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© 1999 American Heart Association, Inc.
Original Contributions |
Expression of Markers of Platelet Activation and the Interpatient Variation in Response to Abciximab
From UMR 5533 CNRS and the Unité des Soins Intensifs (C.D.-J.,
P.C., P.B.), Institut Fédératif de Recherche
"C
ur-Vaisseaux-Thrombose," Hôpital Cardiologique,
Pessac, France.
Correspondence to Alan T. Nurden, Director, UMR 5533 CNRS, Hôpital Cardiologique, 33604 Pessac, France. E-mail alan.nurden{at}cnrshl.u-bordeaux2.fr
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Abstract |
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Abstract—Our study concerns the biological effects of abciximab (c7E3 Fab, ReoPro), a powerful new antiplatelet drug that blocks glycoprotein (GP) IIb-IIIa complexes. Samples were examined from 6 patients with coronary artery disease who received a bolus of abciximab followed by a 10-µg/min infusion for at least 18 hours before percutaneous transluminal coronary angioplasty. Inhibition of ADP-induced PA was maximal for 4 patients but partial (79% and 53%) for 2 others during the infusion. Flow cytometry performed with monoclonal antibodies (PAC-1, AP-6, and F26) specific for the "activated" GP IIb-IIIa complex revealed large decreases in the expression of activation markers on platelets during therapy, but these decreases were less marked when inhibition of ADP-induced PA was incomplete. Residual aggregation was seen for all patients during the infusion when TRAP 14-mer peptide or thrombin was the stimulus. Unblocked GP IIb-IIIa complexes were detected on thrombin-stimulated platelets from the patients by immunoelectron microscopy performed using the monoclonal antibody AP-2. Unblocked GP IIb-IIIa complexes were also detected by flow cytometry when platelets preincubated for 1 hour in vitro with abciximab under saturating conditions were (1) incubated with TRAP 14-mer or (2) permeabilized with Triton X-100. In confirming interpatient variation in the platelet response to a standard dose of abciximab, our results also show that an uninhibited internal pool of GP IIb-IIIa complexes may mediate a residual response to strong agonists.
Key Words: platelet aggregation • activation markers • GP IIb-IIIa complexes • abciximab • arterial thrombosis
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Increased signs of platelet activation have been commonly reported in patients with unstable angina (UA).1 2 3 4 5 Thrombi located in coronary arteries of these patients are white platelet-rich thrombi, therein emphasizing the importance of the platelet contribution to the pathogenesis of UA.6 The occurrence of acute ischemic events during the 24 hours immediately after a high-risk percutaneous revascularization procedure has been related to the platelet activation status measured before the intervention.7 The ex vivo detection of markers localized on the membrane of circulating activated platelets by flow cytometry is a sensitive test designed to indicate the risk of acute cardiac events.7 8 9 10 11 12 13 14 These procedures have been facilitated by the increasing availability of monoclonal antibodies (mAbs) recognizing determinants that are only expressed after platelet activation.15
mAbs that block the function of GP IIb-IIIa (integrin
IIbß3), such as 7E3,
represent a powerful new generation of antithrombotic
agents.16 Administration of abciximab (c7E3 Fab fragments,
ReoPro), a chimeric antibody fragment prepared for in vivo use, is a
potent inhibitor of platelet thrombus formation in
humans.17 The Evaluation of IIb/IIIa Platelet Receptor
Antagonist 7E3 in Preventing Ischemic Complications
(EPIC) and CAPTURE trials, among others, have shown a beneficial
effect of sustained blockade of the glycoprotein (GP)
IIb-IIIa receptor in patients undergoing high-risk
percutaneous transluminal coronary angioplasty
(PTCA), with a reduced rate of acute ischemic
complications.18 19
Our study was designed to investigate the effect of abciximab on the expression of conformation-dependent activation markers on GP IIb-IIIa complexes of platelets of patients with coronary disease. We also evaluated the effect of the drug on platelet aggregation (PA) induced by a range of physiological agonists. Our results showed that flow cytometry permitted the detection of persistent signs of platelet activation in patients in whom inhibition of ADP-induced aggregation was incomplete during the infusion. At the same time, evidence was obtained for the continued presence of unblocked internal pools of GP IIb-IIIa complexes during the infusion that could provide a residual platelet response in the presence of a major hemostatic challenge.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Study Subjects
Studies on 6 adult male patients who underwent PTCA for UA and who received abciximab form the basis of this report. Patients were hospitalized in the coronary care unit of the Hôpital Cardiologique (Pessac, France), each of them exhibiting clinical signs of angina at rest with associated electrical changes. A qualifying coronary angiogram within 48 hours of their most recent episode of myocardial ischemia showed a culprit lesion in a single native coronary vessel suitable for PTCA. Further information on the patients is given in Table 1
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Patients received abciximab as a bolus injection (0.25 mg/kg) followed
by a continuous infusion (10 µg/min) for at least 18 hours (Table 1
). The infusion continued until 1 hour after the completion of
the PTCA, as described for the CAPTURE trial.19 Vascular
sheaths were maintained for at least 24 hours after the end of the
infusion. Each patient received intravenous heparin
adjusted to give an aPTT between 2.0 and 2.5 times normal values.
