(Circulation. 1999;99:2231-2238.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Quantifying GPIIb/IIIa Receptor Binding Using 2 Monoclonal Antibodies
Discriminating Abciximab and Small Molecular Weight Antagonists
From the Centre for Cardiovascular Science (M.Q., A.D., M.S., D.C., D.F.), The Royal College of Surgeons, and St. James's Hospital (B.F.), Dublin, Ireland.
Correspondence to Dr Martin Quinn, The Centre for Cardiovascular Science, 123, St. Stephen's Green, Dublin 2, Ireland. E-mail mquinn{at}rcsi.ie
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Abstract |
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Background—Dosing of glycoprotein (GP) IIb/IIIa receptor antagonists is frequently based on the inhibition of platelet aggregation, which may be influenced by the agonist used or concurrent medications. Here we describe a monoclonal antibody-based technique to quantify total and ligand-occupied GPIIb/IIIa receptors.
Methods and Results—In vitro binding of monoclonal antibodies, LYP18 (Mab1) and 4F8 (Mab2), to the GPIIb/IIIa complex, was characterized using purified receptor and to platelets by flow cytometry. Patients undergoing coronary angioplasty received a single 20 mg dose of the oral GPIIb/IIIa antagonist, xemilofiban, or matching placebo, and antibody binding was compared with inhibition of platelet aggregation. Mab1 and Mab2 were bound to purified GPIIb/IIIa and to unoccupied, inactivated receptor on platelets. Mab2 identified the ß3 subunit, whereas Mab1 was complex-specific. Neither antibody interfered with the other's binding, suggesting that they identified distinct sites. Mab1 identified 53 300±5423 GPIIb/IIIa sites per platelet, whereas Mab2 identified 50 120±5066 sites per platelet. Mab1 binding was inhibited by abciximab in a dose dependent manner (IC50, 0.85±0.1 µg/mL), whereas Mab2 binding was unaffected. In contrast, the 2 small molecular weight antagonists, SC-57101A (IC50, 0.22±0.06 µmol/L) and eptifibatide (IC50, 0.35±0.14 µmol/L) inhibited Mab2 but not Mab1 binding. In patients treated with xemilofiban, Mab1 binding was unaltered but Mab2 binding decreased from 37 930±2061 sites per platelet at baseline to 8318±870 sites per platelet 6 hours after dosing (P<0.0001). Platelet aggregation to adenosine diphosphate (20 µmol/L) fell to 3±3% of baseline in line with the inhibition of Mab2 binding (correlation coefficient 0.8, P<0.0001).
Conclusions—Mab1 and Mab2 bind to GPIIb/IIIa and are differentially displaced by abciximab and small molecular weight antagonists. These antibodies may be used to monitor receptor number and occupancy during administration of a GPIIb/IIIa antagonist.
Key Words: glycoproteins • platelet aggregation inhibitors • abciximab • thrombosis
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Introduction |
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TopAbstractIntroductionMaterials and MethodsStatistical AnalysisResultsDiscussionReferences |
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Glycoprotein (GP) IIb/IIIa is one of a family of integrins that are heterodimers of an
and ß subunit and act as
receptors for adhesion proteins.1 GPIIb/IIIa is the
principle integrin on platelets and is the receptor for fibrinogen,
which mediates platelet aggregation and adhesion.
