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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Thrombocytopenia after second exposure to abciximab is caused by antibodies
that recognize abciximab-coated platelets
Brian R. Curtis, Julia Swyers, Ajit Divgi, Janice G. McFarland, and Richard H. Aster
Thrombocytopenia, often severe, occurs
in 1% to 2% of patients given the fibrinogen receptor antagonist abciximab, a chimeric Fab fragment containing murine
specificity-determining and human framework sequences. The cause of this complication has not yet been defined. Studies
of 9 patients who developed profound
thrombocytopenia (platelets <10 ⴛ 109/L
[10 000/␮L]) within a few hours of being
given abciximab a second time showed
that each had a strong immunoglobulin G
(IgG) antibody that recognized platelets
sensitized with abciximab. Five patients
also had IgM antibodies. IgG antibodies
reactive with abciximab-coated platelets
were also found in 77 (74%) of 104 healthy
subjects. However, the patient antibodies
could be distinguished from “normal”
ones in 2 ways: (1) only the patient antibodies reacted preferentially with platelets sensitized with the intact monoclonal
antibody 7E3 from which the murine sequences in abciximab are derived; and
(2) the “normal” antibodies could be inhibited by Fab fragments derived from normal human IgG, whereas the patient antibodies were relatively resistant to this
treatment. The findings suggest that antibodies from the patients are specific for
murine sequences in abciximab and are
capable of causing life-threatening throm-
bocytopenia upon injection of this drug.
The antibodies commonly found in
healthy subjects are specific for the papain cleavage site of any Fab fragments
and, although they react with abciximabcoated platelets, appear not to cause significant thrombocytopenia. It may be possible to identify patients at risk for
developing thrombocytopenia if given abciximab by screening for antibodies that
recognize 7E3-coated platelets. (Blood.
2002;99:2054-2059)
© 2002 by The American Society of Hematology
Introduction
Agents specific for platelet fibrinogen receptor (GPIIb/IIIa, ␣IIb/␤3
integrin) that block fibrinogen binding (fibrinogen receptor antagonists) offer a promising new approach to antithrombotic therapy.1-3
Abciximab, a chimeric Fab fragment containing specificitydetermining sequences from a murine monoclonal antibody (7E3)
specific for GPIIb/IIIa,3-5 has been shown to reduce significantly
the incidence of urgent repeat percutaneous coronary intervention
and coronary artery bypass surgery in patients undergoing percutaneous transluminal coronary angioplasty (PTCA)2,6-8 and was the
first fibrinogen receptor antagonist to be approved for clinical use.
In each trial of abciximab and in subsequent clinical experience,
about 0.5% to 1.0% of patients experienced severe (platelets
⬍ 50 ⫻ 109/L [50 000/␮L]) or profound (platelets ⬍ 20 ⫻ 109/L
[20 000/␮L]) thrombocytopenia within a few hours of receiving the
drug, usually given in the form of a bolus infusion followed by a
12-hour intravenous drip.9-11 In patients given abciximab a second
time, thrombocytopenia occurred about 4 times as often as after
first exposure12,13 and more often when the interval between
exposures was only 2 to 3 weeks.12 Severe thrombocytopenia can
have serious consequences when the function of platelets remaining in the circulation is impaired by a fibrinogen receptor antagonist.
The pathogenesis of abciximab-associated thrombocytopenia is
not understood. Its acute onset and its higher incidence in patients
exposed to the drug a second time suggests that antibodies may be
responsible, but there is no published evidence to support this
possibility, and alternative, nonimmune mechanisms have been
proposed to explain this complication.14,15 We studied 9 patients
who developed severe or profound thrombocytopenia after being
given abciximab a second time and obtained evidence that antibodies, presumably induced by first exposure to this drug, were the
cause of platelet destruction in these individuals.
From the Blood Research Institute and Diagnostic Laboratories, The Blood
Center of Southeastern Wisconsin; Departments of Medicine and Pathology,
Medical College of Wisconsin; and Department of Medicine, St Luke’s Medical
Center; Milwaukee, WI.
Reprints: Brian Curtis, Technical Director, Platelet/Neutrophil Immunology
Laboratory, The Blood Center of Southeastern Wisconsin, PO Box 2178,
Milwaukee, WI 53201-2178; e-mail: [email protected].