Heparin was continued for 24 hours (patient 1), 48 hours (patients 4,
5, and 6), or 72 hours (patients 2 and 3) after the completion of PTCA.
Each patient then received subcutaneous calciparin, which continued
over the period of our study. Aspirin (250 mg oral daily) was also
initiated before the abciximab bolus was given. Nitrates, calcium
antagonists, and/or ß-blockers were maintained throughout
the infusion and for varying periods after PTCA. Patients 1, 2, 5, and
6 were enrolled in the CAPTURE trial; the 2 remaining patients were
studied after the trial had ended but were treated similarly. Approval
was obtained from the National Ethics Committee before the onset of the
study.
Murine mAbs Used in This Study
PAC-1, a murine IgM-
mAb, binds to a conformation-dependent
determinant on activated GP IIb-IIIa complexes unoccupied with
adhesive protein.20 21 It was purchased from the
University of Pennsylvania (Philadelphia) through the courtesy of Prof
S. Shattil. AP-6, an IgM, obtained by immunization of mice with the
ß3204–227 peptide, was a gift from Dr T.
Kunicki (La Jolla, Calif). The epitope for AP-6 is a ligand-induced
binding site (LIBS) exposed on GP IIb-IIIa complexes after
agonist-induced fibrinogen binding.22 The
anti–receptor-induced binding site (RIBS) IgG murine mAb, F26,
recognizes receptor-bound fibrinogen on platelets23
and was provided by Dr H. Gralnick (Bethesda, Md). AP-2 (from Dr
Kunicki) is an IgG mAb that reacts with a complex-dependent determinant
common to unactivated and activated GP
IIb-IIIa.24 Platelet secretion was assessed using an
anti–P-selectin mAb, VH10, prepared by us.10
Studies on Platelets From Patients
Blood Sampling
Blood was collected on at least 4 occasions from each patient
(see Figure 1) and 2 anticoagulant
systems were used (see below). The initial samples were always taken
before the bolus injection of abciximab but after the onset of heparin
and aspirin. For all patients, samples were taken near the end of the
infusion and before PTCA. Sampling was continued at intervals for up to
5 days after the infusion. For patients 3 and 5, blood was also
collected 3 hours into the infusion. Initially, blood was withdrawn
from the femoral artery via the vascular sheath used for cardiac
catheterization. After removal of the catheter,
peripheral blood was obtained by clean
venipuncture. The initial 3 mL of blood was always
discarded.
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Aggregation Response in Platelet-Rich Plasma
Blood was anticoagulated with 3.8% (wt/vol) sodium citrate (1
vol:9 vol) and platelet-rich plasma (PRP) prepared by
centrifugation at 120g for 10 minutes at
room temperature.25 Platelets were stimulated
with 0.5 to 8 µmol/L ADP, 0.72 mg/mL arachidonic
acid, 25 µmol/L TRAP 14-mer (SFLLRNPNDKYEPF) purchased from
Neosystem, or 1.5 mg/mL ristocetin. Results were expressed as
percentage light transmission at maximum aggregation. Inhibition was
calculated relative to the response of the sample taken before the
onset of abciximab therapy. On occasion, activated
platelets (see below) were also evaluated using citrated PRP that
had been incubated with 10 µmol/L ADP without stirring.
