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Blood, Vol. 94 No. 2 (July 15), 1999:
pp. 587-599
Fibrinogen Receptor Antagonist-Induced Thrombocytopenia in
Chimpanzee and Rhesus Monkey Associated With Preexisting
Drug-Dependent Antibodies to Platelet Glycoprotein IIb/IIIa
By
Bohumil Bednar,
Jacquelynn J. Cook,
Marie A. Holahan,
Michael E. Cunningham,
Patricia A. Jumes,
Rodney A. Bednar,
George D. Hartman, and
Robert J. Gould
From the Departments of Pharmacology and Medicinal Chemistry, Merck
Research Laboratories, West Point, PA.
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ABSTRACT |
Most clinical trials with fibrinogen receptor antagonists (FRAs)
have been associated with thrombocytopenia. This report describes the
occurrence of thrombocytopenia in one chimpanzee and one rhesus monkey
upon administration of potent FRAs. Chimpanzee A-264 experienced profound thrombocytopenia on two occasions immediately upon intravenous administration of two different potent FRAs, L-738,167 and L-739,758. However, an equally efficacious antiaggregatory dose of another potent
antagonist, L-734,217, caused no change in platelet count. These
compounds did not affect platelet count in five other chimpanzees or
numerous other nonhuman primates. Flow cytometric analysis showed
drug-dependent antibodies (DDAbs) in the plasma of chimpanzee A-264
that bound to platelets of chimpanzees, humans, and all other primates
tested only in the presence of the compounds that induced
thrombocytopenia. Rhesus monkey 94-R021 experienced thrombocytopenia upon administration of a different antagonist, L-767,679, and several
prodrugs that are converted into the active form, L-767,679, in the
blood. More than 20 other FRAs, including those that induced thrombocytopenia in chimpanzee A-264, had no effect on platelet count
in this monkey. Flow cytometric measurements again identified DDAbs
that reacted with platelets of all primates tested and required the
presence of L-767,679. Screening for DDAbs in the plasma of 1,032 human
subjects with L-738,167 and L-739,758 demonstrated that the incidence
of these preexisting antibodies in this population was 0.8% ± 0.6%
and 1.1% ± 0.6%, respectively.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
RECENT CLINICAL TRIALS with platelet
fibrinogen receptor antagonists (FRAs) demonstrated that glycoprotein
(GP) IIb/IIIa blockade represents a major new advancement in the
management of patients with coronary disease.1-3 However,
all clinical trials, with one exception,4 have consistently
been associated with an incidence of thrombocytopenia (typically 1% to
2% of patients).3-10 The observed thrombocytopenias have
been of two types: immediate, within hours, or with a delayed onset,
several days after drug administration. Although clinical
thrombocytopenia is defined as a platelet count of less than 100,000 platelets/µL and severe thrombocytopenia is defined as a platelet
count of less than 50,000 platelets/µL, some cases associated with GP
IIb/IIIa antagonist administration have been profound (<20,000
platelets/µL).5-7 The mechanism underlying profound
thrombocytopenia after the initial exposure to FRAs, occurring in some
cases within several hours after administration of a GP IIb/IIIa
blocker,6,7 is poorly understood.3,5
Drug-induced thrombocytopenia was described as early as 1928 during
quinine therapy.11 Since that time, immune-mediated thrombocytopenia associated with drug-dependent antibodies (DDAbs) has
been well documented for quinine, quinidine, and more than 100 other
low molecular weight compounds as well as currently used
therapeutics.5,11-15 The mechanism of immune-mediated
thrombocytopenia remains unknown5; however, it has been
frequently demonstrated that DDAbs recognize epitopes exposed on the
drug-receptor complex upon binding of the drug.5
Interestingly, epitopes for quinine/quinidine-dependent antibodies
described first on the platelet GP Ib/IX complex16,17 were
later also found on the GP IIb/IIIa complex.18-20 Recently, antigens on GP IIb/IIIa have been identified as targets of DDAbs in
patients who experienced pentamidine- and sulfonamide-induced thrombocytopenia.14,15
Numerous methods have been developed for the detection of
platelet-associated DDAbs in patients with drug-induced
thrombocytopenia.5,15,21-23 The recently described flow
cytometric method15 provides a very sensitive technique for
detection of antiplatelet antibodies using whole platelets. This assay
closely mimics the conditions in whole blood, without significantly
disturbing neoepitopes exposed on the platelet surface upon drug
administration.15
In this report, we describe thrombocytopenia in two nonhuman primates
induced by the administration of structurally and functionally diverse
RGD-mimetic, FRAs: L-738,167-(A1-L),24,25
L-739,758-(A2-L),26 and L-767,679-(B).27 We
also provide direct flow cytometric evidence that the plasma of both
primates contains preexisting DDAbs that recognize epitopes on GP
IIb/IIIa of all human and nonhuman primates evaluated. Screening for
DDAbs in the plasma of 1,032 human subjects with L-738,167 and
L-739,758 demonstrated that the incidence of these preexisting
antibodies in this population was 0.8% ± 0.6% and 1.1% ± 0.6%, respectively.
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MATERIALS AND METHODS |
Experimental protocol for in vivo studies in chimpanzees, baboons,
rhesus monkeys, cynomolgus monkeys, African green monkeys, squirrel
monkeys, and dogs.