Submitted May 29, 2001; accepted November 2, 2001.
Supported by grants HL-13629 and HL-44612 from the National Heart, Lung,
and Blood Institute and by a grant from Centocor, Malvern, PA.
2054
Materials and methods
Platelet isolation
Platelets were isolated from blood anticoagulated with acid citrate dextrose
anticoagulant and were suspended in isotonic, phosphate-buffered NaCl,
pH 6.6, containing 0.2% bovine serum albumin (phosphate-buffered saline
[PBS]) as described previously.16
Reactions of antibodies with abciximab-coated platelets
Abciximab was added to washed platelets suspended in PBS at a ratio of 0.7
␮g abciximab per 1.6 ⫻ 107 platelets and was incubated for 20 minutes at
room temperature. By measuring bound abciximab with a fluorescein
isothiocyanate (FITC)–labeled goat antihuman immunoglobulin G (IgG)
(H⫹L) chain reagent (Jackson Immunoresearch Laboratories, West Grove,
PA) it was found that available GPIIb/IIIa receptors are saturated with
abciximab under these conditions. The sensitized platelets were washed
once in PBS and were suspended in PBS at a concentration of 1.6 ⫻ 109/L
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
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BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
ABCIXIMAB-INDUCED THROMBOCYTOPENIA
(1.6 ⫻ 106/␮L). Ten-microliter aliquots of the sensitized platelets were
added to wells of a 96-well polypropylene V-bottom microtiter plate (Nalge
Nunc International, Naperville, IL) Forty microliters of patient serum was
added to each well, and the mixture was incubated for 30 minutes at room
temperature. A total of 150 ␮L PBS, pH 7.2, was then added to each well,
and the trays were centrifuged at 2200 rpm in a swinging bucket rotor. The
supernatants were removed by inverting the plates and tapping gently on
absorbent paper. The platelet buttons were then washed twice with 200 ␮L
PBS. To each button was added 100 ␮L of a 1:100 dilution of FITC-labeled
goat F(ab⬘)2 antibody specific for human IgG or IgM Fc domains (Jackson
Immunoresearch Laboratories). The platelet buttons were resuspended in
this reagent and incubated for 20 minutes at room temperature in the dark. A
total of 100 ␮L PBS was then added, and the mixture was centrifuged for 3
minutes at 2200 rpm. The supernatants were decanted, and the buttons were
resuspended in 150 ␮L PBS and transferred to tubes containing 0.5 mL PBS
for flow cytometric analysis. Platelet fluorescence intensity was recorded in
a FACSCalibur analyzer (Becton Dickinson, Mountain View, CA) as
previously described.16,17 A positive reaction was defined as a mean platelet
fluorescence intensity (MFI) at least twice that of platelets processed
identically except for the absence of abciximab. This value always
exceeded the mean obtained with PBS or negative control serum by at least
3.0 SD. In preliminary studies, it was found that the FITC-labeled
secondary probes (antihuman IgG or IgM Fc) do not recognize platelets
sensitized with abciximab alone.
Reactions of patient antibodies with platelets sensitized with
GPIIb/IIIa-specific murine monoclonal antibodies
IgG1 murine monoclonal antibodies AP3 specific for GPIIIa (from P. J.
Newman, Blood Research Institute) and AP2 specific for the GPIIb/IIIa
complex (from T. J. Kunicki, Scripps Institute, La Jolla, CA) were produced
by the Monoclonal Antibody Core Laboratory of the Blood Research
Institute. The IgG1 monoclonal 7E3 specific for the GPIIb/IIIa complex and
the source of variable region sequences used to create abciximab4 was a gift
from Centocor. Washed platelets in PBS at a concentration of 1.6 ⫻ 109/L
(1.6 ⫻ 106/␮L) were incubated with AP3 (30 ␮g/mL), AP2 (30 ␮g/␮L), or
7E3 (20 ␮g/mL) for 20 minutes at room temperature. In preliminary studies
using FITC-labeled goat antimouse IgG (Jackson Immunoresearch Laboratories), it was found that these quantities of the monoclonals were sufficient
to saturate available GPIIb/IIIa sites. The platelets were washed once in
PBS and were resuspended in PBS at a concentration of 1.6 ⫻ 109/L
(1.6 ⫻ 106/␮L). Patient and normal serum samples were incubated with
25% (final concentration) normal mouse serum (Sigma St. Louis, MO) for
20 minutes at room temperature to neutralize naturally occurring antibodies
reactive with mouse IgG18-20 that were present in some samples. Binding of
human antibodies to platelets coated with 7E3, AP2, or AP3 was determined
by the same protocol used with abciximab-coated platelets.
