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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1622-1633
Labeling of the Internal Pool of GP IIb-IIIa in Platelets by c7E3
Fab Fragments (abciximab): Flow and Endocytic Mechanisms
Contribute to the Transport
By
Paquita Nurden,
Christel Poujol,
Catherine Durrieu-Jais,
Joëlle Winckler,
Robert Combrié,
Laurent Macchi,
Claude Bihour,
Carrie Wagner,
Robert Jordan, and
Alan T. Nurden
From UMR 5533 CNRS, IFR Coeur-Vaisseaux-Thrombose and Unité des
Soins Intensifs, Hôpital Cardiologique, Pessac, France; and
Centocor, Malvern, PA.
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ABSTRACT |
Abciximab is a new antiplatelet therapeutic in ischemic
cardiovascular disease. The drug, chimeric Fab fragments of a murine monoclonal antibody (MoAb) (c7E3), blocks GP IIb-IIIa function. However, its capacity to reach all receptor pools in platelets is
unknown. Electron microscopy and immunogold labeling were used to
localize abciximab in platelets of patients receiving the drug for up
to 24 hours. Studies on frozen-thin sections showed that c7E3 Fab, in
addition to the surface pool, also labeled the surface-connected canalicular system (SCCS) and  -granules. Analysis of gold particle distribution showed that intraplatelet labeling was not accumulative and in equilibrium with the surface pool. After short-term incubations of platelets with c7E3 Fab in vitro, gold particles were often seen in
lines within thin elements of the SCCS, some of which appeared in
contact with -granules. Little labeling was associated with
Glanzmann's thrombasthenia platelets, confirming that the channels
contained bound and not free c7E3 Fab. Endocytosis of abciximab in
clathrin-containing vesicles was visualized by double staining and
constitutes an alternative mechanism of transport. The remaining free
pool of GP IIb-IIIa was evaluated with the MoAb AP-2; flow cytometry
showed it to be about 9% on the surface of nonstimulated platelets but
33% on thrombin-activated platelets. The ability of drugs to block all
pools of GP IIb-IIIa and then to be associated with secretion-dependent
residual aggregation must be considered when evaluating their
efficiency in a clinical context.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
GP IIb-IIIa COMPLEXES (INTEGRIN
 IIb 3), are not only
constituents of the surface membrane of platelets, they are also widely
present in internal membrane compartments, including those of the
surface-connected canalicular system (SCCS) and the
-granules.1-4 The complexes mediate platelet aggregation
through the linking of adjacent platelets by adhesive
proteins.5 Some soluble agonists such as adenosine
diphosphate (ADP) initiate aggregation without directly inducing the
secretory mechanism of platelets. In contrast, strong agonists such as
thrombin additionally promote expression of the internal pool of GP
IIb-IIIa at the platelet surface.6 New-generation
antiplatelet drugs based on the concept of blocking the final common
step of platelet aggregation inhibit the binding of adhesive proteins
to the GP IIb-IIIa complex.7 These drugs include monoclonal
antibodies (MoAbs) such as abciximab, RGD, or KGD-containing peptides
such as integrilin, peptidomimetics, and orally available
inhibitors.7-11 Abciximab is the generic name for the
humanized form of Fab fragments of the murine MoAb, 7E3 (c7E3). It is
used in ischemic cardiovascular disease to prevent arterial thrombosis,
in particular in patients with a high thrombotic risk undergoing
percutaneous transluminal coronary angioplasty (PTCA). It is the most
commonly used anti-GP IIb-IIIa therapy at the current
time.11 It is also the first anti-integrin therapy to be
used on a large scale.7
The first major trial using abciximab was the EPIC study, which showed
that it can reduce by 35% the major complications of PTCA at the
primary end point of 30 days.12 These results were obtained
using a regimen in which a bolus of 0.25 µg/kg was followed by an
infusion of 10 µg/min for 12 hours. In the EPIC study, platelet aggregation induced by ADP was totally inhibited during the perfusion. The lack of platelet aggregation in patients receiving abciximab can be
compared with that observed in the inherited disorder, Glanzmann's
thrombasthenia.13 Nevertheless, if there are similarities in the response of platelets to ADP, the response to thrombin is
different. Whereas in thrombasthenia the platelets do not aggregate to
thrombin, Kleimann et al14 showed that following
administration of a bolus of c7E3, platelets of patients with ischemic
coronary disease continue to show a residual aggregation response to
thrombin, thereby implying the continued presence of unblocked
receptors. Recently, we reported similar results for platelets of
patients receiving a bolus of c7E3 Fab followed by an
infusion.15 An apparently internal pool of GP IIb-IIIa free
of abciximab was detected using a competitive MoAb, AP-2, and the
surface expression of this pool was shown after thrombin
stimulation.15
Our current study investigated the mechanisms whereby abciximab gains
access to the different pools of GP IIb-IIIa in platelets. To do this,
we first used electron microscopy (EM) and immunogold labeling to
detect c7E3 Fab on frozen ultrathin sections of platelets isolated from
patients undergoing antithrombotic therapy. The patients in question
received abciximab for 18 to 24 hours before prescheduled PTCA under
the conditions defined in the CAPTURE protocol.15,16 Using
a previously characterized rabbit antibody monospecific for c7E3 Fab
fragments,17,18 we observed that abciximab not only labeled
the surface membrane but also penetrated within the SCCS and was
detected on the membranes of some -granules. Significantly, a
time-dependent loss was observed from all pools in the days following
the perfusion. Short-term incubation of platelets with abciximab, in
vitro, led to the visualization of many thin SCCS channels lined with
the gold particles representing receptor-bound c7E3 Fab. These
sometimes juxtaposed with the -granules of unstimulated platelets,
raising the question of a direct exchange of GP IIb-IIIa between them.
