binding to alpha(v)beta(3) by Ca. Moreover, these two categories also appear to be distinguished in terms of their primary cross-linking sites. Barbourin, which binds specifically to alphabeta(3)(24) and does not react with alpha(v)beta(3) even in the presence of Mn, represents a Class III ligand. Osteopontin is the known representative of Class IV ligands. Although it binds to alpha(v)beta(3) in the presence of Mn, it does not bind alphabeta(3)(57) . Some caution must be taken in assigning these classifications as cation determined specificity may be somewhat relative; i.e. some binding of nonreactive ligands to beta(3) integrins may occur over extended time periods(37) .



Fibrinogen appears to be a member of Class II, as cHarGD behaved like a low molecular weight mimetic of fibrinogen in virtually all respects. The classification of the fibrinogen -chain peptides presents an interesting challenge. We found that -chain peptides bound to alpha(v)beta(3) in the presence of 1 mM Mn, indicating that -chain peptides behave like cHarGD and belong in Class II. This proposal raises the possibility that soluble fibrinogen may bind to alpha(v)beta(3) in the presence of Mn via its -chain, whereas immobilized fibrinogen appears to be recognized via its RGD sequences(58) . Although the -chain peptides cross-link to alpha(23) rather than beta(3), such cross-linking may be influenced by peptide length as well as by the greater molecular flexibility of alpha as compared to beta(3)(59) .

As a final issue, the reactivity of cHarGD with alpha(v)beta(3) in the presence of Mn bear upon the development of alphabeta(3) antagonists for use as antithrombotic drugs(60) . Binding of cHarGD to alpha(v)beta(3) was observed at Mn concentrations above 1 µM. Tissue concentrations of Mn may be in the 1-14 µM range(37, 61) ; and liver concentrations may reach 30 µM(62) . Therefore, physiological Mn concentrations could support cross-reactivity of Class II ligands with alpha(v)beta(3). The results with barbourin suggest that the design of low molecular weight, high affinity, and specific alphabeta(3) agonists is feasible. Antagonists which maintain a Lys or a faithful substitution for it in XGD mimetics should uphold this specificity.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL-38292 and HL-54924. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Partially supported by the Ryoichi Naito Foundation for Medical Research.

To whom correspondence should be addressed: Joseph J. Jacobs Center for Thrombosis and Vascular Biology/FF20, The Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-8200; Fax: 216-445-8204.

(^1)
The abbreviations used are: Har, homoarginine; cHarGD, cyclic homoarginine-glycine-aspartic acid-containing peptide, KYGC(s-s)HarGDWPC(s-s); BS^3, bis(sulfosuccinimidyl) suberate; IC, concentration required to give 50% inhibition of binding; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Timothy A. Burke for technical assistance and Dr. Alexander Redlitz for helpful discussions. We also thank Jane Rein for help in the preparation of this manuscript.