2055
Results
Clinical observations
Blood samples from 9 patients (6 men and 3 women aged 41 to 75
years, median 60 years) who developed acute, profound thrombocytopenia after being given abciximab a second time were studied
(Table 1). Each patient had undergone a second PTCA procedure
with or without stent implantation for recurrence of coronary
symptoms 2 to 15 weeks (median 3 weeks) after the first procedure.
Patient 5 was known to have had a 30% decrease in platelets after
first exposure to abciximab; the other patients did not experience a
significant drop in platelet level after first exposure to the drug. All
patients were given heparin before and for various times after each
procedure. The platelet count was normal or near normal in all
cases before treatment.
Thrombocytopenia was detected within 12 hours of the time
abciximab infusion was begun (median 4 hours) and was profound,
the platelet count nadir being 6 ⫻ 109/L (6000/␮L) or less in all
cases (Table 1). Two patients (nos. 1 and 9) exhibited symptoms of
hypersensitivity soon after abciximab infusion was begun. Patient
no. 1 experienced nausea, vomiting, and a drop in blood pressure
after about 15 minutes, at which time the infusion was discontinued. For the next 3 days, she had gastrointestinal bleeding and
required red cell transfusions. Patient no. 9 had a frank anaphylactic reaction after about 30 minutes of infusion that was treated
successfully with supportive measures. However, mechanical ventilation was required for several days because of pulmonary
dysfunction resulting from intrapulmonary hemorrhage. The other
7 patients had less severe bleeding symptoms, ranging from
extensive petechial hemorrhages to oozing from venipuncture sites
(Table 1).
All patients were given platelet transfusions, and 3 (nos. 1, 5,
and 9) also received intravenous ␥-globulin. Three patients (nos. 5,
6, and 7) achieved satisfactory increases in platelet levels after
platelets were given. Two (nos. 3 and 4) responded only transiently
and became profoundly thrombocytopenic again within 24 hours.
No significant posttransfusion platelet increments were achieved in
patient nos. 1 and 9 on repeated occasions. Seven patients achieved
a platelet level of at least 100 ⫻ 109/L (100 000/␮L) within 4 to 7
days. In patient nos. 1 and 9, recovery occurred after 1 week.
Table 1. Clinical findings
Patient
no.
Age/sex
Interval
between
abciximab
exposures, wk
1
53/F
3
Platelet level
Pretreatment
Nadir
“Normal”
1 ⫻ 109/L
Days
required to
reach 100
⫻ 109/L
9
Bleeding symptoms
Petechiae, ecchymoses, gastrointestinal
bleeding
Other
Emesis and hypotension 15 minutes
after infusion was started,
abciximab stopped
2
75/F
15
190 ⫻ 109/L
6 ⫻ 109/L
⬎3
Petechiae, oozing from venipuncture sites
3
45/F
2
“Normal”
4 ⫻ 109/L
⬍5
Petechiae, oozing from venipuncture sites
4
60/M
3
“Normal”
1 ⫻ 109/L
4
Petechiae
5
63/M
3
142 ⫻ 109/L
1 ⫻ 109/L
5
Petechiae, oozing from venipuncture sites
Had drop in platelets from 199 ⫻
109/L to 139 ⫻ 109/L after first
treatment
6
48/M
2
139 ⫻ 109/L
6 ⫻ 109/L
6
Petechiae, ecchymoses
7
41/M
6
239 ⫻ 109/L
5 ⫻ 109/L
4
Petechiae
8
55/M
2
205 ⫻ 109/L
3 ⫻ 109/L
7
Petechiae, ecchymoses
9
60/M
4
150 ⫻ 109/L
2 ⫻ 109/L
8
Petechia, oozing from venipuncture sites,
intrapulmonary hemorrhage
Anaphylactic reaction, required
mechanical ventilation
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2056
BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
CURTIS et al
Table 2. Laboratory findings
Patient no.