Aggregation of Washed Platelets by Thrombin and Detection of
Unblocked GP IIb-IIIa Complexes by Immunoelectron Microscopy
Blood samples were collected into acid-citrate-dextrose NIH
formula A (ACD-A) (1 vol anticoagulant: 6 vol blood). Washed
platelets were prepared according to our previously published
procedures.10 Aggregation was tested for each patient at
each time point using 0.5 U/mL human -thrombin (Ortho
Diagnostics). Immunogold staining was performed on
platelets obtained from patients 3 and 4 near the end of the
abciximab infusion to investigate the surface expression of unblocked
pools of GP IIb-IIIa at the time of maximum inhibition of ADP-induced
PA. Here, unstirred suspensions of washed platelets were incubated
with or without 0.5 U/mL -thrombin for 10 minutes before being fixed
and processed for immunogold staining with the mAb AP-2, using
ultrathin cryosections according to the procedures previously detailed
by us.22 Bound AP-2 was located using goat antimouse IgG
adsorbed onto 5-nm gold particles (1:100 dilution, Auroprobe EM GAM G5;
Amersham). Sections were examined in a Jeol JEM-1010 electron
microscope.22
Ex Vivo Detection of Activated Platelets
The procedures for ex vivo detection of activated
platelets have been described previously by us.10
Here, ACD-A was used as anticoagulant because prior studies showed that
it minimizes platelet activation during blood collection and
subsequent experimentation. PRP was prepared at 120g for 10
minutes. Aliquots (10 µL) were immediately added to polystyrene tubes
containing 100 µL 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, and 0.1% (wt/vol) BSA, pH 7.4 (HEPES-buffered modified
Tyrode's, HBMT) and a predetermined concentration of 1 of the
following mAbs: PAC-1 (IgM, 6.25 µg/mL), AP-6 (ascites, 1:1000
vol/vol), F26 (IgG, 5.0 µg/mL), VH10 (IgG, 5.0 µg/mL). Controls
were effected in the absence of primary antibody or using the same
amount of myeloma mouse IgG (or IgM) (Sigma) whose subclass matched
that of the test mAb. Samples were incubated for 15 minutes at room
temperature without stirring and bound antibody was measured using
DTAF-conjugated, affinity-purified, F(ab')2
fragments of donkey anti-mouse IgM (Jackson ImmunoResearch) or
FITC-conjugated affinity-purified F(ab')2
fragments of goat antibody to mouse IgG (Fc
fragment specific,
Jackson ImmunoResearch) as described.10 In vitro
experiments performed by incubating washed platelets (prepared as
above) from normal volunteers with 10 µg/mL abciximab (Lilly-France)
confirmed that the secondary antibodies did not cross-react with
platelet-bound abciximab (c7E3 Fab fragments) in flow cytometry.
Furthermore, the same antibodies did not detect abciximab adsorbed onto
nitrocellulose in a dot-blot assay performed using a chemiluminescence
revelation procedure (data not shown).
Flow Cytometry
After the incubation of the patients' platelets with
antibodies, samples were diluted with 1 mL HBMT and analyzed
using a Becton-Dickinson FACScan flow cytometer as
described.10 25 Fluorescence histograms were
obtained for 10 000 cells, data being analyzed using Lysys II
software (Becton-Dickinson). Histograms were composed from
fluorescence data obtained using a logarithmic mode of
amplification. Antibody binding was expressed as the percentage of
platelets positive for antibody. Mean fluorescence
intensity (MFI, arbitrary units converted to a linear
scale10 ) was a measure of the extent of antibody binding
to individual platelets. The gate for activated
platelets was set so as to include <1% of the events seen when
identical platelet samples were incubated with the control murine
immunoglobulin (IgM or IgG) used at the same concentration as the
murine mAb. Percentages of activated platelets in PRP
prepared from ACD-A–anticoagulated blood taken from 10 age-matched
healthy and medication-free control subjects, given as mean±SD
(range), were as follows: F26, 2±0.7 (0.8 to 3.4); PAC-1, 4.7±3 (1.3
to 9.3); AP-6, 1.1±0.3 (0.1 to 2); and VH10, 3.8±2.4 (1.7 to
9.2).