Antagonists of GPIIb/IIIa, including the monoclonal
antibody abciximab2 and several peptide (eg,
eptifibatide)3 and nonpeptide small
molecules,4 are potent inhibitors of
platelet aggregation; given as short-term infusions, they reduce
the complications of coronary angioplasty when combined with
aspirin.5 6 7 8 Orally active agents have also been
developed9 that will hopefully extend the use of
GPIIb/IIIa receptor antagonists to long-term therapy for
patients with stable coronary disease. Currently, dosing of GPIIb/IIIa receptor antagonists is based on the inhibition of platelet aggregation. This has several disadvantages. Platelet aggregation may vary depending on the agonist used, the platelet count, ingestion of food or alcohol, smoking, and the use of concurrent medications.10 11 12 Platelet aggregation is also insensitive to very high or low levels of receptor occupancy. Aggregation reflects cross-linking of GPIIb/IIIa receptors from adjacent platelets and is unaffected at levels of receptor occupancy <30% to 50%.13 Similarly, as receptor occupancy exceeds 80%, aggregation may be completely inhibited despite the presence of unoccupied receptors. The inhibition of residual unoccupied receptors may have functional effects as evidenced by a further increase in bleeding time with levels of occupancy >80%.14
Here, we describe the binding of 2 monoclonal antibodies to the platelet GPIIb/IIIa, LYP18 (Mab1), previously characterized to bind to the ligand recognition site and to be complex specific,15 and 4F8 (Mab2). Binding of these antibodies can be used in a flow cytometric assay to directly quantify occupied and unoccupied GPIIb/IIIa. We also present data on the application of this assay following administration of an oral GPIIb/IIIa receptor antagonist. This approach may be useful in monitoring drug effects and to follow receptor density in patients on long-term therapy with GPIIb/IIIa antagonists.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsStatistical AnalysisResultsDiscussionReferences |
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Monoclonal Antibodies
Monoclonal antibody 1 (Mab1, clone LYP18), a murine monoclonal antibody to the GPIIb/IIIa complex; monoclonal antibody 2 (Mab2, clone 4F8), a murine monoclonal antibody to the beta III subunit; isotypic control; fluorescein isothiocyanate (FITC) labeled F(ab')2 fragments of human Ig absorbed, sheep anti-mouse IgG antibodies; and calibration beads with 4 different known amounts of antibody per bead were provided by Dr M Canton (Biocytex, Marseille, France). SC-57101A (a HCl salt of the active component of the oral GPIIb/IIIa antagonist, orbofiban) and H3SC- 52021B, a radiolabeled GPIIb/IIIa ligand,16 were gifts of Dr Robert Anders (J.D. Searle, Skokie, Ill). Eptifibatide was a gift of Dr David Phillips (Cor Therapeutics, San Francisco, Calif). The monoclonal antibody abciximab was purchased from Eli Lilly and Co, Ind. GPIIb/IIIa was purified according to the protocol of Phillips et al.17 Ligand dilutions were performed in PBS.
Ligand Binding Studies
Nine parts blood from healthy donors, who had not taken aspirin
or any other anti-platelet agent in the previous 7 days, was
collected to one part sodium citrate (3.8%). This was
centrifuged at 150g for 10 minutes and the
platelet rich plasma (PRP) was aspirated. PRP was diluted to 1x
107 platelets/mL with PBS and incubated with
different concentrations of ligand for 30 minutes at room temperature.
Aliquots of each dilution were incubated with Mab1 or Mab2 (10 µg/mL)
at room temperature for 20 minutes. Antibody binding was determined
using fluorescein isothyocyanate (FITC) labeled
F(ab)2 fragments of human Ig absorbed, sheep
anti-mouse IgG(H+L) antibodies. The samples were fixed with 1 mL of 1%
formaldehyde after 10 minutes incubation and analyzed by flow
cytometry (FACScan, Becton Dickinson) at 488 nm excitation.
Platelet populations were gated according to their forward and side
light scatter. Histograms were generated using 10 000 counts, and
geometric mean fluorescence was calculated using the CELLQUEST
software of the FACScan system (Becton Dickenson). The binding of an
isotypic control antibody was taken as nonspecific binding and was
subtracted from the observed geometric mean fluorescence.
Calibration beads, consisting of a mixture of 4 different populations of 2-µm diameter latex beads, each with a different defined amount of murine antibody per bead, were used to estimate the number of antibodies bound per platelet, similar to the method described by Poncelet et al.18 The beads were analyzed in parallel with the samples, with the same FITC reagent and the same settings as the samples. The singlet bead populations were gated according to their forward and side scatter. Histograms of the geometric mean fluorescence intensity of 10 000 events were recorded and used to plot a log-log graph of the mean fluorescence intensity versus the number of antibodies attached to each bead. The number of platelet-bound Mab1 and Mab2 molecules was estimated from this graph on the basis of the geometric mean fluorescence intensity of the sample. After subtraction of nonspecific binding and assuming monovalent binding, the number of specifically bound antibody molecules was taken as the number of bound sites for either Mab1 or Mab2.
In additional experiments, we examined competition of Mab1 or Mab2 for each other's binding site using FITC labeled Mab1 and Mab2. PRP was incubated with Mab1, Mab2, or isotypic control for 20 minutes. FITC-labeled Mab1 or Mab2 was then added and incubated for 10 minutes and analyzed on the flow cytometer.
