COMMUNICATION:
Agonist-activated alpha vbeta 3 on Platelets and Lymphocytes Binds to the Matrix Protein Osteopontin*

(Received for publication, November 21, 1996, and in revised form, January 27, 1997)

Joel S. Bennett Dagger §, Chia Chan Dagger , Gaston Vilaire Dagger , Shaker A. Mousa and William F. DeGrado par

From the Dagger  Hematology-Oncology Division, Department of Medicine and the par  Department of Biochemistry and Biophysics, the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and  Dupont Merck Pharmaceuticals, Wilmington, Delaware 19880

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The phosphorylated acidic glycoprotein osteopontin is present in the extracellular matrix of atherosclerotic plaques and the wall of injured but not normal arteries. To determine if osteopontin could serve as a substrate for platelet adhesion, we measured the adherence of resting and agonist-stimulated human platelets to immobilized recombinant human osteopontin. Agonist-stimulated but not resting platelets bound to osteopontin by a process that was mediated primarily by alpha vbeta 3. alpha vbeta 3-mediated adherence occurred at physiologic concentrations of calcium and was inhibited by an alpha vbeta 3-selective cyclic peptide. Assays using phorbol myristate acetate-stimulated transfected B lymphocytes expressing both alpha vbeta 3 and alpha IIbbeta 3 confirmed that activated alpha vbeta 3 not activated alpha IIbbeta 3 was responsible for the cellular adherence we measured. These studies indicate that alpha vbeta 3 can reside on the cell surface in an inactive state and can be converted to a ligand binding conformation by cellular agonists. Moreover, they suggest that platelet adherence to osteopontin mediated by activated alpha vbeta 3 could play a role in anchoring platelets to disrupted atherosclerotic plaques and the walls of injured arteries. By inhibiting alpha vbeta 3 function, it may be possible to inhibit platelet-mediated vascular occlusion with a minimal effect on primary hemostasis.


INTRODUCTION

The final event in a variety of cardiovascular diseases is often arterial occlusion by a platelet-rich thrombus. Members of the integrin superfamily of adhesion molecules play a key role in this pathologic process by anchoring platelets to the exposed subendothelium of damaged arteries and by mediating platelet aggregation. The integrin, alpha IIbbeta 3, mediates platelet aggregation when platelet stimulation converts it from a resting to a ligand-binding conformation (1). Platelets contain a second beta 3 integrin, alpha vbeta 3, but whether alpha vbeta 3 plays a role in platelet function is not known. However, a monoclonal antibody that binds to both alpha IIbbeta 3 and alpha vbeta 3 has been shown in clinical trials to have additional efficacy relative to compounds that bind only to alpha IIbbeta 3 by preventing the reocclusion that often occurs months after PTCA1 (2-4).

Formation of an occlusive thrombus or a normal hemostatic platelet plug is initiated when platelets adhere to newly exposed components of the subendothelial extracellular matrix of diseased or damaged blood vessels. The matrix components assumed to function as substrates for platelet adherence include collagen, fibronectin, and von Willebrand's factor because platelets contain receptors for each of these proteins and adhere to these proteins in vitro (2). Nevertheless, the substrates that actually mediate platelet adherence to disrupted atherosclerotic plaques are not known. Osteopontin is an acidic phosphorylated glycoprotein secreted by a number of cells including osteocytes, osteoclasts, macrophages, and smooth muscle cells (5-7). Although not present in the walls of normal arteries, osteopontin is widely distributed throughout the matrix of calcified plaques in arteries involved by atherosclerosis (8-10). Studies in vitro suggest that osteopontin may be involved in the formation of the neointima characteristic of the atherosclerotic process by serving as a substrate for alpha v-integrin-mediated smooth muscle and endothelial cell migration (8, 11). Because it is likely that osteopontin is exposed to circulating blood by the plaque disruption that precedes acute coronary artery occlusion and results from PTCA, we examined the possibility that osteopontin could serve as an adhesive substrate for platelets. We found that activated but not resting platelets adhere to osteopontin and that their adherence is mediated by an activated conformation of alpha vbeta 3.