Confirmation that Unblocked Internal Pools of GP IIb-IIIa Can Be
Detected by AP-2 in the Presence of Abciximab
Here, we made use of our previous observation that platelets
saturated with abciximab fail to bind the mAb AP-2.24 26
Citrated whole blood from control donors was incubated for 1 hour at
37°C in the presence of 5 or 10 µg/mL abciximab. PRP was prepared
and aliquots were incubated with or without 25 µmol/L TRAP
14-mer peptide or 10 µmol/L ADP in the presence of AP-2 (ascites
1:200 vol/vol) or VH10 (as above). After 15 minutes, FITC-conjugated
affinity-purified F(ab')2 fragments of goat
antibody to mouse IgG (Fc fragment specific) were added and the
samples processed for flow cytometry as described in the previous
section. Second, selected samples were fixed with 1% (wt/vol)
paraformaldehyde (PFA), permeabilized
or not with 1% wt/vol Triton X-100, and the accessibility of the
internal pools of GP IIb-IIIa to AP-2 was determined by flow cytometry
as in an earlier study.27
Statistics
Data for PA testing were expressed as mean±SD. The mean values
were compared by paired Student's t test. The mean
percentages of activated platelets were analyzed by
ANOVA using the Kruskal-Wallis nonparametric test.
Wilcoxon's test was used when appropriate to compare the
variation of values as a function of time.
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Results |
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Clinically, each PTCA was successful. None of the patients presented ischemic chest pain after the procedure. Creatinine kinase (CK-MB) isozyme levels were not seen to rise after PTCA. No undue bleeding was observed.
Platelet Aggregation
Before the bolus injection of abciximab, PA with ADP was, as
expected for subjects receiving aspirin, often reversible (particularly
for 2 and 4 µmol/L ADP). Testing of samples taken after the
bolus and 3 hours into the infusion (patients 3 and 5) confirmed that
near-maximal inhibition of PA had already been achieved (Figure 1). ADP-induced PA at all doses tested was virtually totally
inhibited (>98%) for patients 1, 2, and 3 at time points of 12 hours
or more into the abciximab infusion. However, patient 6 showed a
significant residual and irreversible aggregation to 4 µmol/L
ADP. Even at the lowest dose of ADP, complete inhibition was not
observed for this patient. Patients 4 and 5 showed a somewhat
intermediate profile. Considerable intersubject variation was also seen
during the recovery of ADP-induced PA after abciximab infusion had
ended. No correlations were found between the extent of the inhibition
of PA and the platelet count.
The intensity of platelet agglutination with ristocetin was little
changed in patients receiving abciximab (Table 2). Notwithstanding, it was sometimes
reversible during abciximab infusion (data not illustrated). PA with
arachidonic acid was minimal, as expected for patients
receiving aspirin. PA was induced by way of the thrombin receptor,
using 25 µmol/L TRAP 14-mer peptide, the minimum dose that
regularly gives full-scale aggregation with citrated PRP in our
laboratory. Residual platelet responses to TRAP were seen during
the infusion for all patients and ranged from 28% to 71% of
pretreatment levels (Table 2). When the aggregation of washed
platelets was examined with 0.1 U/mL thrombin, residual PA was
again seen for samples taken from all patients during the infusion
(Table 2).
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Activation-Induced Expression of Unoccupied GP IIb-IIIa Receptors
From the Internal Pool
Washed platelets prepared from blood collected from patients 3
and 4 near the end of the abciximab infusion were incubated with
thrombin to give maximum secretion. After fixation, ultrathin
cryosections were incubated with AP-2 and bound mAb was visualized by
immunogold labeling in electron microscopy. Results are shown for
patient 3 in Figure 2. Whereas labeling
with AP-2 was abundant on the surface of platelets taken before the
onset of abciximab (A), it was much reduced by the end of the infusion
(B). Yet, after platelets in the same sample had been stimulated
with thrombin, gold beads representing bound antibody were
again readily detected at the surface (C). The platelet illustrated
in C has a long pseudopod extending back to the surface with
considerable labeling. We conclude that unblocked GP IIb-IIIa complexes
are made available at the platelet surface after thrombin
stimulation. Similar results were obtained for patient 4.
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Experiments were continued using normal subjects' citrated PRP
preincubated with abciximab in vitro. As shown in Figure 3, flow cytometry confirmed that
abciximab blocked the binding of AP-2 to the surface of unstimulated
platelets (compare A and C). In contrast, TRAP 14-mer induced the
appearance of a subpopulation of GP IIb-IIIa complexes able to bind
AP-2 (Figure 3). The normal appearance of P-selectin (B and D)
confirmed that abciximab had little effect on secretion at this
concentration of TRAP. Little increase in AP-2 binding was seen after
the addition of 10 µmol/L ADP (data not shown). Finally, we
looked at the binding of AP-2 to PFA-fixed, Triton
X-100–permeabilized platelets. Samples were again
volumes of citrated PRP preincubated for 1 hour with abciximab. Typical
results are shown in Figure 3 (E and F). As can be seen, a
pool of AP-2 binding sites is exposed after permeabilization, which
renders accessible the internal receptor pools of platelets (see
Reference 2727 ). We point out that in the absence of abciximab, the flow
cytometer calibration did not permit a separation of the histograms,
representing the high-density surface pool of unblocked GP
IIb-IIIa recognized by AP-2 (MFI=549 in the experiment shown in A) and
the increased surface expression seen after TRAP stimulation
(MFI=605).