An occasional presence of vesicles containing both c7E3 and clathrin
also showed that endocytosis was occurring. Thus, monovalent c7E3 Fab
fragments are an interesting tool for following the trafficking of GP
IIb-IIIa complexes in platelets. At the same time, our results
underline that under current conditions of abciximab therapy, unblocked
GP IIb-IIIa complexes in platelets are able to mediate a residual
secretion-dependent aggregation.
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MATERIALS AND METHODS |
Patients and the Abciximab Infusion Protocol
Our report covers studies on 6 patients treated with abciximab in the
Cardiology Department of our hospital. All of the patients suffered
from ischemic coronary disease and were diagnosed as having refractory
unstable angina. The patients received 0.25 mg/kg body weight of
abciximab as a bolus followed by a perfusion of 10 µg/min over a
period of 12 to 24 hours. The details for each patient are shown in
Fig 1. With the exception of patient 3, PTCA was performed and the abciximab infusion continued for 1 hour as
in the CAPTURE study.16 Each patient received intravenous heparin and aspirin as described elsewhere.15 Bleeding
complications were not seen in this group, and none of the patients
developed thrombocytopenia during the period of study. For patient 3, lesions were present in three coronary vessels, and a late decision was taken for coronary artery bypass graft surgery (CABG). This was performed when platelet aggregation with ADP had returned to values higher than 50%. Approval from the local Ethics Committee was obtained
before the onset of the study.
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| Fig 1.
Schema showing the duration of the abciximab infusion
(grey section) following the bolus in each of the patients studied. The
moment at which PTCA was performed is indicated, as is the time at
which patient 3 underwent CABG. The vertical arrows show the times (h)
at which samples were taken for EM and platelet function testing. The
point at which abciximab therapy started is considered as time zero.
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Platelet Preparations
Figure 1 shows the times at which samples were taken for EM and
platelet function testing. During the infusion of abciximab and during
the following 24 hours, samples were obtained from a vascular sheath.
After removal of the catheter, peripheral blood was obtained by clean
venipuncture.15 The initial 3 mL of blood was always
discarded. Blood was collected into acid-citrate-dextrose NIH formula A
(ACD-A) (1 vol of anticoagulant:6 vol of blood) or into 3.8% (wt/vol)
sodium citrate (1 vol:9 vol) (see below). Platelet-rich plasma (PRP)
was prepared by centrifugation at 120g for 10 minutes at room
temperature. To prepare washed platelets, prostaglandin
E1 (100 nmol/L; Sigma, St Louis, MO), 25 µg/mL apyrase (Sigma), and ACD-A (1 vol:9 vol, PRP) were added
immediately to PRP obtained from blood anticoagulated with ACD-A.
Platelets were sedimented by centrifugation at 1,200g for 15 minutes and washed as previously described.19 The platelets
were resuspended at 2 × 108/mL in 137 mmol/L NaCl, 2 mmol/L KCl, 12 mmol/L NaHCO3, 0.3 mmol/L NaH2PO4, 1 mmol/L MgCl2, 5.5 mmol/L
glucose, 5 mmol/L HEPES, 0.1% (wt/vol) bovine serum albumin (BSA), pH
7.4 (HEPES buffer-modified tyrode [HBMT]).
Platelet Aggregation
The platelet response during the abciximab infusion was tested using
citrated PRP in a platelet aggregometer (PAP-4 model; Biodata
Corporation, Wellcome, Paris) with stirring.20 ADP (8 µmol/L; Sigma) or thrombin receptor activating peptide (TRAP)-14 mer
(25 µmol/L; Neosystem, Strasbourg, France) were used as agonists. The
platelet count did not vary significantly before and after the infusion
of abciximab for the patients tested, so the PRP contained
approximately the same number of platelets for each patient. Results
are given as the maximal intensity of aggregation measured as the light
transmission change after three minutes when the plateau had been
reached (expressed as a percentage). The inhibition of platelet
aggregation by abciximab was calculated as a function of the response
measured for the same patient before the abciximab bolus injection.