Chimpanzees were sedated with ketamine (10 mg/kg intramuscularly
[IM]) for compound administration and blood withdrawal
for the determination of platelet count and evaluation of platelet aggregation responses. Before intravenous (IV) bolus (10 µg/kg) administration of A1-L (n = 6) and A2-L (n = 1) or IV infusion (0.4 µg/kg/min for 60 minutes) of A3 (n = 4), a blood sample (sodium citrate; final concentration, 0.38%) was taken by venipuncture from a
saphenous vein. This was used for determination of baseline whole blood
platelet count and ex vivo platelet aggregation response to adenosine
diphosphate (ADP). Additional blood samples were taken at
selected time points after the initiation of treatment. Other nonhuman
primates (squirrel monkey [n = 12], African green monkey
[n = 4], rhesus monkey [n = 88], cynomolgus monkey
[n = 79], and baboon [n = 12]) were also evaluated for
FRA-induced thrombocytopenia. The studies with these primates were
conducted at several different facilities and were, therefore, studied
in either the conscious or sedated state depending on the normal procedures at each site. Conscious animals were placed in primate restraint chairs and transported to the laboratory on the day of the
experiment; sedated animals were chemically restrained with ketamine
for blood withdrawal and compound administration. Blood samples were
taken before compound administration and at selected time points after
initiation of treatment and used for platelet count and platelet
aggregation measurements. A subpopulation of rhesus monkeys (n = 5)
received A1-L after prior treatment with A3 (0.4 µg/kg/min for 60 minutes). A1-L was administered to conscious (n = 157) and
anesthetized (n = 128, 35 mg/kg IV sodium pentobarbital) purpose-bred
mongrel dogs of either sex. Conscious dogs rested comfortably in nylon
slings for administration of A1-L orally (by gastric lavage in aqueous
solution or as solid, crystalline material in capsule) or as an IV
bolus and/or continuous infusion.
Measurement of ex vivo platelet aggregation responses and whole
blood platelet count.
Platelet-rich plasma (PRP) was prepared by centrifugation of whole
blood at 150g for 5 minutes and the platelet concentration was
adjusted to 2 × 108 platelets/mL with time-matched
platelet-poor plasma (PPP). PRP (300 µL, 2 × 108
platelets/mL) was incubated at 37°C for 3 minutes before the addition
of agonist. Platelet aggregation was measured by the change in light
transmittance (PPP represents 100%) under stirring conditions (1,100 rpm) at 37°C in a Biodata Platelet Aggregation Profiler
(Model PAP-4; Biodata Corp, Horsham, PA) and was initiated by the
addition of 20 µmol/L ADP for primates and 10 µmol/L ADP + 1 µmol/L epinephrine for dogs. The extent of aggregation was taken as
the peak percentage of aggregation achieved based on a maximum of
100%, and the effect of treatment on platelet aggregation was
expressed as the percentage of inhibition using the baseline, pretreatment aggregation response as 100%. Whole blood platelet counts were determined using an automated hematology analyzer (Biochem Immunosystems, Allentown, PA) for all experiments except chimpanzees (Coulter Cell Counter; Coulter, Hialeah, FL).
Preparation of gel-filtered platelets (GFP).
GFP were prepared from blood anticoagulated with 13 mmol/L trisodium
citrate. PRP was generated by centrifugation at 180g for 20 minutes in the presence of 1 µmol/L prostaglandin
E1 (PGE1; Sigma Chemical, St
Louis, MO). Platelets were separated from plasma by gel filtration
through a Sepharose 2B column (Pharmacia Biotech, Uppsala,
Sweden) and equilibrated in platelet buffer (138 mmol/L NaCl, 6 mmol/L KCl, 1.7 mmol/L Na2HPO4, 6.3 mmol/L HEPES, 1 mg/mL dextrose, and 2% bovine serum albumin [BSA],
pH 7.4). The platelets were diluted to 2 × 108 cells/mL
in platelet buffer before use.
RGD-mimetics.
RGD-mimetics A1-L,21,22 A3,25,26
A2-L,23 C,27,28 B,24 A2-R (R-isomer
of A2-L), A1-R (R-isomer of A1-L), B-P1, B-P2, and D were synthesized
by the Medicinal Chemistry Department, Merck Research Labs (West Point,
PA). The chemical structures are listed in Table
1.
Flow cytometric assay of DDAbs.
The flow cytometric assay for detection of DDAbs is based on the method
of Curtis et al.15 Platelets at a concentration of 1 × 108 platelets/mL in flow cytometric buffer (10 mmol/L
HEPES, 5 mmol/L KCl, 145 mmol/L NaCl, and 1 mmol/L MgCl2)
were incubated with platelet inhibition cocktail that contained
PGI2 (10 µmol/L), apyrase (2 U/mL), leupeptin (0.5 mmol/L), and hirudin (10 nmol/L) and either FRAs (10 µmol/L) or
buffer (control) for 15 minutes at room temperature. Platelet
suspension (50 µL) was spun at 100g for 4 minutes and
platelets resuspended in 100 µL of plasma containing platelet
inhibition cocktail with or without FRA. The mixture was incubated for
1 hour at room temperature. The platelets were then pelleted
(PGE1 added) as described previously and washed with 500 µL of flow cytometric buffer in the presence or absence of the
appropriate FRA (10 µmol/L). The platelets were resuspended in 100 µL of flow cytometric buffer in the presence or absence (control) of
FRA and 20 µL of platelet suspension was added to tubes containing
200 µL of goat fluorescein isothiocyanate (FITC)-labeled antihuman or
antimonkey IgG (experiments with rhesus monkey plasma; Accurate
Chemical & Scientific Co, Westbury, NY) and incubated for 1 hour at
room temperature. The amount of bound FITC-antihuman or antimonkey IgG
was determined by flow cytometry15 (FACSCalibur; Becton
Dickinson, San Jose, CA).