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Detection of Circulating Activated Platelets
Initial experiments confirmed that abciximab brought about a
dose-dependent inhibition of AP-6 and PAC-1 binding when control
platelets were stimulated with ADP in vitro. Inhibition was
complete at 10 µg/mL, showing that c7E3 Fab either masked the
activation-dependent epitopes on GP IIb-IIIa or prevented their
formation (data not shown). The binding of abciximab to GP IIb-IIIa did
not itself induce the expression of the LIBS epitope recognized by
AP-6. Abciximab did not interfere with VH10 binding to P-selectin (data
not shown).
Assessment of Platelet Activation Markers During and After
Abciximab Infusion
As shown in Figure 4, the percentage
of platelets positive for 2 or more of the mAbs directed against
activation-dependent markers on GP IIb-IIIa was elevated for 4 patients
before the addition of abciximab, whereas for patients 2 and 4 the
results were at the upper limit of the range observed for age-matched
healthy controls. As shown in Figure 4, abciximab therapy had a
marked effect on these levels and the trend was toward reduction. The
highest number of activated platelets detected before the
bolus injection was from patient 6. For this patient, even 15 hours
after the onset of the abciximab infusion, 47% and 13% of
platelets remained positive with PAC-1 and AP-6, respectively.
Interestingly, this was the patient with the highest residual
aggregation response to ADP. Results were not always concordant for all
3 antibodies (see patients 1, 2, and 5). The reason for this
variability is unknown but confirms the value of testing a series of
mAbs in studies such as these. Taking the patients as a group, the
decrease in levels of activated platelets in the samples
taken shortly before the PTCA reached statistical significance with
respect to those present before abciximab for F26
(P<0.001) and AP-6 (P<0.05). Results for PAC-1
(P<0.06) did not, owing to the fact that platelets of 3
patients failed to give elevated values for this antibody before the
onset of abciximab.
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Platelet P-selectin expression as measured using VH10 was low in patients 1 through 4 (<10%) both before and during abciximab infusion (data not illustrated). In contrast, the percentage of platelets that had undergone secretion and that were recognized by VH10 were elevated in patient 6 both before abciximab administration (23%) and at 15 hours into therapy (41%). For patient 5, 14% of platelets were recognized by VH10 at 21 hours into the infusion. Nevertheless, mean changes in P-selectin levels did not reach statistical significance within the infusion period.
Extent of the Observed Activation
The heterogeneity in the extent to which
individual platelets within the total platelet population bind
the activation-dependent mAbs can be considerable. This is illustrated
in histograms shown for patient 3 in Figure 5. Levels of bound AP-6 and F26 differed
widely within the activated subpopulation observed before
abciximab therapy was started; however, the MFI values had both fallen
to baseline levels just before the PTCA. Also shown are histograms
obtained after PRP from blood collected into citrate was
activated in vitro with ADP. Now the expression of the
activation-dependent epitopes was complete. This selected result
emphasizes how the levels of platelet activation seen in vivo are
often partial. As a general rule in our study, a fall in the number of
activated platelets was accompanied by a decrease in the
MFI (data not given). This finding suggests that measuring numbers of
activated platelets indeed reflects the extent of the
hemostatic challenge.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Our results highlight interpatient variability in biological tests performed during and after the administration of abciximab to patients with coronary artery disease and selected for PTCA. The six patients studied received abciximab administered according to the CAPTURE guidelines. Platelet activation is known to occur episodically in coronary syndromes, and the pre-abciximab levels seen here confirm this trend and ongoing platelet reactivity before the onset of therapy. The pretherapy values obtained for both the PA response and the numbers of activated platelets served as a reference for subsequent measurements on the same patient. The initial levels of activated platelets were globally much greater for the patients than those seen for healthy age-matched control donors not taking aspirin. Although aspirin prevents thromboxane production in platelets, our results confirm that aspirin treatment clearly does not stop platelet activation in patients with coronary artery disease. Thus, at the time that the abciximab bolus was administered platelet activation was occurring in at least 4 of the patients. Not least, our study highlights that by performing platelet function testing and/or measuring platelet activation levels before and during therapy, it is possible to identify cases in which the inhibitory effect of abciximab is less than total.