Short-Duration Incubations of Abciximab With Platelets In Vitro
Blood from normal subjects or from a patient with type I
Glanzmann's thrombasthenia (see George et al,21
patient no. 9) with a homozygous mutation 1063G'A in exon 12 of
the gene coding for GP IIb giving rise to a E355K
mutation22 was taken into ACD-A anticoagulant and washed
platelets prepared as described above. The platelets suspended at
250,000/µL in HBMT were incubated with abciximab (ReoPro; Centocor,
Malvern, PA) at 10 µg/mL for times ranging between 1 and 60 minutes.
Platelets were then fixed directly.
Electron Microscopy
Sample preparation.
Washed platelets from patients receiving abciximab or platelets from
subjects incubated with abciximab in vitro were fixed in the presence
of a mixture of 1% (wt/vol) paraformaldehyde (PFA; Sigma) and 0.1%
(wt/vol) glutaraldehyde (Fluka AG, Buchs, Switzerland) or with 1.25%
(wt/vol) glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.2, for 30 minutes at room temperature. Fixed platelets were washed 3× in
phosphate-buffered saline (PBS), pH 7.2.23 The pellet was
subdivided into small blocks that were cryoprotected with 2.3 mol/L
sucrose before being frozen in liquid propane using the Reichert KF 80 freezing system (Leica, Vienna, Austria).
Preparation of ultrathin sections and immunogold staining.
Procedures were basically as previously described by
us.23,24 In brief, ultrathin sections of platelets were cut
with a Reichert-Jung Ultracut E ultramicrotome equipped with a FC 4E cryokit attachment (Reichert-Jung, Vienna, Austria) and placed on
collodion-coated nickel grids. In subsequent steps, the grids were
transferred onto drops containing antibodies or washing buffer as
described below. Rabbit antibody prepared against c7E3 was used at a
dilution of 1/100 of affinity-purified IgG (a gift from Centocor).17,18 Anti-clathrin heavy-chain MoAb (mouse IgG1 purchased from MedGene Science, Pantin, France) was used at 5 µg/mL.
All dilutions were in PBS, pH 7.2, containing 0.1% (wt/vol) BSA
(Sigma). The grids were kept floating on the antibody-containing solution for 1 hour at room temperature. After 3 washes, bound antibody
was revealed by incubating for 1 hour with gold-labeled anti-rabbit or
anti-mouse IgG (1/100 dilution of Auroprobe EM GAR or GAM G5 or G10
[Amersham, Les Ulis, France]). After 3 more washes, the cryosections
were stained by uranyl acetate and embedded in a thin film of
methylcellulose before being observed at 80 kV in a Jeol JEM-1010
transmission electron microscope (Jeol, Croissy-sur-Seine,
France).24 In the double-labeling experiments, sections
were serially incubated with rabbit anti-c7E3, GAR G5, murine
anti-clathrin MoAb and GAM G10 with three washings between the addition
of each new antibody. Controls involved the substitution of primary
antibodies with nonimmune rabbit or mouse IgG during the labeling of
the sections. Preliminary experiments established that GAM G10 failed
to detect bound abciximab and did not cross-react with rabbit IgG. In
studies on platelets from the patients, control sections were also
prepared from platelets taken from each patient before their receiving
abciximab. Sections of platelets from the patient with Glanzmann's
thrombasthenia were tested with the anti-c7E3 antibody only.
Computer analysis.
The distribution of gold particles revealing bound abciximab was
evaluated for patients 1, 2, and 3 on platelet sections photographed using a Sony XC 077C camera (Sony, France, Paris) digitalized in 640 × 480 pixels coded in 256 levels of gray. Image analysis was
performed using a program constructed by C. Allasia (Faculté de
Médecine, Université de la Mediterranée, Marseille,
France) and installed on an IBM PC Pentium 166 computer equipped with Image Series 640 cards (Matrox France, Paris) driven by the IPS program
(Unilog, Grenoble, France). Gold particles located on the surface
membrane or within the internal compartment of platelets (SCCS + -granules) were counted separately. A minimum of 10 sections were
evaluated for each time point. The density of gold particles on the
surface membrane was calculated by relating the number of gold
particles to the perimeter length. The latest time for which the
quantitation was performed was 7 days after the infusion of abciximab
for patient 3.
Flow Cytometry
Platelets of patients receiving c7E3.
Samples of washed platelets (108/mL) were fixed with 2%
(vol/vol) PFA for 30 minutes at room temperature. The samples were washed 3× in PBS, pH 7.2, containing 0.1% (wt/vol) BSA and then incubated for 30 minutes at room temperature with 100 µL of
fluorescein isothiocyanate (FITC)-conjugated F(ab')2
fragments of a goat antibody monospecific for human IgG
F(ab')2 (Jackson ImmunoResearch, West Grove, PA)
diluted 1/100 in HBMT. Preliminary studies had confirmed that the
murine sequences were largely inaccessible to the anti-mouse IgG
antibodies in flow cytometry when c7E3 Fab was bound to
platelets.15,19 Control measurements were made using
FITC-labeled F(ab')2 fragments of a goat antibody
monospecific for the human IgG Fc fragment (Jackson ImmunoResearch).