Sample
MFI
(IgG)
1
Posttreatment†
502
160
2
Pretreatment
82
16
0.2
Posttreatment
68
NT
⫺1.1
3
Pretreatment
102
32
14.1
Posttreatment
75
NT
NT
4
Posttreatment
331
640
340
5
Posttreatment
102
32
5.6
NT
6
Posttreatment
199
64
5.2
NT
7
Posttreatment
201
64
3.5
NT
8
Posttreatment
112
32
20.6
4
9
Pretreatment
374
NT
16.4
NT
106
16
Posttreatment
Negative control
0.0 ⫾ 1.5
Titer
(IgG)*
MFI
(IgM)
396
Titer
(IgM)*
40
NT
was even stronger than the one found in the posttreatment sample.
The drop in antibody strength after starting abciximab was
especially pronounced in patient no. 9 (Table 2). IgG antibody titers
in the posttreatment samples (obtained within 24 hours of the onset
of thrombocytopenia) ranged from 1:16 (patient no. 2) to 1:640
(patient no. 4).
2
40
3.9
0.0 ⫾ 1.4
MFI indicates MFI obtained with abciximab-coated platelets minus that obtained
with uncoated platelets (arbitrary fluorescence units); and NT, not tested.
*Reciprocal of the dilution yielding a signal at least twice that obtained with
unsensitized platelets.
†Posttreatment samples were drawn within 24 hours of the onset of thrombocytopenia.
Because all patients were treated with unfractionated heparin
during and for various periods of time after PTCA, heparin-induced
thrombocytopenia was suspected in some cases. However, only
serum from patient no. 6 gave positive results in a solid-phase
enzyme-linked immunosorbent assay test in which complexes of
heparin and platelet factor 4 are used as targets for antibody
detection.21 Although this assay is not absolutely specific for
heparin-induced thrombocytopenia, it is extremely sensitive and a
negative test argues strongly against that diagnosis.21-23
Antibodies reactive with abciximab-coated platelets were also
found in serum from healthy individuals
IgG antibodies reactive at least weakly with abciximab-coated
platelets were also found in 77 (74%) of 104 of serum samples from
104 randomly selected healthy subjects (Figure 2). Two of the 77
also had weak IgM antibodies. In general, antibody activity found
in normal serum was weaker than that found in patients. However,
a few of the “normal” antibodies were as strong or stronger than
those from the patients with abciximab-associated thrombocytopenia (Figure 3).
The patient antibodies could be distinguished from those
found in healthy subjects in 2 ways
Many healthy individuals have “naturally occurring” antibodies
that recognize the C-terminus (papain cleavage site) of Fab
Each patient had an antibody that reacted with platelets
sensitized with abciximab
When tested by flow cytometry, each patient’s serum was found to
contain IgG antibodies reactive with abciximab-coated platelets
(Table 2). Representative histograms are shown in Figure 1. Patient
nos. 1 and 4 also had strong IgM antibodies, and patient nos. 3, 8,
and 9 had weaker ones. Very weak IgM antibodies were detected in
patient nos. 5, 6, and 7. Pretreatment samples were available from
patient nos. 2, 3, and 9. In each case, an antibody was detected that
Figure 1. Reactions of patient sera against platelets sensitized with abciximab.
The strong IgG antibody from patient no. 1 (A) and the weaker one from patient no. 2
(B) reacted with abciximab-coated platelets but not with uncoated platelets.
Figure 2. Reactions of serum from 104 unselected healthy subjects against
abciximab-coated platelets. Values shown are MFI values obtained with abciximabcoated platelets minus the MFI obtained with uncoated platelets. A difference of 5.0
MFI units or more, indicating that the signal against abciximab-coated platelets was
at least twice that obtained with uncoated platelets, was considered indicative of
significant binding of IgG to the sensitized platelets. By this criterion, antibodies were
present in 77 subjects (74%).
