(Received for publication, November 21, 1996, and in revised form, January 27, 1997)
From the 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 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,
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 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.
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.
pREP vectors containing cDNAs for 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.
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
There are 50-fold (21) to 500-fold (22) fewer copies of
Because the
The divalent cation dependence of platelet The ability of
Our results have a number of important implications. First, The
affinity state of 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.
Hematology-Oncology Division,
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
v
3.
v
3-mediated
adherence occurred at physiologic concentrations of calcium and was
inhibited by an
v
3-selective cyclic peptide. Assays using phorbol
myristate acetate-stimulated transfected B lymphocytes expressing both
v
3 and
IIb
3 confirmed that activated
v
3 not activated
IIb
3 was responsible for the cellular adherence we measured.
These studies indicate that
v
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
v
3 could play a role in anchoring platelets to disrupted atherosclerotic plaques and the walls
of injured arteries. By inhibiting
v
3 function, it may be
possible to inhibit platelet-mediated vascular occlusion with a minimal
effect on primary hemostasis.
IIb
3, mediates platelet aggregation when platelet stimulation
converts it from a resting to a ligand-binding conformation (1).
Platelets contain a second
3 integrin,
v
3, but whether
v
3 plays a role in platelet function is not known. However, a
monoclonal antibody that binds to both
IIb
3 and
v
3 has been shown in clinical trials to have additional efficacy relative to
compounds that bind only to
IIb
3 by preventing the reocclusion that often occurs months after PTCA1
(2-4).
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
v
3.
Synthesis of Recombinant Human Osteopontin
IIb and
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.
Platelet Adherence to Osteopontin and Fibrinogen
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×).
[View Larger Version of this Image (48K GIF file)]
IIb
3 and against
v
3, the only
v-containing integrin
present in platelets (Fig. 2). Although agonist-mediated
platelet adherence is generally mediated by
IIb
3 (1), platelet
adherence to osteopontin was consistently inhibited by 84-93% by the
v
3-specific mAb LM609 (19) and 96-99% by the
3-integrin-specific mAb 7E3 (20). In contrast, saturating
concentrations of the
IIb
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
v
3 to some extent
(15), whereas an antibody specific for
5
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
IIb
3, the receptor
primarily mediating platelet adherence to osteopontin is
v
3. The
data also indicate that the ability of platelet
v
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.
[View Larger Version of this Image (13K GIF file)]
v
3
compared with
IIb
3 on the platelet surface. Moreover,
v
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
v
3 rather than
IIb
3, we used a B lymphocyte model of platelet integrin function. B lymphocytes constitutively express
v
3 but express
IIb
3
after transfection (15, 24). Following exposure to PMA, only the transfected cells expressing
IIb
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
IIb
3, adherence to osteopontin was inhibited by the mAb LM609, indicating it was mediated by
v
3. Next, we
measured the adherence of the parental line GM1500 that expresses
v
3 but not
IIb
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
v
3 on lymphocytes, like
v
3
on platelets, resides on the cell surface in an inactive state and
confirm that agonist-generated intracellular signals can induce
v
3 binding to osteopontin.
Fig. 3.
Adherence of human B lymphocytes expressing
IIb
3 and/or
v
3 to immobilized osteopontin and
fibrinogen. The adherence of stable B cell lines expressing either
IIb
3 and
v
3 (
IIb
3) or
v
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.
[View Larger Version of this Image (24K GIF file)]
v
3 on activated platelets and lymphocytes interacts
with osteopontin, whereas
IIb
3 on these cells interacts with
fibrinogen, the
subunit of
3 integrins regulates their ligand
binding specificity. The heavy chains of
v and
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
IIb and
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
v encompassing amino acids 139-349
(27) and a site in
IIb that contains amino acids 294-314 (28). A
major difference between the proximal end of the putative
v ligand
binding domain and the corresponding region of
IIb is the presence
of a stretch of 10 additional amino acids in
IIb
(Gly148-Glu157) (25), perhaps accounting for
the different ligand preference of the integrins containing these
subunits.
v
3-mediated cell adhesion to osteopontin occurs
in the presence of Mg2+, although Ca2+ can
support osteopontin binding to purified
v
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
v
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.
[View Larger Version of this Image (20K GIF file)]
v
3 deviates
significantly from that of
v
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
v
1-mediated adherence of 293 cells to osteopontin occurs in media
containing Ca2+ but only when
v
1 is exposed to an
activating monoclonal antibody, suggesting that the affinity state of
v
1 determines its ability to interact with Ca2+ (30).
We found that only
v
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
v
1 and to
v
3
expressed by cells other than platelets, platelet
v
3 is not
constitutively active in the presence of Mg2+.
v
3
v
3 to
mediate platelet adherence to osteopontin at physiologic
Ca2+ concentrations suggests that RGD-based peptides with
selectivity for
v
3 over
IIb
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
v
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
v
3 integrin can discriminate
between osteopontin and fibrinogen and accordingly could inhibit
platelet adhesion to the wall of injured arteries without impairing the
IIb
3-mediated platelet aggregation responsible for primary
hemostasis.
Fig. 5.
Inhibition of ADP-stimulated platelet
adherence to osteopontin and fibrinogen by the cyclic
v
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.
[View Larger Version of this Image (32K GIF file)]
v
3 on platelets and lymphocytes, like that of
IIb
3, is regulated by cellular agonists. Moreover, because agonist-generated signals interact with the
3 cytoplasmic tail to
up-regulate
IIb
3 function (24), it is reasonable to postulate that a similar mechanism is involved in up-regulating the function of
v
3. Second, because
v
3, but not
IIb
3, binds to
osteopontin, our results establish that the
subunit regulates the
selectivity of
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
v
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
v
3 have been shown to be effective in animal models for
restenosis (33), and we have shown that XJ735 has significant,
IIb
3-independent, in vivo antithrombotic efficacy but
does not increase bleeding times in dogs and
swine.2 Thus,
v
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.
*
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.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.