The platelet suspension was then diluted with 750 µL HBMT before
analysis using a Becton Dickinson FACScan flow cytometer (Becton
Dickinson, Le Pont de Claix, France). The fluorescence intensity was
determined for the platelet population identified by gating on light
scattering parameters (forward scatter versus side
scatter).20,23 Ten thousand cells were analyzed for each
sample and the data were registered as log fluorescence. Mean
fluorescence intensities (MFI) as a measure of antibody binding were
expressed using the LYSYS II conversion software in an arithmetic mode.
Short-duration incubations of abciximab with platelets in vitro.
Unblocked GP IIb-IIIa complexes were located using AP-2 (a gift from Dr
Thomas Kunicki, Scripp's Research Institute, La Jolla, CA), a murine
monoclonal antibody that we have shown to be competitive with c7E3 Fab
for GP IIb-IIIa.15,19 Isolated platelets were incubated or
not with 10 µg/mL c7E3 Fab for 30 minutes as described above. After
being washed twice, samples were then incubated in the presence or
absence of 0.5 U/mL thrombin for 10 minutes. Samples were immediately
fixed with PFA as described above. Aliquots of fixed platelets (10 µL
at a concentration of 100,000/µL) were added to polystyrene tubes
containing 100 µL HBMT and 7 µg/mL AP-2. Controls were performed in
the absence of primary antibody or in the presence of purified myeloma
mouse IgG (Sigma). All samples were incubated for 15 minutes at room
temperature without stirring. Then 10 µL of a predetermined optimal
concentration of FITC-conjugated affinity-purified
F(ab')2 fragments of sheep antibody to mouse IgG
(Silenus, Eurobio, Les Ulis, France) were added. The tubes were left in
the dark at room temperature for an additional 15 minutes before
analysis with the FACScan as described above.
Statistical analysis.
Data were analyzed using the Student's t-test for
unpaired data. A value of P < .05 was considered to be
statistically significant.
| |
RESULTS |
Studies on Platelets Obtained From Patients Receiving Abciximab
Platelet function testing.
The effects of abciximab on platelet aggregation induced by ADP and by
TRAP-14 mer were studied for each patient at the sampling times shown
in Fig 1. A greater than 80% inhibition of ADP-induced platelet
aggregation was obtained for each patient during the infusion and was
seen as early as 3 hours after the onset of therapy for the three
patients tested at this time point. This consistency allowed us to
group the results for the patients together
(Fig 2). The recovery of the aggregation
response had begun within 24 hours after the infusion was stopped, and
was extensive after 48 hours, although the difference remained
statistically significant compared with the pretreatment value. With
TRAP-14 mer, which mimics thrombin and induces a secretion-dependent
aggregation in platelet-rich plasma, the inhibition was more modest
during the infusion, and residual aggregation of at least 50% of
initial levels was observed for each patient. The aggregation response to TRAP-14 mer was no longer statistically lower 48 hours after the
infusion had ended. The results with TRAP-14 mer were compatible with
the presence of a pool of GP IIb-IIIa complexes inaccessible to
abciximab even after a combined therapy of bolus and infusion.
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| Fig 2.
Platelet aggregation induced by ADP (8 µmol/L) or 25 µmol/L TRAP-14 mer peptide in citrated PRP prepared from patients
receiving abciximab and tested at the sample times shown in Fig 1.
Results are expressed as percent light transmission at 3 minutes of
aggregation (maximal values) and are grouped together for samples taken
during the infusion, and at periods less than 48 hours and greater than
48 hours after the drug administration ended. The P values for
the observed inhibition were calculated with respect to the
aggregation intensity before the onset of abciximab.
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Localization of abciximab on platelet sections by immunoelectron
microscopy.
Initial controls performed using platelets isolated from each patient
before therapy showed that the rabbit anti-c7E3 antibody did not label
the cryosections in the absence of abciximab (data not shown). For
platelets taken from the patients during the abcximab infusion, we
observed a regular labeling of the platelet surface, which was often
dense (Fig 3). In Fig 3A, the sample was
taken 3 hours after the onset of the infusion to patient 1. The
illustration is typical of the results obtained for each of the three
patients tested at this early time point. Significantly, labeling can
already be seen inside the platelet, where it was localized both to the SCCS and to the luminal surface of the membranes of some but not all
-granules. Figure 3B through E illustrates platelets taken 19 hours
after the start of abciximab infusion to patient 1. The results
resembled those seen at 3 hours with a maximum labeling of the surface
membrane and also staining inside the platelets. Figure 3C shows a
high-power section of an -granule with several membrane-associated
gold particles. We have previously shown similar labeling for
P-selectin, a known -granule protein.6 Figure 3D shows a
thin channel arriving inside an -granule; a thin opening can be seen
in the membrane of the -granule. Figure 3E is a high-power section
from Fig 3B and highlights the thin SCCS channels interwinding within
the platelet and decorated by gold particles. The labeling of SCCS and
-granules during the infusion was a consistent finding for the
platelets of all patients. Nevertheless, the extent of the labeling of
-granules varied considerably from one platelet to another.