Saturation of platelet receptors with DDAbs by repeat incubation
with chimpanzee A-264 plasma.
Gel-filtered platelets (200 µL at 2 × 108
platelets/mL) were incubated with FRA for 15 minutes as described
previously. Platelets were pelleted and then resuspended in 200 µL of
chimpanzee plasma containing platelet inhibition cocktail and incubated
for 30 minutes at room temperature. The platelets were then either
pelleted and resuspended with 200 µL of chimpanzee plasma again or
washed with 500 µL of flow cytometric buffer and analyzed in the flow
cytometer for DDAbs as previously described.
Enzyme-linked immunosorbent assay (ELISA) measurements.
Microtiter plate wells were coated overnight at 4°C with 5 µg/mL of
purified GP IIb/IIIa29 diluted in
phosphate-buffered saline (PBS) either in the presence or absence of 10 µmol/L A2-L. The wells were washed four times with PBS containing
0.05% Tween 20 (PBS/Tween 20) and incubated for 1 hour with either 100 µL of chimpanzee A-264 or control chimpanzee plasma diluted 1:20 in
PBS/Tween 20 in the presence or absence of 10 µmol/L A2-L. The wells
were then washed four times with PBS/Tween 20 and incubated with sheep
antihuman IgG-peroxidase conjugate (Amersham, Cleveland, OH) diluted
1:1,000 in PBS/Tween 20 for 1 hour at room temperature. After washing
the wells four times with PBS/Tween 20, the ABST peroxidase substrate
(Kirkeqaard & Perry, Gaithersburg, MD) was added and the change in
absorbance at 405 nm was measured after 15 minutes.
Screening for DDAbs in human plasma samples.
Human plasma samples (1,032 different anonymous blood donor subjects)
were screened for A1-L- and A2-L-dependent antibodies using the
previously described flow cytometric assay and human gel-filtered
platelets from healthy donors. A plasma sample from chimpanzee A-264
was used as a positive control, and plasma samples from a control
chimpanzee and a human subject (negative for DDAbs) were used as
negative controls. The values of relative fluorescence (fluorescence of
plasma in the presence of drug/fluorescence in the absence of drug)
were 1.02 ± 0.17 and 1.05 ± 0.15 (mean ± SD, n = 60) for
control chimpanzee plasma and control human plasma, respectively.
Samples were considered positive when the value of relative
fluorescence was  2, similar to Curtis et al.15 Whenever
the plasma sample was found positive in the presence of either A1-L or
A2-L, it was tested again using these two compounds and two other FRAs,
A3 and B, on platelets from three different human donors. A plasma
sample was reported positive if it was found positive using at least
two different platelet donors. Positive samples tested positive on
platelets of all tested platelet donors; however, the amount of
detected bound DDAbs, as measured by the fluorescence ratio, varied for
different platelet donors. We repeated testing of 200 samples that were
found to be negative in the first evaluation. All of these samples
remained negative with repeat testing.
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RESULTS |
Effect of administration of FRAs on platelet count in nonhuman
primates.
One of six chimpanzees (A-264) experienced immediate profound
thrombocytopenia upon IV bolus infusion of 10 µg/kg of a potent FRA,
A1-L.24,25 As shown in Fig 1A,
platelet aggregation was completely inhibited in all six chimpanzees
(including A-264) for approximately 12 hours after IV administration of
10 µg/kg of A1-L. The antiaggregatory effect gradually decreased from
12 to 56 hours after A1-L treatment. As shown in Fig 1B, the platelet count for chimpanzee A-264 began to decrease (153,000, 132,000, 119,000, and 84,000 platelets/µL at baseline, 5, 15, and 30 minutes after treatment, respectively) immediately upon drug administration and
reached a minimum (<40,000 platelets/µL) at 2 hours after dosing.
The platelet count remained at that level for the next 6 hours and then
gradually increased from 10 to 96 hours (108,000 platelets/µL). The
increase of platelet count was practically linear, with a rate
consistent with the life span of platelets of about 6 to 7 days. The
apparently small reduction in the initial platelet count in the five
control chimpanzees is within experimental error of the measurement,
although the trend seems to be similar for all animals (Fig 1B). This
observation could be procedural or physiologically based; however, we
have no direct evidence to support either. After full recovery, and
more than 30 days after administration of A1-L, chimpanzee A-264
received an infusion of 0.4 µg/kg/min for 60 minutes of another FRA,
A3.28,30 As shown in the Fig 2,
A3 inhibited ex vivo platelet aggregation during 1 hour of infusion and
for 2 hours after the end of infusion; however, there was no effect on
platelet count. By 24 hours after treatment with A3, ex vivo platelet
aggregation had returned to normal (Fig 2A). Chimpanzee A-264 was then
challenged with an IV bolus of 10 µg/kg of A2-L, an FRA26
functionally and structurally similar to A1-L. As shown in Fig 2B, the
platelet count of chimpanzee A-264 decreased precipitously within 1 hour to less than 20,000 platelets/µL and then recovered with a
similar time course to the recovery after treatment with A1-L.