Platelet Aggregation
For ex vivo platelet aggregation studies, blood was
collected in 3.8% sodium citrate at a final dilution of 1:10 and was
centrifuged at 850g for 3 minutes to procure PRP.
Following PRP removal, the remaining plasma was centrifuged at
2500g for 5 minutes to obtain platelet poor plasma.
Platelet aggregation was studied following the addition of
adenosine diphosphate (20 µmol/L) to PRP at 37°C by light
transmission (Biodata PAP-4, Biodata Corporation). Aggregation was not
adjusted for the platelet count of each sample; however, the
aggregation at the different time-points was expressed as a percentage
of baseline aggregation, before administration of the drug. There was
no significant difference in whole blood platelet count at the
different time-points with a mean±SEM
(x103/µL) of 213±16 at baseline, 201±10
preprocedure, 215±13 post procedure and 189±9 at the 6 hour
time-point.
3HSC- 52021B-Binding
3HSC- 52021B is a triatiated form of a
potent benzamidine GPIIb/IIIa receptor
antagonist.16 Earlier experiments (not shown)
demonstrated that 3HSC-52021B binds in a
dose-dependent saturable manner in whole blood from healthy volunteer
donors, with a Kd of 93.4 nmol/L. Ligand
binding was determined using a modification of the technique described
by Wang et al.19 Whole blood was incubated with a
3HSC-52021B (5 nmol/L) for 30 minutes. Bound
ligand was separated from unbound using a cell harvester (Brandel Inc).
Each sample was eluted through Whatmann GFB microfiber filters
(Whatmann International Ltd) with 3.5 mL of Tris (10 mmol/L)
buffer at 4°C. The filters were washed 3 times with 3.5 mL of
ice-cold buffer and placed in scintillation fluid (Ecoscint A, National
Diagnostics). Filters were analyzed using a
scintillation counter (Wallac, 1214 RACKBETA). Each sample was
analyzed in triplicate with one aliquot containing excess
unlabelled compound (500 µmol/L). This was taken to
represent nonspecific binding. Results are expressed as the
percentage of baseline radioactivity before administration of the
GPIIb/IIIa receptor antagonist.
Ex Vivo Studies
The protocol was reviewed and approved by the Irish Medicines
Board and the Ethics Committee at St. James's Hospital and all
patients gave written, informed consent.
Blood samples were drawn, into 3.8% sodium citrate, from patients receiving a single dose of the oral GPIIb/IIIa receptor antagonist, xemilofiban 20 mg, or a matching placebo administered in a randomized, blinded fashion 30 to 90 minutes before coronary balloon angioplasty or stent placement. All patients received aspirin (150 to 300 mg) before the procedure and heparin at the time of intervention to achieve an ACT of >300 s. Blood sampling was performed from a peripheral vein at baseline before administration of the drug. The intervention was performed no sooner than 30 minutes but within 90 minutes of randomized drug administration. Blood samples were obtained again from the femoral venous sheath after insertion and immediately postangioplasty, discarding the initial 5 mL of blood in each case. Peripheral venous blood was also obtained just before the next dose of study medication, 4 to 7 hours following the initial dose. Samples were collected into 3.8% sodium citrate for platelet studies. Platelet aggregation studies and monoclonal antibody binding by flow cytometry were performed within 2 hours of blood sampling. 3HSC-52021B binding studies were performed within 24 hours. Previous experiments had confirmed stability of the 3HSC- 52021B binding assay for up to 7 days, when samples were stored at 4°C (data not shown). For Mab1 and Mab2 binding, whole blood was diluted 1 in 4 with platelet poor plasma from the corresponding aggregation studies at each time-point. This was performed in order to ensure that Mab1 and Mab2 (5 µg/mL) were at saturating concentrations. Platelet poor plasma is used to prevent dilution of the ligand. Diluted samples were incubated with antibody for 20 minutes at room temperature, stained with secondary antibody, and analyzed by flow cytometry.