REFERENCES

  1. Hynes, R. O. (1987) Cell 48, 549-550 [Medline] [Order article via Infotrieve]
  2. Ginsberg, M. H., Loftus, J. C., and Plow, E. F. (1988) Thromb. Haemostasis 59, 1-6 [Medline] [Order article via Infotrieve]
  3. Luscinskas, F. W., and Lawler, J. (1994) FASEB J. 8, 929-938 [Abstract/Free Full Text]
  4. Shattil, S. J. (1995) Thromb. Haemostasis 74, 149-155 [Medline] [Order article via Infotrieve]
  5. Plow, E. F., D'Souza, S. E., and Ginsberg, M. H. (1992) J. Lab. Clin. Med. 120, 198-204 [Medline] [Order article via Infotrieve]
  6. Smyth, S. S., Joneckis, C. C., and Parise, L. V. (1993) Blood 81, 2827-2843 [Medline] [Order article via Infotrieve]
  7. Calvete, J. J. (1994) Thromb. Haemostasis 72, 1-15 [Medline] [Order article via Infotrieve]
  8. Felding-Habermann, B., and Cheresh, D. A. (1993) Curr. Opin. Cell Biol. 5, 864-868 [Medline] [Order article via Infotrieve]
  9. Brooks, P. C., Clark, R. A., and Cheresh, D. A. (1994) Science 264, 569-571 [Medline] [Order article via Infotrieve]
  10. Liaw, L., Skinner, M. P., Raines, E. W., Ross, R., Cheresh, D. A., Schwartz, S. M., and Giachelli, C. M. (1995) J. Clin. Invest. 95, 713-724 [Medline] [Order article via Infotrieve]
  11. Fitzgerald, L. A., Poncz, M., Steiner, B., Rall, S. C., Jr., Bennett, J. S., and Phillips, D. R. (1987) Biochemistry 26, 8158-8165 [Medline] [Order article via Infotrieve]
  12. Plow, E. F., McEver, R. -P., Coller, B. S., Woods, V. L., Marguerie, G. A., and Ginsberg, M. H. (1985) Blood 66, 724-727 [Abstract]
  13. Thiagarajan, P., and Kelley, K. L. (1988) J. Biol. Chem. 263, 3035-3038 [Abstract/Free Full Text]
  14. Lawler, J., Weinstein, R., and Hynes, R. O. (1988) J. Cell Biol. 107, 2351-2361 [Abstract]
  15. Charo, I. F., Nannizzi, L., Smith, J. W., and Cheresh, D. A. (1990) J. Cell Biol. 111, 2795-2800 [Abstract]
  16. Smith, J. W., Ruggeri, Z. M., Kunicki, T. J., and Cheresh, D. A. (1990) J. Biol. Chem. 265, 12267-12271 [Abstract/Free Full Text]
  17. D'Souza, S. E., Ginsberg, M. H., Burke, T. A., Lam, S. C.-T., and Plow, E. F. (1988) Science 242, 91-93 [Medline] [Order article via Infotrieve]
  18. Smith, J. W., and Cheresh, D. A. (1988) J. Biol. Chem. 263, 18726-18731 [Abstract/Free Full Text]
  19. Loftus, J. C., O'Toole, T. E., Plow, E. F., Glass, A., Frelinger, A. L., and Ginsberg, M. H. (1990) Science 249, 915-918 [Medline] [Order article via Infotrieve]
  20. Bajt, M. L., Ginsberg, M. H., Frelinger, A. L., III, Berndt, M. C., and Loftus, J. C. (1992) J. Biol. Chem. 267, 3789-3794 [Abstract/Free Full Text]
  21. Lanza, F., Stierlé, A., Fournier, D., Morales, M., André, G., Nurden, A. T., and Cazenave, J.-P. (1992) J. Clin. Invest. 89, 1995-2004 [Medline] [Order article via Infotrieve]
  22. Bajt, M. L., and Loftus, J. C. (1994) J. Biol. Chem. 269, 20913-20919 [Abstract/Free Full Text]
  23. D'Souza, S. E., Ginsberg, M. H., Burke, T. A., and Plow, E. F. (1990) J. Biol. Chem. 265, 3440-3446 [Abstract/Free Full Text]
  24. Scarborough, R. M., Rose, J. W., Hsu, M. A., Phillips, D. R., Fried, V. A., Campbell, A. M., Nannizzi, L., and Charo, I. F. (1991) J. Biol. Chem. 266, 9359-9362 [Abstract/Free Full Text]
  25. Scarborough, R. M., Naughton, M. A., Teng, W., Rose, J. W., Phillips, D. R., Nannizzi, L., Arsten, A., Campbell, A. M., and Charo, I. F. (1993) J. Biol. Chem. 268, 1066-1073 [Abstract/Free Full Text]
  26. Ruoslahti, E., and Pierschbacher, M. D. (1987) Science 238, 491-496 [Medline] [Order article via Infotrieve]
  27. Haas, T. A., and Plow, E. F. (1994) Curr. Opin. Cell Biol. 6, 656-662 [Medline] [Order article via Infotrieve]
  28. Smith, J. W., and Cheresh, D. A. (1990) J. Biol. Chem. 265, 2168-2172 [Abstract/Free Full Text]
  29. Smith, J. W., and Cheresh, D. A. (1991) J. Biol. Chem. 266, 11429-11432 [Abstract/Free Full Text]
  30. D'Souza, S. E., Haas, T. A., Piotrowicz, R. S., Byers-Ward, V., McGrath, D. E., Soule, H. R., Cierniewski, C. S., Plow, E. F., and Smith, J. W. (1994) Cell 79, 659-667 [Medline] [Order article via Infotrieve]
  31. Gailit, J., and Ruoslahti, E. (1988) J. Biol. Chem. 263, 12927-12932 [Abstract/Free Full Text]
  32. Staatz, W. D., Rajpara, S. M., Wayner, E. A., Carter, W. G., and Santoro, S. A. (1989) J. Cell Biol. 108, 1917-1924 [Abstract]
  33. Elices, M. J., Urry, L. A., and Hemler, M. E. (1991) J. Cell Biol. 112, 169-181 [Abstract]