EXPERIMENTAL PROCEDURES

Synthesis of Recombinant Human Osteopontin

Recombinant human osteopontin was synthesized as a histidine-tagged fusion protein using the pET system (Novagen). A cDNA for human osteopontin was inserted into the plasmid pET16b, and recombinant protein was synthesized as insoluble inclusion bodies in Escherichia coli BL21(DES)pLysS. Following lysis of the pelleted bacteria in 20 mM Tris-HCl buffer, pH 7.9, containing 0.5 M NaCl, 1 mg/ml lysozyme, and 0.1% Triton X-100, osteopontin was solubilized using 6 M guanidine HCl and isolated by metal chelate affinity chromatography on a Ni2+ nitrilotriacetic acid resin (His·Bind Resin, Novagen). Recombinant osteopontin was eluted from the resin using 20 mM Tris-HCl buffer, pH 7.9, containing 0.5 M NaCl and 500 mM imidazole and renatured by dialysis against phosphate-buffered saline, pH 7.4. 0.1% SDS-7.5% polyacrylamide gel electrophoresis of the renatured protein revealed a single band with an apparent molecular weight of 58,000. The mass of the recombinant protein as determined by electrospray mass spectroscopy was 35,518, consistent with the calculated mass of the full-length osteopontin amino acid backbone (12) plus the polyhistidine tag and Factor Xa cleavage site contributed by pET16b.

Measurement of Platelet Adherence to Osteopontin and Fibrinogen

96-well flat bottom microtiter plates (Immulon 2, Dynatech) were coated with 5 µg/ml recombinant osteopontin, purified human fibrinogen, or bovine serum albumin, each dissolved in 50 mM NaHCO3 buffer, pH 8.0, containing 150 mM NaCl. Unoccupied protein binding sites on the wells were blocked with 5 mg/ml bovine serum albumin dissolved in the same buffer. Human platelets were isolated from blood anticoagulated with 0.1 volume of 3.8% sodium citrate by gel filtration using a 4 mM HEPES buffer, pH 7.4, containing 135 mM NaCl, 2.7 mM KCl, 5.6 mM glucose, 3.3 mM NaH2PO4, 0.35 mg/ml bovine serum albumin, and various concentrations of CaCl2 or MgCl2 according to the experiment (13). 100-µl aliquots of the gel filtered platelet suspension containing ~2-5 × 106 platelets were added to the protein-coated wells in the absence or the presence of a platelet agonist. Following an incubation for 30 min at 37 °C without agitation, the plates were washed four times with the gel filtration buffer, and the number of adherent platelets was measured using the colorimetric assay reported by Bellavite et al. (14). Briefly, 150 µl of a 0.1 M citrate buffer, pH 5.4, containing 5 mM p-nitrophenyl phosphate (Sigma) and 0.1% Triton X-100 was added to the wells after washing. After an incubation for 60 min at room temperature in the dark, color was developed by the addition of 100 µl of 2 N NaOH and read in a microtiter plate reader at 405 nm.

Measurement of Lymphocyte Adherence to Osteopontin and Fibrinogen

pREP vectors containing cDNAs for alpha IIb and beta 3 were introduced into 7.5 × 106 GM1500 B lymphocytes by electroporation, and stable cotransfectants were selected by growth in media containing G418 and hygromycin as described previously (15). The phorbol myristate acetate (PMA)-stimulated adherence of transfected and untransfected lymphocytes to osteopontin and fibrinogen was measured as described previously (15). Briefly, 1.5 × 105 B lymphocytes, metabolically labeled overnight with [35S]methionine, were suspended in 100 µl of 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl, 0.5 mM CaCl2, 0.1% glucose, and 1% bovine serum albumin and added to the wells of microtiter plates coated with osteopontin or fibrinogen, either in the presence or the absence of 200 ng/ml PMA. Following an incubation at 37 °C for 30 min without agitation, the plates were washed four times with the lymphocyte suspension buffer, and adherent cells were dissolved using 2% SDS. The SDS solutions were then counted for 35S in a liquid scintillation counter.


RESULTS AND DISCUSSION

Platelet Adherence to Osteopontin and Fibrinogen

To determine if osteopontin could serve as a substrate for platelet adherence, we used a solid phase assay to measure the adherence of gel filtered human platelets to either purified recombinant human osteopontin or purified human fibrinogen (13), a known adhesive ligand for platelets (16). In the presence of Mg2+, there was substantial adherence of unstimulated platelets to fibrinogen but little adherence to osteopontin (Fig. 1A). Platelet stimulation with 10 µM ADP resulted in a dramatic increase in the number of platelets adherent to osteopontin, as well as a smaller increase in the number of platelets adherent to fibrinogen. Inspection of the assay plates by light microscopy confirmed these results and revealed that ADP stimulation resulted in both platelet adherence and spreading on the fibrinogen and osteopontin-coated surfaces (Fig. 1B). Similar results were observed when the platelets were stimulated by 20 µM epinephrine, 0.1 unit/ml thrombin, or 200 ng/ml PMA, and the presence of 25 µM indomethacin had no effect on the adherence of ADP-stimulated platelets. The addition of EDTA prevented platelet adherence to either substrate. To verify that activated platelets adhere to native osteopontin, as well as to recombinant protein, assays were repeated using purified osteopontin isolated from human urine (uropontin). We found no difference in the ability of uropontin and recombinant osteopontin to support platelet adherence (data not shown). Thus, stimulated but not unstimulated human platelets are able to use immobilized osteopontin as an adhesive substrate.