It is interesting to speculate as to why patient 6 appeared to be more resistant to the effects of abciximab, although it cannot be excluded that inhibition was maximal early in the therapy and that aggregation to ADP had begun to recover by 15 hours (despite the continued infusion and the fact that for patients 3 and 5 the inhibition seen at 3 hours was maintained at 19 hours and 21 hours, respectively). One possibility is that a significant number of activated GP IIb-IIIa complexes are occupied by adhesive protein and unable to bind abciximab. This hypothesis would fit with a continued recognition of some platelets by the anti-RIBS and anti-LIBS antibodies. Previous studies on patients with UA have demonstrated marked heterogeneity in the extent and duration of inhibition of PA after a single bolus injection of this drug.17 28 A preliminary report has described how residual platelet function in the presence of a GP IIb-IIIa inhibitor varies with respect to the coronary disease state and the level of platelet activation.29 Patient 6 confirms that this is so and points to the personalization of the abciximab regimen for the benefit of individual patients. It has previously been described that thrombocytosis can lead to a decreased efficiency of abciximab.30 However, patient 6 had a platelet count between 139 000/µL pretreatment and 142 000/µL 12 hours postinfusion, whereas binding studies with AP-2 performed during a post-PTCA checkup showed a normal GP IIb-IIIa density on his platelets (data not given). A recent investigation demonstrated an association between the PlA2 polymorphism of the GP IIIa gene and the risk for acute coronary thrombosis.31 We considered that patient 6 may possess the PlA2 allele. However, analysis of his PlA genotype in our laboratory by molecular biology procedures32 revealed that he is homozygous for PlA1 (A.T.N., unpublished data, 1998), so this is not the explanation. Notwithstanding, it is plausible that occasional patients may possess GP IIb-IIIa complexes in which structural differences mean that abciximab binds with an altered affinity. A much larger series of patients will now have to be studied to assess the frequency of situations in which, as for patient 6, significant levels of platelet activation and a residual PA response to ADP also continued during the abciximab infusion.
Kleiman et al28 reported that PA in response to TRAP was
significantly less inhibited after a bolus injection of 0.25 mg/kg
abciximab than PA induced by ADP. Our data substantiate this report and
show that this was also the case for the patients that we have studied
despite the additional 10 µg/mL post–bolus infusion. Using
platelets coated with another anti–GP IIb-IIIa, Niiya et
al33 showed that subsequent stimulation with 0.1 U/mL
thrombin permitted the binding of 39 000 fibrinogen molecules per
platelet as a result of the activation-dependent expression of GP
IIb-IIIa complexes from within the platelet. We therefore
considered it highly probable that the residual PA to TRAP observed
during abciximab infusion was mediated by GP IIb-IIIa receptors coming
from an internal platelet compartment. To prove this, we made use
of the fact that abciximab inhibits the binding to GP IIb-IIIa of
another mAb, AP-2.26 Three lines of evidence supported our
theory. First, a regular surface labeling by AP-2 was located by
immunoelectron microscopy after thrombin stimulation of platelets
isolated from patients near the end of the abciximab infusion. Then,
using flow cytometry and platelets preincubated with abciximab in
vitro under saturating conditions, AP-2 was shown to detect (1)
unblocked GP IIb-IIIa newly exposed after TRAP 14-mer stimulation and
(2) an internal pool of unblocked GP IIb-IIIa complexes in
platelets permeabilized with Triton X-100. Whether
these complexes can be expressed on the surface occupied with
fibrinogen from the -granules22 has to be determined.
Other experiments confirmed that under these conditions, aspirin had
little effect on thrombin-induced secretion from platelets (data
not shown)
Bhattacharya et al34 reported that patients receiving a
single dose of 0.25 mg/kg of abciximab showed a partial recovery (50%
of baseline) of ADP-induced PA 4 hours postinjection. Tcheng et
al35 emphasized donor variability in the recovery of
ADP-PA after treatment in studies performed on 11 patients who received
a bolus combined with abciximab infusion during 12 hours.