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| Fig 3.
Detection of abciximab by immunogold labeling using an
affinity-purified rabbit antibody specific for c7E3. Examined were
frozen ultrathin sections of platelets taken from patient 1 (A) 3 hours
or 19 hours (B through E) into the infusion. High-power magnifications
of a labeled -granule and of thin tortuous channels of the SCCS are
shown in (C) and (E), respectively. In (D) a very thin aperture in the
membrane of -granule at a point of junction of a thin channel is
shown and indicated between two arrows. Bars = 0.2 µm.
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The distribution of gold particles during the 48 hours after the
infusion was stopped and closely resembled that seen on platelets taken
during the therapy. Abciximab was detected on the platelet surface and
inside the platelets; labeling again concerned both the SCCS and the
membranes of some but not all -granules. In Fig 4A, it is the surface membrane that is
primarily labeled; in Fig 4B there is considerable labeling of the
internal pool. Such types of image were seen for all patients and
underline the heterogeneity observed in the platelet response to this
drug. For the illustrated final samplings, taken for patient 1 after 85 hours (Fig 4C) and patient 3 after 1 week (Fig 4D), labeling both at
the platelet surface and in the interior was generally much more
sparse. Although a considerable number of new platelets would have been
released into the circulation since the stopping of therapy, most of
the platelets continued to show some labeling (not illustrated).
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| Fig 4.
Immunogold labeling for abciximab on ultrathin sections
of platelets taken (A) 20 hours after the end of the abciximab infusion
(patient 1), (B) 15 hours after the end of the infusion (patient 3),
(C) 67 hours after the end of the infusion (patient 1), (D) 1 week
after the end of the infusion (patient 3). There is a notable decrease
in the labeling at the longer time points. Bars = 0.2 µm.
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Computer analysis.
The number of gold particles was quantified on sections of a minimum of
10 platelets for each sample taken during and after abciximab infusion
for patients 1, 2, and 3 (Table 1). A
computer program also compared the distribution of gold particles
between the surface and internal membrane systems. During the infusion, the mean gold particle density on the surface of platelets of each
patient was consistently high. Particles associated with internal
membrane systems represented 24%, 28%, and 31% of the total number
counted for each patient. These values changed little in the 48 hours
that followed the end of the infusion, although there was slightly more
intersubject variation. After 48 hours, labeling both at the surface
and within the platelet had decreased, and although the percentage of
gold particles inside the platelet was marginally greater, these
measurements confirmed that there was not a progressive and
unidirectional accumulation of abciximab within the platelets.
Flow cytometry.
As a supplement to the EM studies, we used flow cytometry to evaluate
the levels of c7E3 Fab bound to the platelet surface. A rapid and large
increase in the MFI was observed during the infusion of abciximab. The
grouped MFI values during the infusion (266.4 ± 74.3; n = 7) were
the maximum obtained; levels remained high at 48 hours (218.3 ± 90.8, P = .031; n = 6) but fell progressively at time points
greater than 48 hours (162 ± 20.8, P = .024; n = 4).
Interestingly, a single peak of fluorescence was always observed on the
histograms, showing that new platelets produced after the end of the
abciximab infusion possessed c7E3 Fab, seemingly confirming an exchange
between platelets via the plasma pool as suggested by Christopoulos et
al.17,18 The positivity was not related to the binding of
plasma immunoglobulins, either to prebound c7E3 or to exposed platelet
neoantigens, as the binding of FITC-labeled anti-human Fc fragments
antibody remained negative.
Short-Duration Incubations of Platelets With Abciximab In Vitro
Immunogold staining for abciximab.
Platelets from normal subjects were incubated with 10 µg/mL c7E3 Fab
for periods of 1 minute, 5 minutes, or 1 hour. The results showed that
some abciximab was already seen inside the cell after a 1-minute
incubation but that labeling was greater after 1 hour. Figure 5A illustrates a platelet incubated
for 1 minute with c7E3 Fab. In Fig 5B, normal platelets have been
incubated for 1 hour with c7E3; a long, thin channel of SCCS located
parallel to the plasma membrane is strongly labeled; and gold beads
were associated with some -granules. As was found for platelets from
patients receiving abciximab, not all the membranes located inside the platelets were labeled. Figure 5C illustrates the labeling of platelets
from a patient with Glanzmann's thrombasthenia incubated for 1 hour
with 10 µg/mL of c7E3. In the absence of GP IIb-IIIa complexes,
little or no staining was present on the platelet surface at all time
points tested and only rare gold particles were present inside the
platelets. This result confirms that we were not locating free c7E3
that had simply diffused within the channels.
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| Fig 5.
Incubation of platelets from a control donor (A and B)
and a patient with Glanzmann's thrombasthenia (C) with abciximab in
vitro. Platelets were fixed after 1-minute (A) or 1-hour staining (B
and C). Arrows highlight the presence of gold particles within the thin
channels of the SCCS, arrow heads indicate gold particles associated
with -granules. In (C) almost no labeling was seen. Bars = 0.2 µm.