  34. Dobrina, A., Menegazzi, R., Carlos, T. M., Nardon, E., Cramer, R., Zacchi, T., Harlan, J. M., and Patriarca, P. (1991) J. Clin. Invest. 88, 20-26 [Medline] [Order article via Infotrieve]
  35. Altieri, D. C. (1991) J. Immunol. 147, 1891-1898 [Abstract/Free Full Text]
  36. Dransfield, I., Cabanas, C., Craig, A., and Hogg, N. (1992) J. Cell Biol. 116, 219-226 [Abstract]
  37. Smith, J. W., Piotrowicz, R. S., and Mathis, D. (1994) J. Biol. Chem. 269, 960-967 [Abstract/Free Full Text]
  38. 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]
  39. Yatohgo, T., Izumi, M., Kashiwagi, H., and Hayashi, M. (1988) Cell Struct. Funct. 13, 281-292 [Medline] [Order article via Infotrieve]
  40. Pytela, R., Pierschbacher, M. D., Ginsberg, M. H., Plow, E. F., and Ruoslahti, E. (1986) Science 231, 1559-1562 [Medline] [Order article via Infotrieve]
  41. Scarborough, R. M., Rose, J. W., Naughton, M. A., Phillips, D. R., Nannizzi, L., Arfsten, A., Campbell, A. M., and Charo, I. F. (1993) J. Biol. Chem. 268, 1058-1065 [Abstract/Free Full Text]
  42. D'Souza, S. E., Ginsberg, M. H., Lam, S. C.-T., and Plow, E. F. (1988) J. Biol. Chem. 263, 3943-3951 [Abstract/Free Full Text]
  43. Coller, B. S. (1985) J. Clin. Invest. 76, 101-108 [Medline] [Order article via Infotrieve]
  44. Charo, I. F., Nannizzi, L., Phillips, D. R., Hsu, M. A., and Scarborough, R. M. (1991) J. Biol. Chem. 266, 1415-1421 [Abstract/Free Full Text]
  45. Shadle, P. J., Ginsberg, M. H., Plow, E. F., and Barondes, S. H. (1984) J. Cell Biol. 99, 2056-2060 [Abstract]
  46. Ginsberg, M. H., Lightsey, A. L., Kunicki, T. J., Kaufman, A., Marguerie, G. A., and Plow, E. F. (1986) J. Clin. Invest. 78, 1103-1111 [Medline] [Order article via Infotrieve]
  47. Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C., and Ginsberg, M. H. (1991) Cell 65, 409-416 [Medline] [Order article via Infotrieve]
  48. Santoro, S. A., and Lawing, W. J., Jr. (1987) Cell 48, 867-873 [Medline] [Order article via Infotrieve]
  49. Marguerie, G. A., Plow, E. F., and Edgington, T. S. (1979) J. Biol. Chem. 254, 5357-5363 [Medline] [Order article via Infotrieve]
  50. Kirchhofer, D., Gailit, J., Ruoslahti, E., Grzesiak, J., and Pierschbacher, M. D. (1990) J. Biol. Chem. 265, 18525-18530 [Abstract/Free Full Text]
  51. Masumoto, A., and Hemler, M. E. (1993) J. Cell Biol. 123, 245-253 [Abstract]
  52. Bazzoni, G., Shih, D.-T., Buck, C. A., and Hemler, M. E. (1995) J. Biol. Chem. 270, 25570-25577 [Abstract/Free Full Text]
  53. Pfaff, M., Tangemann, K., Müller, B., Gurrath, M., Müller, G., Kessler, H., Timpl, R., and Engel, J. (1994) J. Biol. Chem. 269, 20233-20238 [Abstract/Free Full Text]
  54. Calvete, J. J., Henschen, A., and González-Rodríguez, J. (1991) Biochem. J. 274, 63-71 [Medline] [Order article via Infotrieve]
  55. Ramsamooj, P., Lively, M. O., and Hantgan, R. R. (1991) Biochem. J. 276, 725-732 [Medline] [Order article via Infotrieve]
  56. Calvete, J. J., McLane, M. A., Stewart, G. J., and Niewiarowski, S. (1994) Biochem. Biophys. Res. Commun. 202, 135-140 [CrossRef][Medline] [Order article via Infotrieve]
  57. Hu, D. D., Hoyer, J. R., and Smith, J. W. (1995) J. Biol. Chem. 270, 9917-9925 [Abstract/Free Full Text]
  58. Cheresh, D. A., Berliner, S. A., Vicente, V., and Ruggeri, Z. M. (1989) Cell 58, 945-953 [Medline] [Order article via Infotrieve]
  59. Calvete, J. J., Schäfer, W., Mann, K., Henschen, A., and González-Rodríguez, J. (1992) Eur. J. Biochem. 206, 759-765 [Abstract]
  60. Lefkovits, J., Plow, E. F., and Topol, E. J. (1995) N. Engl. J. Med. 332, 1553-1559 [Free Full Text]
  61. Brandt, M., and Schramm, V. L. (1986) in Manganese in Metabolism and Enzyme Function (Schramm, V. L., and Wedler, F. C., eds) pp. 3-16, Academic Press, New York
  62. Baruthio, F., Guillard, O., Arnaud, J., Pierre, F., and Zawislak, R. (1988) Clin. Chem. 34, 227-234 [Abstract/Free Full Text]
  63. McLane, M. A., Kowalska, M. A., Silver, L., Shattil, S. J., and Niewiarowski, S. (1994) Biochem. J. 301, 429-436 [Medline] [Order article via Infotrieve]
  64. Lu, X., Williams, J. A., Deadman, J. J., Salmon, G. P., Kakkar, V. V., Wilkinson, J. M., Baruch, D., Authi, K. S., and Rahman, S. (1994) Biochem. J. 304, 929-936 [Medline] [Order article via Infotrieve]
  65. Marcinkiewicz, C., Rosenthal, L. A., Mosser, D. M., and Niewiarowski, S. (1995) Thromb. Haemostasis 73, 1123a

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