Chimpanzee A-264 has not been challenged with any other FRA since these
thrombocytopenic episodes.
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| Fig 1.
The effect of 10 µg/kg IV bolus of A1-L on ex vivo
platelet aggregation in PRP stimulated with 20 µmol/L ADP (A) and
platelet count in whole blood (B) in ketamine-sedated chimpanzees.
( ) Inhibition of platelet aggregation as measured by a change in
light transmittance (PPP 100%) under stirring conditions (1,100 rpm)
at 37°C in Biodata Platelet Aggregation Profiler for chimpanzee A-264
and five control chimpanzees (mean ± SEM, n = 6). Platelet
count measured in whole blood using a Coulter Cell Counter for ( )
control chimpanzees (mean ± SEM, n = 5) and ( ) chimpanzee
A-264.
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| Fig 2.
The effect of the infusion of A3 (0.4 µg/kg/min for 1 hour) followed 24 hours later by 10 µg/kg IV bolus of A2-L on ex vivo
platelet aggregation in PRP stimulated with 20 µmol/L ADP (A) and
platelet count in whole blood (B) in ketamine-sedated chimpanzee A-264.
() Inhibition of platelet aggregation as measured by a change in
light transmittance (PPP 100%) under stirring conditions (1,100 rpm)
at 37°C in Biodata Platelet Aggregation Profiler. () Platelet
count measured in whole blood using a Coulter Cell Counter.
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Part of our overall strategy for the investigation of the mechanism of
thrombocytopenia induced by GP IIb/IIIa inhibitors as well as for
assessing the risk for thrombocytopenia with other compounds was the
identification of other primates susceptible to thrombocytopenia upon
exposure to a potent FRA (A1-L). As described in the methods, A1-L was
administered as an IV bolus of 10 µg/kg to numerous primate species,
including chimpanzee (n = 6), rhesus monkey (n = 88), cynomolgus
monkey (n = 79), African green monkey (n = 4), squirrel monkey
(n = 12), and baboon (n = 12). The whole blood platelet count at 2 and 4 hours after dosing did not decrease to the level of
thrombocytopenia in any animal (with or without pretreatment with A3)
except chimpanzee A-264. Therefore, thrombocytopenia occurred in 0.5% ± 0.5% of the primates in this population (1/201).
Binding characteristics of selected FRAs in binding to chimpanzee and
human platelets.
Because all described FRAs are highly selective24-28
ligands for GP IIb/IIIa versus other integrins, it was of interest to compare their binding affinities to GP IIb/IIIa on platelets from chimpanzee A-264, a control chimpanzee, and human platelets. As shown
in Table 2, dissociation constants of the
fluorescein-labeled FRA31,32 C as well as A1-L and A2-L in
binding to chimpanzee A-264, control chimpanzee, and human platelets
are identical within experimental error. We also considered the
possibility that the unique response in chimpanzee A-264 versus other
primates could be due to a dissimilar number of receptors per platelet.
However, the number of GP IIb/IIIa receptors per platelet was found
identical for both chimpanzees and within the range found for 100 human subjects (Table 2). We further found, using flow cytometry, that the
size as well as the distribution in the size of platelets for
chimpanzee A-264 and control chimpanzee was identical and comparable
with data for human platelets (data not shown). The binding of FRA
(used in our report) to the receptors on resting platelets does not
result in the activation of platelets as measured by standard methods
(calcium flux, serotonin, and P-selectin secretion), although all
compounds induce more ligand-induced binding sites (LIBS) and expansion
upon binding to GP IIb/IIIa (data not shown).
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Table 2.
Binding Characteristics of FRAs and Number of GP
IIb/IIIa Receptors Per Platelet on Chimpanzee A-264, Control
Chimpanzee, and Human Platelets
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Flow cytometric measurement of DDAbs in plasma from chimpanzee A-264.
It is also possible that the observed thrombocytopenia in chimpanzee
A-264 could be mediated by an immune response. To determine whether
plasma from chimpanzee A-264 contained drug-dependent antiplatelet
antibodies, we used a slightly modified flow cytometric method
described by Curtis et al15 (for details, see Materials and
Methods). Figure 3A shows histograms from
the flow cytometric measurements of DDAbs using plasma and platelets
from chimpanzee A-264 and A2-L. The shape of the flow cytometric
histogram in Fig 3 (+ A2-L) is indistinguishable from the histogram
obtained by size measuring light scattering detector (not shown),
indicating that DDAbs bind equally well to the receptors on all
platelets present. Figure 3B depicts results of flow cytometric
measurements on chimpanzee A-264 and control chimpanzee plasma using
platelets from both animals. As shown in Fig 3B, DDAbs from chimpanzee
A-264 plasma selectively reacted with platelets from both chimpanzees (control or A-264) in the presence of A1-L and A2-L. However, the
amount of antibody bound to platelets as indicated by the relative
fluorescence was significantly lower for A1-L in all measurements. No
DDAbs were detected in the presence of A3, an FRA that did not have an
effect on platelet count in chimpanzee A-264. Similar results were
obtained when, instead of chimpanzee platelets, platelets from 45 human
subjects (see Fig 3C) and several other nonhuman primates (data not
shown) were used in incubation with chimpanzee plasma. Thus, chimpanzee
A-264 plasma appears to contain DDAbs that recognize an epitope on
chimpanzee, human, and several other nonhuman primate platelets.