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Statistical Analysis |
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Continuous data are presented as mean±SEM. Clinical data from xemilofiban- and placebo-treated patients was compared by ANOVA. Within the groups of treated and untreated patients, data from the different time-points was compared by an initial ANOVA with subsequent Dunnett's t test (when P<0.05) to compare each time-point to baseline. Percent of baseline Mab2 binding and percent of baseline aggregation were correlated using Pearson's correlation coefficient.
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Results |
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Binding of Mab1 and Mab2 to GPIIb/IIIa
Mab1 and Mab2 were bound to purified GPIIb/IIIa in a dot blot assay, where no binding was detected with an isotypic antibody. Following protein gel electrophoresis of the purified receptor (performed under reducing conditions), Mab2 identified GPIIb/IIIa, binding to the ß3 subunit (Figure 1
). As Mab1 binding is complex
specific,15 it did not identify the receptor under these
reducing conditions.
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Binding of Mab1 and Mab2 to Platelets In Vitro
In undiluted PRP and in PRP diluted to
1x107 platelets/mL from healthy donors, Mab1
and Mab2 bound in a concentration-dependent and saturable manner. Mab1
had a slightly lower affinity than Mab2 with Kd
of 5.3±1.3 µg/mL and 3.8±1.1 µg/mL (n=3), respectively, in PRP
and 1.52±1.4 µg/mL and 1.4±1.5 µg/mL (n=3), respectively, in PRP
diluted to 1x107/mL (Figure 2). Maximum binding occurred at 6 µg/mL
in dilute PRP and at 15 µg/mL in undiluted PRP. Mab1 identified a
mean of 53 300±5423 GPIIb/IIIa receptors sites per platelet at
maximum binding. The number of sites determined by Mab2 was slightly
lower at 50 120±5066 sites. Mab1 binding was reduced to background
fluorescence in platelet preparations incubated with EDTA
(10 mmol/L at 37°C for 30 minutes), whereas Mab2 binding was
reduced but was not completely abolished and the antibody still
recognized 13 041±577 sites per platelet. In competition studies
using FITC-labeled Mab1 and Mab2, Mab1 did not interfere with Mab2
binding and Mab2 did not inhibit Mab1 binding, suggesting that they
bind to separate sites (Figure 3).
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) and Mab2 (
) to
platelets. PRP was diluted to 1x107 platelets/mL
and incubated with increasing concentrations of antibody. Mab2 binds
with a Kd of 1.4±1.5 µg/mL and recognizes 50 120±5066
sites at saturation, whereas Mab1 binds with a Kd of
1.52±1.4 µg/mL and recognizes a maximum of 53 300±5423
sites.
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Repeated analysis of a sample from a single donor drawn at the same time revealed a coefficient of variation of 0.55% for Mab1 and 1.3% for Mab2 in the number of sites identified. Analysis of samples from 4 different healthy donors revealed a range for Mab1 binding of 35 815 to 47 721 and of 35 175 to 42 556 for Mab2. Reanalysis of samples from the same healthy donors one month later revealed a mean coefficient of variation of 14% for the number of sites identified by Mab1 and 5.4% for Mab2.
Displacement of Mab1 and Mab2 by Antagonists to the
Platelet GPIIb/IIIa In Vitro
Abciximab inhibited Mab1 binding in a dose-dependent manner with
an IC50 of 0.85±0.1 µg/mL, reducing the number
of binding sites for Mab1 from a maximum of 49 691±1016 in the
absence of abciximab to 1297±154 sites at 10 µg/mL (Figure 4). The number of binding sites detected
by Mab2 remained constant at a mean of 47 874±746 sites per
platelet. In contrast, the synthetic ligand eptifibatide displaced
Mab2 in a dose-dependent manner (IC50,
0.35±0.14 µmol/L) and had little effect on Mab1 binding.
Increasing concentrations of eptifibatide reduced Mab2 binding to
7455±1173 sites from a maximum of 44 328±2033 sites, whereas Mab1
binding remained constant at 50 412±480 sites. Similarly,
SC-57101A (IC50, 0.22±0.06 µmol/L),
reduced Mab2 binding to a minimum of 9118± 1,835 sites at a
concentration of 30 µmol/L from a maximum of 43 182±2373,
whereas Mab1 binding remained constant at a mean of 54 109±2605.