Fig. 1. Adherence of gel filtered human platelets to immobilized osteopontin and fibrinogen. Platelet adherence to the wells of microtiter platelets coated with either recombinant human osteopontin or purified human fibrinogen was measured as described under "Experimental Procedures." A, quantitation of platelet adherence using a colorimetric based on measurement of platelet acid phosphatase activity (14). The data shown are the mean and standard error of measurements made in triplicate and are representative of 16 separate experiments. B, visualization of adherent platelets adherent by light microscopy (200×).
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Identification of the Receptor Responsible for Platelet Adherence to Osteopontin

To identify the receptor on activated platelets that mediates platelet adherence to osteopontin, we repeated the adherence assays in the presence of monoclonal antibodies against alpha IIbbeta 3 and against alpha vbeta 3, the only alpha v-containing integrin present in platelets (Fig. 2). Although agonist-mediated platelet adherence is generally mediated by alpha IIbbeta 3 (1), platelet adherence to osteopontin was consistently inhibited by 84-93% by the alpha vbeta 3-specific mAb LM609 (19) and 96-99% by the beta 3-integrin-specific mAb 7E3 (20). In contrast, saturating concentrations of the alpha IIbbeta 3-selective mAbs A2A9 (17) and 10E5 (18) inhibited platelet adherence to osteopontin by only 30-40%, perhaps because these antibodies also cross-react with alpha vbeta 3 to some extent (15), whereas an antibody specific for alpha 5beta 1 (mAb 16, Becton Dickinson) was not inhibitory (data not shown). Inhibition by the tetrapeptide RGDS was nearly complete, consistent with platelet adherence to osteopontin being an integrin-mediated process. Conversely, there was nearly complete inhibition of platelet adherence to fibrinogen by A2A9, 10E5, and 7E3 and only minimal inhibition by LM609 (data not shown). Thus, these data indicate that whereas platelet adherence to fibrinogen is mediated by alpha IIbbeta 3, the receptor primarily mediating platelet adherence to osteopontin is alpha vbeta 3. The data also indicate that the ability of platelet alpha vbeta 3 to recognize osteopontin requires platelet stimulation.


Fig. 2. Identification of the receptor mediating platelet adherence to osteopontin. Adherence of ADP-stimulated platelets to osteopontin was measured as described in the legend to Fig. 1. The inhibitory effect of saturating concentrations of the following monoclonal antibodies was then compared with that of 5 mM RGDS: A2A9 (50 µg/ml), 10E5 (20 µg/ml), 7E3 (20 µg/ml), and LM609 (30 µg/ml). The data shown are the means and standard errors of triplicate determinations and were normalized to 100% binding in the absence of inhibitors.
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There are 50-fold (21) to 500-fold (22) fewer copies of alpha vbeta 3 compared with alpha IIbbeta 3 on the platelet surface. Moreover, alpha vbeta 3 is generally considered to reside on the surface of most cells in a constitutively active state (23). To verify that agonist-stimulated platelet adherence to osteopontin is mediated by alpha vbeta 3 rather than alpha IIbbeta 3, we used a B lymphocyte model of platelet integrin function. B lymphocytes constitutively express alpha vbeta 3 but express alpha IIbbeta 3 after transfection (15, 24). Following exposure to PMA, only the transfected cells expressing alpha IIbbeta 3 bind soluble fibrinogen (24) or adhere to immobilized fibrinogen (15). We found little adherence of transfected lymphocytes to either osteopontin or fibrinogen in the absence of PMA stimulation (Fig. 3). Following PMA stimulation, lymphocyte adherence to osteopontin and fibrinogen increased by 13.6- and 8.1-fold, respectively. Nevertheless, whereas adherence to fibrinogen was inhibited by the mAb A2A9, indicating it was mediated by alpha IIbbeta 3, adherence to osteopontin was inhibited by the mAb LM609, indicating it was mediated by alpha vbeta 3. Next, we measured the adherence of the parental line GM1500 that expresses alpha vbeta 3 but not alpha IIbbeta 3 to both substrates (Fig. 3). As anticipated, the parental cells did not adhere to fibrinogen; however, their adherence to osteopontin was identical to that of the transfected cells. These data indicate that alpha vbeta 3 on lymphocytes, like alpha vbeta 3 on platelets, resides on the cell surface in an inactive state and confirm that agonist-generated intracellular signals can induce alpha vbeta 3 binding to osteopontin.