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In Fig 6, a series of high-power insets
illustrate the presence of gold beads associated with -granules and
the thin tortuous channels of the SCCS. Examples have been chosen where
the channels arrive in close proximity to the -granules. Most of the
labeling of the -granules was associated with the membrane. However,
intense labeling of the entire -granule membrane was rare. The
channels sometimes are directed toward the -granule and, on
occasion, appear to be in contact with them (see Fig 6B and C).
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| Fig 6.
High-power magnifications showing the incorporation of
abciximab into control platelets in vitro. Incubation periods with
abciximab before fixation were 1 minute (A, B, and C), 5 minutes (D),
30 minutes (E), and 1 hour (F). Thin channels in close association and
apparently directed toward -granules are labeled with arrows. Bars
= 0.2 µm.
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Colocalization with clathrin.
Because translocation of bound ligands from the surface to the internal
compartment is classically believed to involve the formation of
endocytic vesicles containing clathrin, we performed double staining of
sections with the rabbit anti-c7E3 antibody and a murine MoAb to
clathrin. As shown in Fig 7, colocalization was observed (A) in vesicular structures within the platelets and (B
and C) within -granules. Colocalization was not observed in the
SCCS. In Fig 7D a vesicle appears to be fusing with an -granule.
These results show for the first time that internalization of abciximab
can occur through the formation of endocytic vesicles, although it
remains to be shown what proportion of abciximab is incorporated in
this way.
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| Fig 7.
Double-staining for c7E3 and clathrin on platelet
sections after the incubation of platelets with abciximab for 5 minutes
in vitro. Clathrin was detected with a mouse MoAb, in turn localized
with goat anti-mouse IgG adsorbed to 10 nm gold particles (heavy
arrows). Abciximab was detected using rabbit antibody itself detected
using anti-rabbit IgG adsorbed onto 5 nm gold particles (thin arrows).
In (A) a vesicle in close proximity to a granule contains both
abciximab and clathrin, in (B and C) clathrin and abciximab are both
associated with the membrane of -granules, and in (D) a small
vesicle containing clathrin and abciximab appears to be fusing with an
-granule. Bars = 0.2 µm.
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Flow cytometry.
To analyze the free pool of GP IIb-IIIa, we have used AP-2, a murine
MoAb that is competitive for c7E3 as described
previously.15,19 Incubation of normal platelets for 30 minutes with 10 µg/mL of c7E3 Fab in vitro resulted in a severe
reduction in the binding of AP-2 to the surface of unstimulated
platelets. We found that the MFI after incubation was 9.20 ± 4.3%
of the initial values. After stimulation of untreated platelets with
thrombin 0.5 U/mL for 10 minutes, the MFI for AP-2 binding was 202 ± 20.48% (n = 5) of that seen for unstimulated platelets. After
incubation with c7E3, the MFI after thrombin treatment was 33.80 ± 10.91% (n = 5) of the value obtained in the absence of drug. The
results, illustrated in Fig 8, therefore
confirm that an appreciable pool of unblocked GP IIb-IIIa appeared on
the platelet surface after thrombin stimulation and c7E3 incubation.
The conclusion from these experiments is that only part of the internal
pool, which can represent about half of the total pool of GP IIb-IIIa,
was in contact with abciximab at any one time. Quantitatively, the values obtained in flow cytometry in the in vitro experiments were very
close to the results found for patients receiving c7E3 and where
antibody distribution was assessed by computer analysis of labeled
electron micrographs.
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| Fig 8.
Detection by flow cytometry of AP-2 binding to platelets
after their incubation with abciximab for 30 minutes in vitro. In (A),
AP-2 binding to untreated and unstimulated platelets was considered as
100%. This value increased twofold after incubation of the platelets
with 0.5 U/mL thrombin for 10 minutes. In (B), AP-2 binding to
unstimulated platelets was again considered as 100%. AP-2 binding was
severely decreased after a 30-minute preincubation with 10 µg/mL of
abciximab. In (C), AP-2 binding to untreated thrombin-stimulated
platelets was now taken as 100%; when platelets were incubated with
c7E3 Fab before stimulation with thrombin, AP-2 binding was 33.8 ± 10.9% of that obtained in the absence of drug. Tests were performed on
platelets from five donors.
|
|
| |
DISCUSSION |
In this study, we have localized c7E3 Fab fragments within the
different membrane compartments of platelets using two model systems:
(1) platelets from patients receiving abciximab for ~24 hours during
antithrombotic therapy and (2) control platelets given a short-duration
challenge with abciximab in vitro. In view of their small size,
monovalent nature, and therapeutic use, c7E3 Fab are interesting probes
for following GP IIb-IIIa movements in platelets. The initial detection
of c7E3 Fab within the SCCS and on the membrane of some -granules
within 3 hours of the start of the infusion implied rapid trafficking
toward the internal membrane systems, and this was confirmed when
control platelets were incubated for shorter times with abciximab in vitro.