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| Fig 3.
Flow cytometric measurement of antibody binding from
chimpanzee A-264 and control chimpanzee plasma alternatively using
chimpanzee A-264 and control chimpanzee platelets (A and B) and human
platelets (C) in the presence of 10 µmol/L drug. (A) depicts the
histograms from flow cytometric measurements of DDAb binding in the
presence (+) and in the absence ( ) of A2-L. Data in (B) represent
the mean ± SD from three measurements. Data in (C) using A2-L and
A1-L represent the mean ± SD from measurements using platelets of 45 human subjects; for other compounds, data represent the mean ± SD
from measurements using platelets of three human subjects. Binding of
DDAbs is plotted as relative fluorescence of an FITC-labeled secondary
antibody. The right two sets of bars in (C) represent results with
human platelets pretreated at 37°C for 1 hour with 10 mmol/L EDTA.
A2-R (Kd, 0.45 nmol/L) and A1-R (Kd, 0.28 nmol/L) are R-isomers of A2-L
and A1-L, respectively.
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It is interesting that the R-isomer of A2-L (compound A2-R), which is
also a potent FRA, induced binding of DDAbs to human platelets similar
to A2-L, whereas the R-isomer of A1-L (A1-R) had no effect (Fig 3C).
Because the concentration of FRAs used in the flow cytometric assay (10 µmol/L; ~10-fold higher than the maximum in vivo concentration) is
much higher than the possible maximal concentration of receptors (~10
nmol/L) and higher than the dissociation constants (see Table 2), all
GP IIb/IIIa receptors on the surface of platelets can be assumed to be
occupied by FRAs. Results of flow cytometric measurements of DDAbs
presented in Fig 3 were obtained with FITC-labeled goat antihuman IgG
secondary antibody. When goat antihuman IgM or IgA secondary antibodies were used, no DDAbs in chimpanzee A-264 were detected (data not shown).
Treatment of platelets with 10 mmol/L EDTA at 37°C causes dissociation of the GP IIb/IIIa complex on platelets, resulting in a
complete loss33,34 of RGD-ligand binding as well as the dissociation of complex or conformational dependent antibodies. Flow
cytometric measurements on human platelets (Fig 3C) indicated that the
reaction of DDAbs from chimpanzee A-264 plasma with human platelets was
abolished when the platelets were treated for 1 hour with 10 mmol/L
EDTA at 37°C. An activation of platelets by either ADP or TRAP before
incubation with plasma from chimpanzee A-264 did not induce binding of
DDAbs (data not shown).
Both the plasma concentration of DDAbs as well as the affinity of the
antibodies for platelets may play critical roles in the induction of
thrombocytopenia. Flow cytometric measurements with different dilutions
of chimpanzee A-264 plasma indicated that, upon dilution of plasma to
one fifth the fluorescence as a measure of platelet-bound
antibodies decreased approximately to the level found with control
chimpanzee plasma. Flow cytometric measurement of DDAbs in chimpanzee
A-264 plasma collected over 30 months showed that the titer of DDAbs
remained constant. Figure 4 shows that the
fluorescence, as a measure of bound DDAbs in flow cytometric
measurements, increased with increasing concentration of A2-L from 1 to
100 nmol/L and then remained constant. Results presented in Fig
4 were obtained with GFP from a single
human donor with 2 × 108 cells/mL and an average of
105,000 GP IIb/IIIa receptors per platelet, suggesting a concentration
of receptors of about 35 nmol/L. The Kd value for the
A2-L in binding to GP IIb/IIIa on platelets is approximately 0.1 nmol/L
(see Table 2); therefore, most of the receptors should be occupied at
equilibrium when the concentration of the drug reaches the
concentration of receptors (~35 nmol/L). Thus, the maximum binding of
DDAbs should be reached at an A2-L concentration of 35 nmol/L. However,
during the wash step of the experimental procedure, equilibrium is not
maintained. Thus, the receptor occupancy is always lower than it would
be at equilibrium conditions. This results in a shift of the
concentration necessary for full receptor occupancy to higher
concentrations. As a result, the maximum fluorescence in Fig 4 is
shifted also to higher concentrations of A2-L, whereas at the
concentration 35 nmol/L, approximately 90% of DDAb is bound. Data in
Fig 4 further indicate that the drug structure is not likely a major
part of the epitope, because more than 100-fold excess of the free drug over the receptors does not lower DDAb binding.
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| Fig 4.
The effect of the concentration of A2-L on the binding of
DDAbs from chimpanzee A-264 plasma to human GFP (100 µL,
2 × 108 platelets/mL) as measured by the fluorescence
of the FITC-labeled secondary antibody using flow cytometry. The
concentration of GP IIb/IIIa receptors was 35 nmol/L.