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BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
ABCIXIMAB-INDUCED THROMBOCYTOPENIA
2057
Figure 3. Reactions of patient antibodies (left) and antibodies in 104 healthy
subjects (right) against abciximab-coated platelets. Values shown are MFI
obtained with abciximab-coated platelets minus that obtained with uncoated platelets. Brackets depict mean ⫾ 1 SD.
fragments prepared from normal human IgG by digestion with
papain.24-26 Because abciximab is produced by papain cleavage of a
hybrid IgG molecule containing human IgG1 constant domains and
murine (7E3) variable sequences,26 it seemed possible that either
the patient or normal antibodies were specific for the papain
cleavage site of abciximab. We therefore determined whether the
antibodies identified in patients and healthy subjects could be
inhibited by Fab fragments prepared from normal IgG. For these
studies, antibody-containing sera, diluted to a point at which they
produced a mean signal of about 25 arbitrary fluorescence units
when tested against abciximab-coated platelets, were incubated
with pooled normal human Fab fragments at a concentration of
500 ␮g/mL for 60 minutes before testing. As shown in Figure 4,
antibodies from 13 healthy subjects were inhibited by 70% to 98%
(average 84.7%) by this treatment. In contrast, antibodies from the
9 patients were inhibited by 0% to 83% (average 36.3%). Two of
the diluted patient antibodies (patients no. 4 and 7) behaved like
antibodies from the healthy subjects, being inhibited by more than
80%, but the other 7 were only minimally affected. These findings
suggested that the major antibody activity in patients no. 4 and 7
and in each of the 13 healthy subjects tested were mainly specific
for IgG Fab fragments, whereas those present in the other 7 patients
recognized a different target on abciximab or on GPIIb/IIIa.
It was then determined whether antibodies not inhibited by
normal Fab fragments might recognize epitopes expressed on
murine (Ig variable) sequences in abciximab by comparing the
reactions of patient and normal antibodies against platelets saturated with the IgG1 murine monoclonal 7E3 (from which abciximab is derived) or IgG1 monoclonals AP3 and AP2, specific for
other sites on GPIIIa and GPIIb/IIIa, respectively. Results were
expressed as the ratio of the signal obtained with 7E3-coated
platelets to that obtained with AP3- or AP2-coated platelets. As
shown in Figure 5, serum from each of the 9 patients reacted more
strongly with platelets sensitized with 7E3 than with those sensitized with AP3, yielding ratios (7E3:AP3) that ranged from 5.4 to
91.6 (mean 29.1). In contrast, antibodies from healthy subjects,
Figure 4. Inhibition of patient antibodies (left) and antibodies from 13 healthy
subjects (right) by soluble Fab fragments derived from pooled normal human
IgG. Seven of the 9 patient antibodies were inhibited by less than 50%, and 2 (from
patient nos. 4 and 7) were inhibited by more than 80%. Antibodies from the 13 healthy
subjects were inhibited by 68% to 98% (average 85%). Brackets depict mean ⫾ 1 SD.
including the one that gave the strongest reaction with abciximabcoated platelets (Figure 3), failed to discriminate between the 2
target monoclonals, yielding ratios ranging from 0.7 to 2.4 (mean
1.3) (P ⬍ .001). Essentially the same results were obtained when
Figure 5. Reactions of 9 patient antibodies (left) and 13 antibodies from healthy
subjects (right) against platelets saturated with monoclonal antibodies 7E3
and AP3. Values shown are the ratios of MFI obtained with 7E3-coated platelets to that
obtained with AP3-coated platelets (7E3:AP3). Reactions of the patient antibodies against
7E3-coated platelets were significantly stronger (up to 100 times) than those obtained with
AP3-coated platelets (P ⬍ .001). In contrast, antibodies from the healthy subjects failed to
discriminate between the 2 preparations. Similar results were obtained when monoclonal
AP2 was substituted for AP3 (data not shown). Brackets depict mean ⫾ 1 SD.
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2058
CURTIS et al
the study was repeated using platelets sensitized with 7E3 or AP2
(data not shown). These findings suggest that the patients who
experienced thrombocytopenia had antibodies specific for 7E3
variable region sequences present in abciximab but not in AP3 or
AP2. Reactions of serum from patients no. 4 and 7, whose diluted
antibodies were largely inhibited by normal human Fab (Figure 4),
can be explained by the presence of 2 antibodies, a high-titer one
specific for IgG Fab and one with a lower titer specific for murine
sequences in abciximab.