The labeling of the SCCS channels and -granule membranes in the in
vitro studies where uptake was seen as early as 1 minute after the
addition of abciximab suggested a rapid diffusion of c7E3 into the
internal platelet compartments. Zucker-Franklin in a pioneering study
nicely showed that the channels of the SCCS were sites of entry for
exogenous substances.25 When platelets from a patient with
type I Glanzmann's thrombasthenia were incubated with c7E3, only
occasional gold particles were associated with platelets when
ultracryosections were incubated with the anti-c7E3. The absence of
labeling within the SCCS proves that we were detecting bound rather
than free abciximab in the studies on platelets of normal subjects. The
rare gold particles seen on the membrane of -granules of the
patient's platelets may correspond to the labeling of the vitronectin
receptor. This receptor has been found to be present at low density in
the -granule membrane.24 The patient studied has a
homozygous mutation in the IIb
gene,22 thus allowing the vitronectin receptor to be present.
In our studies on platelets of patients receiving an abciximab infusion
or with platelets incubated with abciximab in vitro, we repeatedly
observed the presence of thin channels labeled for abciximab in close
proximity to the -granules. Although the opening of the SCCS into
the external medium has been previously shown,25,26 a
direct interaction between these channels and the membrane of -granules in unstimulated platelets is unknown. The apparent presence on rare sections of a gap in the membrane of an -granule with a channel present in this position lends weight to the hypothesis that the thin channels may provide a facilitated route for transport from the exterior to the granules. The existence of a direct
interaction between granules and the SCCS was hypothesized by
Zucker-Franklin,25 who underlined that modest secretion
from platelets can sometimes occur without detectable morphological
evidence of granule fusion or exocytosis. Recently, "worm-like
structures" containing MoAbs prebound to platelets were visualized
after platelet activation with ADP.27 Such structures may
correspond to the thin SCCS seen here and represent routes between the
surface and the platelet interior.
Performing immunogold labeling on ultrathin cryosections without
dehydration, as in the current study, allows an improved preservation
of platelet ultrastructure, whereas the highlighting of the thin
channels by the gold particles lining them may also help to explain why
we see such structures where others using standard EM have failed.
Fusion of -granule membranes with the intraplatelet channels after
platelet secretion has been deduced from the observed presence of
granule-proteins within the SCCS.28,29 In the cited
studies, the SCCS channels were dilated helping their visualization.
Just as the juxtaposition of the SCCS with granules can facilitate
secretion, it can also help to explain how plasma proteins are able to
rapidly find their way to the granules. The abundance of the SCCS
network spreading through the cytoplasm26 can also be a
factor in this regard. Whereas specific proteins such as gap junction
proteins30 or docking proteins31 are known to
be involved in secretory-dependent fusion processes and endocytosis in
other cells, their involvement in the uptake of ligands through the
SCCS and the formation of points of contact with the granules will be a
matter for further study. Visualization of c7E3 inside the SCCS must be
distinguished from the previously described activation-dependent
translocation of GP IIb-IIIa-bound fibrinogen conjugated to gold
particles or to biotin.32,33 In this situation, fibrinogen
is progressively cleared from the platelet surface to dilated elements
of the SCCS. These processes differ from the uptake described here for
unactivated platelets. The speed at which labeling of the thin channels
occurred would suggest that diffusion or flow of c7E3 fab fragments
into them was followed by binding. The fact that the surface remains
covered with gold particles is not in favor of an unidirectional
translocation of complex-bound c7E3 Fab from the platelet surface.
During in vitro incubations, we observed vesicles containing both
abciximab and clathrin in the intracytoplasmic compartment and
sometimes in close association with -granules. Thus c7E3 may also
reach the membrane of -granules by the endocytic pathway. The
formation of coated pits containing ligand-bound receptors followed by
the formation of endocytic vesicles is a classic pathway for the
internalization of ligands. Behnke first visualized coated vesicles
fusing with -granules when platelets were incubated with either
colloidal thorium dioxide or a colloidal gold suspension.34 Clathrin-coated endocytic vesicles containing adhesive proteins including fibrinogen have since been identified in
platelets.35,36 Their association with Src-related tyrosine
kinases further suggests that they constitute part of an active uptake
process.37 Endocytosis has been previously invoked to
explain the accumulation of immunoglobulin G inside platelets, the IgG
being stored together with fibrinogen and other proteins in the
-granules, although it is unknown whether IgG uptake is receptor
linked.38 Intact murine or human antibodies that bind to GP
IIb-IIIa are known to be internalized by platelets.27,39,40 In one study, where an anti-GP IIb-IIIa MoAb was coupled directly to
gold particles, the latter reached the membranes of -granules although no explanation was provided concerning the mechanism responsible for the internalization.40
Notwithstanding, little evidence was found in our study for
unidirectional transport leading to an eventual surface clearance of
the Fab fragments accompanied by the accumulation of c7E3 in the
-granules either during or after the infusion. A quantitative analysis showed that only about 25% of the gold particles were associated with internal pools during the infusion of abciximab to the
patients and that the labeling of all pools decreased in parallel after
the infusion was stopped. One interpretation of this finding is that
the monovalent Fab fragments predominantly accompany the natural
movements of GP IIb-IIIa complexes to and from the surface. Two small
snake venom peptides, kristin and applagin, have both been shown to
reach the internal compartment of platelets after binding to GP
IIb-IIIa,41,42 and the pool of applagin inside the
platelets was estimated to be 17% of the total platelet-associated
peptide.42 Kristin, like c7E3 Fab, inhibits the uptake and
storage of fibrinogen by platelets when administered in
vivo.41,43 The fact that the surface content of GP IIb-IIIa
was maintained despite the transport of applagin to the -granules
led Wencel Drake et al42 to propose that GP IIb-IIIa
complexes recycle to the platelet surface. Continual exchange of
glycoproteins between external and internal membrane systems in cells
is a well-known phenomenon44 and would explain our
findings. In a recent study, Mascelli et al45 also showed that the surface distribution of c7E3 was homogeneous for the whole
platelet population not only during the infusion but also during the
next 15 days. This is also in favor of a lack of unidirectional uptake
of abciximab into platelets.