Fig. 3. Adherence of human B lymphocytes expressing alpha IIbbeta 3 and/or alpha vbeta 3 to immobilized osteopontin and fibrinogen. The adherence of stable B cell lines expressing either alpha IIbbeta 3 and alpha vbeta 3 (alpha IIbbeta 3) or alpha vbeta 3 alone (GM1500) to osteopontin and fibrinogen in the presence or the absence of 200 ng/ml PMA was measured as described under "Experimental Procedures." Identity of the receptor mediating lymphocyte adherence was determined by performing the assay in the presence of A2A9 or LM609. The data shown are the means and standard errors of quadruplicate determinations.
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Because the alpha vbeta 3 on activated platelets and lymphocytes interacts with osteopontin, whereas alpha IIbbeta 3 on these cells interacts with fibrinogen, the alpha  subunit of beta 3 integrins regulates their ligand binding specificity. The heavy chains of alpha v and alpha IIb exhibit 38% overall homology, but homology increases to 57% when their amino-terminal halves containing their putative calcium binding domains are compared (25). Recent studies of alpha IIb and alpha v concluded that the amino-terminal one-third of each protein is involved in ligand recognition (26), a conclusion consistent with peptide cross-linking studies implicating two sites in alpha v encompassing amino acids 139-349 (27) and a site in alpha IIb that contains amino acids 294-314 (28). A major difference between the proximal end of the putative alpha v ligand binding domain and the corresponding region of alpha IIb is the presence of a stretch of 10 additional amino acids in alpha IIb (Gly148-Glu157) (25), perhaps accounting for the different ligand preference of the integrins containing these alpha  subunits.

Role of Divalent Cations in Platelet Adherence to Osteopontin

alpha vbeta 3-mediated cell adhesion to osteopontin occurs in the presence of Mg2+, although Ca2+ can support osteopontin binding to purified alpha vbeta 3 (29). To verify that Ca2+ can support platelet adherence to osteopontin, we measured the adherence of ADP-stimulated platelets suspended in buffer containing either Ca2+ or Mg2+ (Fig. 4). In the presence of EDTA, there was no adherence of ADP-stimulated platelets to osteopontin. However, both Ca2+ and Mg2+ supported platelet adherence to osteopontin in a concentration-dependent manner. At cation concentrations up to 1 mM, there was little difference in the ability of Ca2+ and Mg2+ to support platelet adherence; although at higher concentrations, the ability of Ca2+ to support adherence declined relative to that of Mg2+. Nevertheless, at a physiologically relevant concentration of 1 mM, Ca2+ supported platelet adherence to osteopontin nearly as well as Mg2+. Moreover, adherence in either Ca2+- or Mg2+-containing buffer was inhibited by LM609, indicating that it was mediated by alpha vbeta 3 regardless of the divalent cation present (data not shown).


Fig. 4. Effect of Ca2+ or Mg2+ on ADP-stimulated platelet adherence to osteopontin. Gel filtered platelets were suspended in buffer containing various concentrations of either Ca2+ or Mg2+. ADP-stimulated platelet adherence to osteopontin was measured as described in the legend to Fig. 1 and under "Experimental Procedures." The baseline for the colorimetric assay was determined by measuring platelet adherence in the presence of 5 mM EDTA. The data shown are the means and standard errors of triplicate determinations.
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The divalent cation dependence of platelet alpha vbeta 3 deviates significantly from that of alpha vbeta 3 in certain cell lines where Ca2+ does not support cell adherence to osteopontin and can even be inhibitory (29). It has been reported that the alpha vbeta 1-mediated adherence of 293 cells to osteopontin occurs in media containing Ca2+ but only when alpha vbeta 1 is exposed to an activating monoclonal antibody, suggesting that the affinity state of alpha vbeta 1 determines its ability to interact with Ca2+ (30). We found that only alpha vbeta 3 on activated platelets mediates adherence to osteopontin in the presence of Ca2+, suggesting that the affinity of this integrin also determines its ability to interact with Ca2+. However, in contrast to alpha vbeta 1 and to alpha vbeta 3 expressed by cells other than platelets, platelet alpha vbeta 3 is not constitutively active in the presence of Mg2+.