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| Fig 5.
The effect of repeated incubations of chimpanzee A-264
(A) and human platelets (B) with chimpanzee A-264 plasma on binding of
DDAbs in the presence of 10 µmol/L drug as measured by relative
fluorescence of FITC-labeled secondary antibody using flow cytometry.
(A) One hundred microliters of 2 × 108 platelets/mL was
repeatedly incubated with 60 µL of chimpanzee A-264 plasma. ()
A2-L, () A1-L using chimpanzee A-264 plasma; () A2-L, ( ) A1-L
using control chimpanzee plasma. (B) Fifty microliters of 1 × 108 platelets/mL was repeatedly incubated with 100 µL of
chimpanzee A-264 plasma. () A2-L, () A1-L using chimpanzee A-264
plasma; () A2-L using control chimpanzee plasma.
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The overall fluorescence intensity of the secondary FITC-labeled
antihuman antibody in the flow cytometric assay was threefold to
fivefold lower than expected if all GP IIb/IIIa receptors were occupied
with DDAbs. Therefore, it was of interest to determine if either
repeated incubation of the same platelets with chimpanzee A-264 plasma
or incubation of the same number of platelets with a higher volume of
plasma would result in an increase of the DDAb signal. As shown in Fig
5A, when the original 100 µL of chimpanzee A-264 platelets was
repeatedly incubated with 60 µL of chimpanzee A-264 plasma, the
relative fluorescence of the secondary antibody increased and leveled
off after eight incubations. This indicates a roughly fourfold increase
in the amount of bound A2-L-dependent antibody when compared with a
single incubation. Interestingly, in a parallel experiment with A1-L,
relative fluorescence was unaffected after repeated incubations with
fresh chimpanzee A-264 plasma. A similar experiment using 50 µL of
human platelets in repetitive incubations with 100 µL of chimpanzee
A-264 plasma (Fig 5B) showed that the maximum bound A2-L-dependent
antibody was reached after four incubations. This finding agrees well
with the previous experiment with chimpanzee A-264 platelets, because the amount of platelets in the experiment with human platelets was one
half, and it was incubated with 100 µL of plasma instead of 60 µL
used in the experiment with chimpanzee A-264 platelets. Thus,
saturation of receptors by DDAbs was achieved after four incubations
instead of eight with chimpanzee A-264 platelets. When the plasma of
chimpanzee A-264 that was used in the experiment was used for a second
time on fresh platelets, no DDAbs were found. Similar to the
experiments with chimpanzee A-264 platelets, repetitive incubation of
human platelets with chimpanzee A-264 plasma had little effect on the
amount of bound A1-L-dependent antibodies. Repeated incubations of
platelets with control chimp plasma had no effect on relative
fluorescence, indicating that there were no DDAbs present.
FRA-induced thrombocytopenia in rhesus monkey 94-R021.
After testing numerous FRAs in various animal species (including 285 dogs dosed with A1-L alone), we identified only one primate besides
chimpanzee A-264 that experienced FRA-induced thrombocytopenia. After
oral administration of 0.3 mg/kg of B-P1 and 0.5 mg/kg of B-P2, rhesus
monkey 94-R021 showed complete inhibition of platelet aggregation (Fig
6A) and an approximately 40% to 80%
decrease in platelet count, respectively (Fig 6B). These compounds are both ester prodrugs that convert to the active metabolite
B27 in the blood. The time course of the onset of
thrombocytopenia in the rhesus monkey correlates with the conversion of
prodrugs to the active acid B. To further characterize the
thrombocytopenic response, this monkey received B as an IV bolus of 20 µg/kg. Upon administration of B, this rhesus monkey demonstrated a
precipitous decrease in platelet count (517,000 platelets/µL baseline
to 144,000 platelets/µL at 30 minutes postdose; Fig 6C). However, in
contrast to chimpanzee A-264, the platelet count in rhesus monkey
94-R021 started to increase at 60 minutes (187,000 platelets/µL)
after dosing B and nearly recovered (441,000 platelets/µL) within 6 hours (Fig 6C). The recovery of the platelet count started when the
receptor occupancy by B (see Fig 6D) decreased to less than approximately 40% (40,000) of the total GP IIb/IIIa receptors. When
rhesus monkey 94-R021 was challenged with 5 µg/kg IV bolus of A2-L,
which completely inhibited platelet aggregation for several hours and
induced thrombocytopenia in chimpanzee A-264, no change in platelet
count was observed (Fig 6C). Similarly, 20 other FRAs (including A1-L
and A3) that completely inhibited platelet aggregation had no effect on
platelet count in 94-R021 (data not shown). When rhesus monkey 94-R021
was dosed with 10 µg/kg IV bolus of the FRA D, which is structurally
and functionally similar to A1-L, platelet aggregation was completely
inhibited with no effect on platelet count (Fig
7). Interestingly, the administration of B after the administration of D had no effect on platelet count. Thus,
the binding of D to GP IIb/IIIa on rhesus monkey 94-R021 platelets
prevented thrombocytopenia otherwise induced by the administration of
B.
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| Fig 6.