Another aspect of our work deserves mention. Abciximab binding was
associated with a greater than 80% inhibition of ADP-induced platelet
aggregation during the infusion, results that were comparable to those
reported in other studies.8,15,46 Nevertheless, despite the
clinical success of abciximab,43,47 our study confirms that
it is difficult to completely inhibit all of the GP IIb-IIIa complexes
in platelets, and residual aggregation was observed after stimulation
of platelets in PRP with TRAP-14 mer peptide. This confirms another
study by us15 for TRAP-14 mer and thrombin and the previous
results of Kleimann et al.14 A twofold increase in the
binding of radiolabeled MoAbs to GP IIb-IIIa after thrombin-induced activation of platelets48,49 is strong evidence that about half of the total population of GP IIb-IIIa complexes are innaccessible to intact IgG in unstimulated platelets. Our current flow cytometry studies with AP-2 strongly agree with this finding. Using radiolabeled 7E3 Fab fragments in direct binding studies, Wagner et al found 92,900 ± 13,100 binding sites on unstimulated
platelet.50 This is much larger than the 35,000 to 50,000 sites as reported in studies with intact MoAbs,50 a result
that can be explained by the fact that each intact antibody in fact
binds to two adjacent molecules of GP IIb-IIIa, although a facilitated
diffusion of the smaller Fab fragments into the SCCS may also in part
account for this high number. Using AP-2, we have shown elsewhere by
immunogold labeling and standard transmission EM that platelets from
patients receiving abciximab express unblocked GP IIb-IIIa at their
surface when stimulated by thrombin.15 Here, we have shown
that following the incubation of platelets in vitro with 10 µg/mL of
c7E3, thrombin stimulation significantly increased the amounts of AP-2
which bound, showing that about 33% of the total pool of GP IIb-IIIa was innaccessible to c7E3. Globally, our results obtained in both in
vitro and in vivo studies show that despite the rapid trafficking of
abciximab into the platelets, not all GP IIb-IIIa complexes participate
in this process and that, at any one time, an appreciable subpopulation
of GP IIb-IIIa is not occupied by c7E3 Fab. As shown by Tcheng et
al8 during studies on the pharmacodynamics of the
inhibition of platelet aggregation by abciximab, the frontier between a
normal and an impaired aggregation response is close and a critical
number of GP IIb-IIIa complexes is needed for platelet aggregation to
occur. Thus, during treatment with abciximab, it is only when more than
80% of the surface pool of GP IIb-IIIa complexes are blocked that
ADP-induced aggregation is inhibited. Our studies imply that with
thrombin, this threshold may be difficult to reach in vivo in the time
scale of the aggregation response. In this situation, the internal
pools of GP IIb-IIIa conserve their own characteristics and are capable
of supporting at least a partial aggregation.This residual response to
thrombin might explain the absence of hemorrhagic syndromes for
patients receiving abciximab when using a patient-adapted dose of
heparin.51 With the development of a range of new compounds
to GP IIb-IIIa, including orally bioavailable prodrugs,52
their access or not to the different intracellular pools of receptors
may well have an important bearing on their efficacity.
| |
FOOTNOTES |
Submitted February 24, 1998; accepted October 22, 1998.
Supported by the CNRS, Université de Bordeaux II (DRED), the
Ministère de l'Enseignement Supérieur et de la Recherche
(ACC-SV No. 9), and Lilly-France. C.P. received doctoral grants from
the GEHT (Synthelabo) and the Société Française
d'Hématologie.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Paquita Nurden, MD, UMR 5533 CNRS, Hôpital Cardiologique, 33604 Pessac, France; e-mail:
Paquita.Nurden{at}cnrshl.u-bordeaux2.fr.
| |
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Top
Abstract
Introduction