Selective Inhibition of Platelet Adherence to Osteopontin by a Cyclic Peptide Selective for alpha vbeta 3

The ability of alpha vbeta 3 to mediate platelet adherence to osteopontin at physiologic Ca2+ concentrations suggests that RGD-based peptides with selectivity for alpha vbeta 3 over alpha IIbbeta 3 could be of clinical utility. To test this possibility in vitro, we compared the ability of XJ735 (cyclo(Ala-Arg-Gly-Asp-Mamb), where Mamb is meta-aminomethyl benzoic acid), a cyclic RGD-based peptide selective for alpha vbeta 3 (31), to inhibit ADP-stimulated platelet adherence to osteopontin and fibrinogen (Fig. 5). XJ735 abolished platelet adherence to osteopontin with an IC50 of ~6 µM, whereas its effect on adherence to fibrinogen was incomplete with an IC50 that was >1 mM. Thus, peptides selective for the alpha vbeta 3 integrin can discriminate between osteopontin and fibrinogen and accordingly could inhibit platelet adhesion to the wall of injured arteries without impairing the alpha IIbbeta 3-mediated platelet aggregation responsible for primary hemostasis.


Fig. 5. Inhibition of ADP-stimulated platelet adherence to osteopontin and fibrinogen by the cyclic alpha vbeta 3-selective peptide XJ735. ADP-stimulated platelet adherence to osteopontin and fibrinogen was measured in the presence of increasing concentrations of XJ735 as described in the legend to Fig. 1 and under "Experimental Procedures." The data were normalized to 100% adherence in the absence of XJ735 to facilitate comparisons. The data are the means of triplicate determinations.
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Our results have a number of important implications. First, The affinity state of alpha vbeta 3 on platelets and lymphocytes, like that of alpha IIbbeta 3, is regulated by cellular agonists. Moreover, because agonist-generated signals interact with the beta 3 cytoplasmic tail to up-regulate alpha IIbbeta 3 function (24), it is reasonable to postulate that a similar mechanism is involved in up-regulating the function of alpha vbeta 3. Second, because alpha vbeta 3, but not alpha IIbbeta 3, binds to osteopontin, our results establish that the alpha  subunit regulates the selectivity of beta 3 integrins for natural ligands. Third, because osteopontin is a major constituent of atherosclerotic plaques (8-10), is absent from the endothelium of normal arteries (8-10), and is strongly up-regulated in areas of endothelial damage (6, 8, 32), it may be possible to impair the formation of platelet thrombi in arteries by preventing the interaction of platelet alpha vbeta 3 with osteopontin. A potential advantage of this approach is that it may be less prone to impair hemostasis than current therapies. Indeed, peptides that bind to alpha vbeta 3 have been shown to be effective in animal models for restenosis (33), and we have shown that XJ735 has significant, alpha IIbbeta 3-independent, in vivo antithrombotic efficacy but does not increase bleeding times in dogs and swine.2 Thus, alpha vbeta 3-mediated platelet adherence to osteopontin could be involved in the pathogenesis of acute arterial occlusion, and inhibitors of this integrin may be useful pharmaceutical agents for treating arterial thrombotic disorders.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants HL40387 (to J. S. B.) and HL51258 (to J. S. B.) and National Science Foundation Grant CHE-9634646 (to W. F. D.).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.
§   To whom correspondence should be addressed: Hematology-Oncology Division, Stellar-Chance Laboratories, Rm. 1005, 422 Curie Blvd., Philadelphia, PA 19104. Tel.: 215-662-4028; Fax: 215-662-7617; E-mail: bennetts{at}mail.med.upenn.edu.
1   The abbreviations used are: PTCA, percutaneous transluminal coronary angioplasty; PMA, phorbol myristate acetate; mAb, monoclonal antibody.
2   S. Mousa, unpublished observations.

ACKNOWLEDGEMENTS

We are indebted to Dr. Barry Coller for supplying mAbs 7E3 and 10E5, Dr. John Hoyer for supplying human uropontin, Dr. Ellen Rollo for the generous gift of a cDNA for human osteopontin, and Dr. John Lawson for the electrospray mass spectroscopy measurement of osteopontin mass.


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