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To determine the time course of displacement of the ligand by the antibodies, samples were spiked with abciximab (1.5 µg/mL) and stored at 4°C. Mab1 and Mab2 binding remained constant for over 2 hours in unfixed dilute PRP. Mab1 binding progressively increased after 2 hours, indicating displacement of the abciximab from its binding site, whereas Mab2 binding remained constant. In dilute unfixed PRP incubated with eptifibatide (12.5x10-6M) or Sc-57101A (6.3x10-6M), Mab2 binding increased after 2 hours indicating the displacement of ligand. Fixing samples in 1% formaldehyde, after incubation with Mab1 and Mab2 prevented this displacement, and binding remained constant for up to 15 days (data not shown).
Clinical Studies
Figure 5 shows the effect of
xemilofiban (20 mg) or placebo on platelet aggregation,
3HSC-52021B binding and number of sites detected
by Mab1 and Mab2. In patients receiving placebo (n=7), Mab2 binding
remained stable with 44 652±4189 sites, 38 013±2681 sites, and
40 047±1986 sites detected preprocedure, postprocedure, and at 6
hours, respectively (P=0.41). The corresponding number of
sites identified by Mab1 were 59 230±4892, 56 679±5332, and
56 955±4537 at each time-point (P=0.98). Platelet
aggregation was largely unchanged at 94±4%, 87±6%, and 97±2%, at
the corresponding time points (P=0.15). Similarly,
radioligand binding was uninhibited at 123±24%
preprocedure, 162±24% postprocedure, and 115±8% at 6 hours
(P=0.12).
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In patients receiving xemilofiban (n=9), there was significant
inhibition of platelet aggregation, radiolabeled ligand binding,
and Mab2 binding (P<0.002). This occurred progressively
over the period of observation, which is consistent with a peak
plasma concentration of 2 to 6 hours.20 At baseline,
Mab1 and 2 identified 50 910±1932 and 37 930±2061 sites per
platelet, respectively. Mab1 binding was unchanged following drug
at 50 795±2808 sites just before the procedure, 50 260±2737
immediately postprocedure, and 48 485±3228 sites at the 6-hour
time-point (P=0.92). In contrast, Mab2 binding decreased to
19 540±4819 (51% of baseline) sites just before the procedure,
13 373±4866 (32% of baseline) immediately postprocedure (at an
average of 92 minutes after the initial drug dose), and reached a
minimum at the 6-hour time-point of 8318±870 sites per platelet
before the next dose of drug (P<0.0001). Aggregation was
reduced to 61±8%, 30±15%, and 3±3% of baseline at the
corresponding time-points (P<0.0001).
Radioligand binding was also inhibited to 79±20% before
the procedure, 77±16% immediately postprocedure, and 57±10% at 6
hours (P<0.05 for postprocedure and 6-hour sample compared
with baseline). Mab2 expressed as a percent of baseline correlated with
platelet aggregation, also expressed as a percent of baseline
(r=0.8; P<0.0001, Figure 6).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsStatistical AnalysisResultsDiscussionReferences |
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Binding of Mab1 and Mab2 to GPIIb/IIIa
Previous studies have shown that Mab1 binds to GPIIb/IIIa on platelets and the vitronectin receptor,
vß3, on
endothelial and melanoma cells.21 22 23 Its
binding to GPIIb/IIIa requires an intact heterodimer because it fails
to bind to either subunit under reducing conditions or in crossed
immunoelectrophoresis.15 The binding site is at or close
to the ligand recognition sites of GPIIb/IIIa, as Mab1 inhibits
fibrinogen binding and platelet aggregation. Mab2 binds to the ß3
subunit of the platelet GPIIb/IIIa, but further characterization is
required to define the precise epitope. Mab2 recognizes the receptor
under reducing conditions, suggesting that it recognizes a linear
epitope. This may explain the continued recognition of the epitope when
the receptor complex is disrupted by chelating
Ca2+. Mab2 also binds to
endothelial cells. This is consistent with its
epitope also being present on the ß3 subunit of the
vitronectin receptor
vß3 (data not
shown).