Significantly, posttreatment heterogeneity was less
when abciximab was given as a bolus combined with a 36-hour infusion.
In our study, in which the infusion was for up to 24 hours, the
recovery of ADP-induced PA was delayed beyond 24 hours for patients 1
and 2 but was more rapid for patients 3 through 6. Among the factors
that may influence the recovery rate are (1) variations in plasma
levels of free abciximab, (2) the rate at which surface-bound abciximab
dissociates from circulating platelets,36 and (3) the
extent to which GP IIb-IIIa receptors exchange between surface and
internal pools.37 As a separate part of our study, we
examined the distribution of abciximab within the different
platelet membrane systems at each time point by immunogold staining
using a rabbit antibody specific for c7E3 (provided by Drs R. Jordan
and C. Wagner, Centocor, Malvern, Pa). The results indicated that
abciximab gains access to the internal membrane pools of GP IIb-IIIa
both through thin channels of the surface-connected
canalicular system and by endocytosis but that there is
not a continued accumulation and storage of the antibody within
-granules, as is observed for fibrinogen (P.N., unpublished data,
1998, and Reference 3838 ). Thus, it is probable that recycling of
GP IIb-IIIa receptors within platelets is continuous and that this
recycling includes complexes both occupied and unoccupied by
abciximab. Furthermore, as shown by Wagner et al,36
abciximab also exchanges from one platelet to another through
dissociation and reassociation. All of these factors probably
contribute to the heterogeneity in the recovery rate.
Measuring P-selectin expression was a less sensitive measure of platelet activation in our study. Nevertheless, the binding of a mAb to this marker was significantly elevated in patients 5 and 6 during the infusion. Previously, high levels of platelets recognized by an anti–P-selectin mAb have been shown to be associated with a higher thrombotic risk during PTCA.7 We have ourselves shown in a much larger series of patients undergoing angioplasty (but without abciximab) that activated platelets expressing P-selectin were maximal at around 24 to 48 hours after the PTCA.39 This finding confirms that angioplasty itself can have an influence on platelet reactivity. Whether the inhibition of delayed PTCA-induced platelet activation would reduce the risk of thrombosis and/or restenosis is an interesting point for further study. Such a delayed response also shows that the onset of heparin therapy (before the PTCA) is not the primary factor initiating platelet activation in these patients, although both heparin (or calciparin) and other medications received by UA patients (nitrates, calcium antagonists, and/or ß-blockers) may influence the recovery rate of PA and the levels of activated platelets observed before and after the removal of the obstruction.14 40
Logic implies that inhibition of GP IIb-IIIa should be optimal before PTCA-induced vessel injury and extend throughout the period when the injured vessel wall is at its most thrombogenic. Limiting the initial platelet-to-platelet interactions through abciximab may also reduce the expression of the internal pool of GP IIb-IIIa complexes on the surface of platelets, a process that also occurs within the aggregate and can help bring fibrinogen onto the surface and thereby promote platelet thrombogenicity.22 41 The finding by Reverter et al42 that abciximab has a dampening effect on platelet-mediated thrombin generation may also help explain the reduced signs of platelet activation during therapy. Finally, our studies highlight a major difference in the functional response between platelets from patients receiving abciximab and those from patients with Glanzmann's thrombasthenia. In the latter, an inherited GP IIb-IIIa deficiency extends to all membrane systems,43 whereas here we have provided evidence for a pool of functional GP IIb-IIIa complexes that can be expressed after platelet stimulation with TRAP 14-mer or thrombin. Quite simply, it appears that despite the infusion, free abciximab levels are insufficient to inhibit the functioning of internal pools in the face of a strong hemostatic challenge. Whether this limits the efficacy of abciximab or provides a protective backup to limit the tendency for hemorrhage is another important question to resolve.
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Acknowledgments |
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This work was financed by funding from the CNRS, Université de Bordeaux II (DRED), the Conseil Régional d'Aquitaine, the Ministère de l'Enseignement Supérieur et de la Recherche (ACC-SV No. 9), and Lilly-France. AP-6 and AP-2 were gifts from Dr T. Kunicki, La Jolla, Calif. The F26 mAb was provided by Dr H. Gralnick, Bethesda, Md, and the c7E3-specific rabbit antibody was provided by Drs R. Jordan and C. Wagner, Centocor, Malvern, Pa.
Received February 2, 1998; accepted June 2, 1998.
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