Discussion
Each of the 9 patients studied experienced profound thrombocytopenia within a few hours of receiving abciximab followed by a
return of platelet levels to at least 100 ⫻ 109/L (100 000/␮L) within
3 to 8 days. They were also treated with unfractionated heparin
during and for up to 1 day after angioplasty, but a sensitive
serologic assay for antibodies characteristic of heparin-induced
thrombocytopenia was positive only in patient no. 6. Patient no. 6
had been given heparin 2 weeks earlier, and it is likely that his
heparin-dependent antibody was stimulated by that exposure.27 A
drop in the platelet level from normal to 6 ⫻ 109/L (6000/␮L)
would be extremely unusual in heparin-induced thrombocytopenia.23 However, it is possible that heparin sensitivity contributed to
the thrombocytopenia in patient no. 6. “Thrombocytopenia” following abciximab treatment can be artifactual (pseudothrombocytopenia), being the result of clumping of platelets in blood collected in
ethylenediaminetetraacetic acid anticoagulant through mechanisms
not yet fully understood.28,29 However, pseudothrombocytopenia
rarely leads to platelet counts as low as the ones observed in the
subjects of this report. Moreover, it is not generally associated with
bleeding, whereas the patients we studied had extensive petechial
hemorrhages (all patients), bleeding from venipuncture sites (patients no. 2, 3, and 5), gastrointestinal hemorrhage requiring red
cell transfusions (patient no. 1), and life-threatening intrapulmonary hemorrhage (patient no. 9). These considerations make it
extremely unlikely that any of the patients studied had
pseudothrombocytopenia.
Our finding that serum from each of the 9 patients contained
IgG—and in 8 cases IgM antibodies that recognized abciximabcoated platelets—suggested that their thrombocytopenia might
have been antibody mediated. However, similar but usually weaker
antibodies were detected in a high percentage of healthy individuals (Figure 2). A partial distinction between patient and “normal”
antibodies was obtained by showing that the “normal” antibodies,
appropriately diluted, could be inhibited at least 65% by soluble
Fab fragments derived from pooled human IgG, whereas all but 2
of the patient antibodies (patient nos. 4 and 7) were relatively
resistant to this treatment (Figure 4). This suggested that the
healthy subjects had naturally occurring antibodies specific for the
papain cleavage site at the C-terminus of abciximab. IgM antibodies were identified in only 2 of 104 healthy subjects. The extent to
which IgM antibodies detected in most of the patients contributed
to platelet destruction requires further study.
A sharp distinction between the patient and “normal” antibodies
was achieved by showing that each of the former reacted preferentially with platelets sensitized with monoclonal 7E3, whereas the
latter reacted about equally well with platelets sensitized with
monoclonals 7E3 and AP3 (Figure 5) or AP2, also IgG1 monoclonals specific for GPIIb/IIIa. Because 7E3 and abciximab share
only the murine Ig variable region sequences incorporated into the
BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
hybrid abciximab molecule, it is likely that the patient antibodies
recognize epitopes determined by these sequences. It has been
suggested that binding of abciximab, and by implication 7E3,
induces secondary conformational changes in the GPIIb/IIIa complex that could be immunogenic.30,31 Therefore, an alternative
explanation for our findings is that the patient antibodies recognize
structural changes induced by abciximab in the GPIIb/IIIa heterodimer. Further studies are needed to distinguish between these 2
possibilities, but in either case our findings suggest that it may be
possible to use platelets sensitized with 7E3 as targets to identify
antibodies capable of causing profound thrombocytopenia.
The literature contains little information about the immunogenicity of abciximab. In early trials in which unmodified (nonchimeric) 7E3 Fab was used as an antiplatelet agent, human antimouse
antibodies were produced by 15%,32 36%,33 and 52%34 of the
recipients, leading to the suggestion that immunogenicity of the
7E3 Fab fragment might limit its therapeutic application.33 The
chimeric Fab fragment was developed to overcome this problem.