Differential Displacement of Mab1 and Mab2 Binding to Platelets
by Antagonists
Both antibodies bound to platelets in a concentration
dependent and saturable manner with equivalent affinities. The number
of sites identified by the antibodies is consistent with
previously reported numbers of GPIIb/IIIa receptors per platelet
identified by bivalent monoclonal antibodies and labeled fibrinogen
binding.24 25 26 27 28 However, it is possible that our antibodies
underestimate the true number of receptors per platelet; Wagner et
al have demonstrated discrepancy between bivalent and monovalent
antibody binding.29 This will not, though, effect the
ability of the assay to estimate the percentage occupancy. On whole
platelets, Mab1 binding was displaced entirely by disruption of the
complex using EDTA and by the monoclonal antibody, abciximab. Abciximab
is directed at the fibrinogen binding site and prevents fibrinogen
binding, platelet aggregation, and platelet
adhesion.30 31 In contrast, abciximab had no effect on
Mab2 binding, even at very high concentrations. This suggests that the
Mab1 and Mab2 recognize different epitopes. Indeed, using prelabeled
Mab1 and Mab2, we have shown that the antibodies did not compete with
each other for binding, again suggesting that they identified different
epitopes.
In contrast to abciximab, 2 small molecule antagonists, the peptide eptifibatide and the nonpeptide SC-57101A, displaced Mab2 but not Mab1. It is not clear why these compounds failed to displace Mab1. As with abciximab, SC-57101A and eptifibatide prevent platelet aggregation and fibrinogen binding, suggesting that they bind at or close to the ligand recognition site. However, there may be differences in the regions recognized by antagonists despite their similar effects on fibrinogen binding. Cross-linking studies using iodinated GPIIb/IIIa antagonists KYGRGDS and the cyclic compound, KYGC(s-s)HarGDWPC(s-s) show that these compounds cross-link to different sites on the ß3 subunit.32 Thus, antagonists may differ in where they bind within the active pocket and this may explain the differential displacement of Mab1 binding.
Similarly, it is unclear why some compounds and not others displace
Mab2. Mab2 binding does not interfere with the binding of the
radiolabeled GPIIb/IIIa antagonist,
3HSC-52021B,33 in contrast with
Mab1, suggesting that Mab2 identifies a site remote from the active
site and remote from where antagonists bind. Therefore,
reduction of Mab2 binding may not reflect a direct interaction between
antibody and antagonist. The displacement of Mab2 may be
the result of a conformational change in the receptor with a loss or
attenuation of the epitope recognized by the antibody. Ligand binding
is known to induce conformational changes in the GPIIb/IIIa, usually
detected as the expression of neoepitopes.34 Ligand
attenuation of a binding site or epitope has also been described in the
fibronectin receptor (5ß1), where the epitope is present on
unbound receptors but disappears from ligand bound
receptors.35
Assay of Total and Occupied GPIIb/IIIa
The differential displacement of the 2 monoclonal antibodies to
the GPIIb/IIIa receptor, Mab1 and Mab2, by abciximab and the synthetic
GPIIb/IIIa antagonists provides a means of calculating
total receptor number and receptor occupancy by GPIIb/IIIa ligand. This
may be useful in following the receptor-ligand interaction, as
demonstrated in our patient population. The differential effects of the
compounds may also be applied to monitor drug receptor interactions
following oral GPIIb/IIIa antagonists in patients
previously treated with abciximab. Currently available methods to
assess the biological activity of GPIIb/IIIa receptor
antagonists are limited by their inability to differentiate
between receptor inhibition by abciximab and small molecular weight
antagonists.36 37 This is important as
abciximab can remain bound to platelets for days following a single
administration, enhancing the effects of oral GPIIb/IIIa
antagonists.10 Finally, by estimating total as
well as bound receptors, this assay provides a means to follow receptor
kinetics during long-term drug administration. Thus, changes in
receptor number as a result of ligand occupancy or following drug
withdrawal may be detected.
In conclusion, Mab1 and Mab2 are 2 monoclonal antibodies that bind to different epitopes of the platelet GPIIb/IIIa and are differentially displaced by abciximab and the small molecule antagonists, eptifibatide, xemilofiban, and SC-57101a. The displacement of Mab1 and Mab2 may be used to monitor drug receptor interactions in vivo and potentially to discriminate between the effects of abciximab and other antagonists in man.
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Acknowledgments |
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This work was supported by grants from the Wellcome Trust, the Charitable Infirmary Charitable Trust, and the Health Research Board of Ireland.
Received June 1, 1998; revision received February 9, 1999; accepted February 10, 1999.
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References |
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