The effect of 0.5 mg/kg (administered orally) of B-P2
(phenyl ester of B) and 0.3 mg/kg (administered orally) of B-P1 (ethyl
ester of B) on ex vivo platelet aggregation in PRP upon stimulation
with 20 µmol/L ADP (A) and platelet count in whole blood (B) in
rhesus monkey 94-R021. The effect of 5 µg/kg IV bolus of A2-L and 20 µg/kg IV bolus of B on ex vivo platelet aggregation in PRP upon
stimulation with 20 µmol/L ADP and platelet count in whole blood (C)
and number of receptors occupied by B (D) in rhesus monkey 94-R021. (A
and B) Inhibition of platelet aggregation by B-P1 ( ) or B-P2 ()
as measured by a change in light transmittance (PPP 100%) under
stirring conditions (1,100 rpm) at 37°C in Biodata Platelet
Aggregation Profiler. Platelet count measured in whole blood of rhesus
monkey 94-R021 using an automated hematology analyzer (Biochem
Immunosystems) upon dosing B-P1 () and B-P2 (). (C and D)
Platelet count measured in whole blood of rhesus monkey 94-R021 using
an automated hematology analyzer (Biochem Immunosystems) upon dosing
A2-L () and B (). Inhibition of platelet aggregation by B ()
or A2-L ( ) as measured by a change in light transmittance (PPP
100%) under stirring conditions (1,100 rpm) at 37°C in Biodata
Platelet Aggregation Profiler. The number of GP IIb/IIIa receptors
occupied by B as measured by competition displacement measurements with
[125I]-L-692,884 ().
|
|
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| Fig 7.
The effect of 10 µg/kg IV bolus of D, 10 µg/kg IV
bolus of B, and 10 µg/kg IV bolus of B administered 30 minutes after
10 µg/kg IV bolus of D on ex vivo platelet aggregation in PRP
stimulated with 20 µmol/L ADP (A) and platelet count in whole blood
(B) in rhesus monkey 94-R021. Inhibition of platelet aggregation by B
() or D () and sequential dosing of these compounds ( ) as
measured by a change in light transmittance (PPP 100%) under stirring
conditions (1,100 rpm) at 37°C in Biodata Platelet Aggregation
Profiler. Platelet count measured in whole blood of rhesus monkey
94-R021 upon dosing D (), B (), and B 30 minutes after D ( ).
|
|
Figure 8 shows the results of flow
cytometric measurements of DDAbs in rhesus monkey and chimpanzee
plasma. DDAbs were identified in rhesus monkey 94-R021 plasma only in
the presence of B, using both rhesus monkey and human platelets. The
FRA A2-L, which induced thrombocytopenia in chimpanzee A-264, had no
effect on antibody binding in rhesus plasma. Similar results (data not
shown) were obtained with 10 other FRAs, including A1-L, which induced
thrombocytopenia in chimpanzee A-264. Figure 8 also demonstrates that B
had no effect on binding of DDAbs from chimpanzee A-264 plasma. No
DDAbs were detected in plasma of a control chimpanzee and rhesus
monkey. When platelets were incubated with plasma samples of rhesus
94-R021 pretreated with 10 mmol/L EDTA for 1 hour at 37°C, the
reaction of DDAbs with platelets was abolished (data not shown), as
described above for chimpanzee A-264 plasma (Fig 3C).
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| Fig 8.
Flow cytometric measurement of DDAbs in chimpanzee A-264,
control chimpanzee, rhesus monkey 94-R021, and control rhesus monkey
plasma alternatively using rhesus 94-R021 and human platelets in the
presence or absence of 1 µmol/L of B or A2-L. Data represent the mean ± SD from three measurements. Binding of DDAbs is plotted as a
relative fluorescence of FITC-labeled secondary antibody.
|
|
Reaction of DDAbs from chimpanzee A-264 and rhesus 94-R021 plasma
with platelets from type I Glanzmann's thrombasthenia patient.
As shown in Table 3, DDAbs from chimpanzee
A-264 as well as rhesus monkey 94-R021 plasma failed to react with
platelets from a patient with type I Glanzmann's thrombasthenia. This
further supports the critical role of GP IIb/IIIa in binding of DDAbs from chimpanzee A-264 and rhesus 94-R021 plasma to platelets.
View this table:
[in this window]
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|
Table 3.
Reaction of DDAbs From Chimpanzee A-264, Rhesus Monkey
94-R021, and Control Chimpanzee Plasma With Platelets From a
Glanzmann's Thrombasthenic Patient and a Control Human Subject
|
|
Detection of DDAbs using purified GP IIb/IIIa coated on ELISA
microplates.
DDAbs from chimpanzee A-264 plasma reacted in the presence of A2-L with
purified GP IIb/IIIa coated on ELISA microplates in the presence of
A2-L, providing a ratio of 7.4 (average of 2 independent measurements)
for absorbance in the presence over the absorbance in the absence of
A2-L. DDAbs failed to react with purified GP IIb/IIIa coated in the
absence of A2-L. This indicates that the epitopes recognized by DDAbs
are exposed on the surface of coated GP IIb/IIIa only in the presence
of the drug.
Effect of rabbit antihuman GP IIb/IIIa polyclonal antibody (RPAb).