The effectiveness of this modification was confirmed in phase 1
clinical trials in which the immunogenicity of 7E3 Fab (87 patients)
and abciximab (70 patients and 18 healthy subjects) was compared.26 In this study, 15 subjects (17%) treated with 7E3 Fab but
only 6 (7%) treated with abciximab (c7E3 Fab) produced human
antimouse antibodies, all of which were of the IgG class. In
solid-phase assays, it was shown that the 15 antibodies made by
patients given 7E3 Fab were mainly directed toward murine Ig
variable sequences. In contrast, only 1 of the 6 antibodies made by
individuals given abciximab had this specificity; the others recognized the C-terminus of Fab derived from human IgG1. It was
suggested that the human (constant region) domains of abciximab
modulate the immune response to the protein in such a way that the
immunogenicity of the murine sequences is greatly diminished.26
In the study by Knight and coworkers,26 no conclusions could
be reached as to whether antibodies specific for murine (variable
region) sequences in abciximab might be more likely to cause
thrombocytopenia than those specific for the C-terminus of Fab.26
However, a report by Christopolous is relevant to this question.35 In
this study, abciximab was given for about 48 hours to 9 patients
with stable angina, and platelet-associated IgG (PAIgG) was
measured by flow cytometry. Within 24 hours of treatment, there
was a variable but statistically significant rise in PAIgG that was
accentuated several days after the infusion was terminated, despite
an exponential fall in the amount of abciximab carried on the
surface of circulating platelets. PAIgG returned to normal within 2
weeks, and none of the patients developed thrombocytopenia. The
timing of the recruitment of IgG to the platelet surface suggested
that some of the subjects studied had pre-existing antibodies that
reacted with abciximab-coated platelets in vivo without causing
thrombocytopenia. Support for this possibility was obtained by
showing that 14 of 21 healthy subjects had naturally occurring IgG
antibodies that reacted with abciximab-coated platelets in vitro and
that 1 of the 21 had an antibody that recognized platelets coated
with 7E3 Fab. In light of our findings, it seems likely that the
naturally occurring antibodies identified by Christopolous in two
thirds of his study group were specific for human IgG Fab. The
results of his in vivo studies suggest that, although these ubiquitous
Fab-specific antibodies are recruited to platelets following abciximab treatment, they do not cause thrombocytopenia. Clinical
experience showing that significant thrombocytopenia is a rare
complication of abciximab also supports this conclusion.
The observation of Knight and coworkers that only 1 (1.1%) of
88 individuals challenged with abciximab produced antibodies
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BLOOD, 15 MARCH 2002 䡠 VOLUME 99, NUMBER 6
ABCIXIMAB-INDUCED THROMBOCYTOPENIA
specific for murine variable sequences,26 together with our finding
that each of 9 patients with abciximab-induced thrombocytopenia
had antibodies with this apparent specificity, strongly suggests that
these antibodies are capable of causing platelet destruction in vivo
when abciximab is readministered. It is known that abciximab
remains bound to circulating platelets in detectable quantities for
up to 2 weeks after treatment, probably a consequence of plateletto-platelet transfer of drug.5,35 Persistence of the drug in the
circulation for this length of time would offer the immune system
ample opportunity to process it and, on occasion, mount a humoral
immune response against its murine components. We have initiated
a prospective study to obtain a better estimate of how often
antibodies potentially able to cause thrombocytopenia upon second
exposure develop in patients given abciximab. Because severe
thrombocytopenia appears to be more common when re-exposure
2059
occurs within 2 to 3 weeks,12 a determination of how long such
antibodies persist in the circulation may be helpful in assessing the
risk of thrombocytopenia upon re-exposure to the drug.
Our findings suggest that screening of patients about to receive
abciximab a second time for antibodies that recognize its murine
sequences could permit identification of patients at risk for
developing profound thrombocytopenia. Severe thrombocytopenia
also occurs in 0.5% to 1% of patients given abciximab for the first
time.9-11 Our survey of 104 healthy subjects failed to identify
naturally occurring antibodies with presumptive specificity for Ig
variable sequences in 7E3. However, Christopolous identified one
such antibody among 21 healthy individuals.35 Prospective studies
are needed to determine whether persons who have naturally
occurring antibodies specific for murine sequences in abciximab
are at risk for thrombocytopenia when given the drug for the first time.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2002 99: 2054-2059
doi:10.1182/blood.V99.6.2054
Thrombocytopenia after second exposure to abciximab is caused by
antibodies that recognize abciximab-coated platelets
Brian R. Curtis, Julia Swyers, Ajit Divgi, Janice G. McFarland and Richard H. Aster
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