RPAb raised against purified human GP IIb/IIIa blocks platelet
aggregation stimulated by ADP and the binding of fibrinogen and the
monoclonal antibody PAC1 to activated platelets.35 RPAb abolished binding of DDAbs from both chimpanzee A-264 and rhesus 94-R021 plasma to all primate platelets tested (data not shown), thus
indicating that the DDAbs from chimpanzee and rhesus monkey platelets
recognize epitopes on primate GP IIb/IIIa.
Screening for preexisting DDAbs in human plasma samples.
The discovery of pre-existing DDAbs in plasma samples of chimpanzee
A-264 and rhesus monkey 94-R021 and the consequent drug-induced thrombocytopenia in these primates stimulated a screening effort to
determine the incidence of such DDAbs in humans. We screened 1,032 human plasma samples from healthy, anonymous blood donors using the
flow cytometric assay described earlier and the FRAs that induced
thrombocytopenia in chimpanzee A-264. Results are summarized in Table
4. This screening identified 11 (1.1% ± 0.6%) plasma samples positive for A2-L-dependent and 8 (0.8% ± 0.6%) samples with A1-L-dependent antibodies. Only four
positive plasma samples were positive for DDAbs using both FRAs (Table
4). All positive plasma samples were then tested using other FRAs. As shown in Table 4, B and A3 stimulated binding of DDAbs to human platelets in some of the positive plasma samples. The flow cytomeric method could underestimate the incidence of DDAbs in human plasma if
low-affinity DDAbs are lost during the wash step.
| |
DISCUSSION |
These studies document the sudden appearance of profound
thrombocytopenia in one chimpanzee and one rhesus monkey upon
administration of the highly GP IIb/IIIa selective, potent FRAs A1-L,
A2-L, or B at doses that completely inhibited platelet aggregation.
Flow cytometric analysis identified DDAbs in the plasma of both
primates. The antibodies from these animals reacted with platelets from all primates tested only in the presence of the compound that induced
thrombocytopenia in that particular animal. Although these two cases of
thrombocytopenia are similar in the precipitous decrease in platelet
count immediately after drug administration, there is an obvious
difference in the rate of platelet recovery.
Kinetic studies of platelets in dogs with profound thrombocytopenia
induced by the infusion of an antiglycoprotein IIb/IIIa monoclonal
antibody demonstrated a dose-dependent precipitous decrease in platelet
count after administration of antibody.36 The recovery of
platelet count was also dose-related, with 3 hours required after 200 µg/kg and 12 hours after 300 µg/kg of antibody.32 The
onset of drug-induced thrombocytopenia in both primates described in
this report immediately followed the dosing of the drug.
The DDAbs identified in plasma of both primates were found in samples
collected before the dosing of the drug as well as in samples collected
several months after the drug-induced thrombocytopenia. These findings
suggest that the drug-dependent antiplatelet antibodies present in the
plasma of both primates before the first exposure to FRAs could have
mediated the observed thrombocytopenia.
FRAs A1-L and A2-L have high affinity for GP IIb/IIIa, and at the
concentrations used in chimpanzee A-264, they had an identical effect
on ex vivo platelet aggregation. Both compounds induced profound
thrombocytopenia in chimpanzee A-264; however, flow cytometric measurements of DDAbs indicate that the amount of antibody bound to the
platelets was lower when A1-L was used. Repeat incubation of platelets
with chimpanzee plasma in the presence of A2-L increased the amount of
bound antibody, whereas repeat incubation in the presence of A1-L had
little effect. The titer of DDAbs is low; however, the affinity of the
antibodies for the receptor must be high, because they survived the
washing step in the flow cytometric procedure. Thus, one can speculate
that the concentration and also the dissociation constant of
A2-L-dependent antibodies in chimpanzee A-264 plasma should be much
lower than the concentration of GP IIb/IIIa receptors. Platelets were
incubated with plasma at concentrations of 0.5 to 1.0 × 108 platelets/mL, which, assuming that the number of
receptors per platelet is 57,000 ± 12,000 (see Table 1), gives a
receptor concentration of approximately 4 to 10 nmol/L. The estimated
dissociation constants of A2-L-dependent antibodies should then be
subnanomolar, and the concentration of such antibodies in chimpanzee
plasma would be in the range of several nanomolar. The lower amount of
platelet-bound A1-L-dependent antibodies found by flow cytometry
likely indicates higher dissociation constants of these antibodies,
which prevented titration of the receptors after repeat incubations of
platelets with chimpanzee plasma. The cause of these differences in
binding affinities of DDAbs may stem from different effects of A1-L and A2-L on the conformation of the receptor and thus variability in the
exposed neoepitopes. We have recently demonstrated subtle differences
in the exposure of neoepitopes on GP IIb/IIIa upon ligand binding as
measured by monoclonal antibodies.37 The lack of binding of
DDAbs from chimpanzee A-264 plasma in the presence of A1-R, the
R-isomer of A1-L, highlights the sensitivity of neoantigen exposure to
minor changes in the structure of FRAs. The fact that neither of the
drugs that induced thrombocytopenia in chimpanzee A-264 affected the
platelet count or induced binding of DDAbs in rhesus 94-R021 also
underscores the importance of specific drug structure on the exposure
of LIBS for specific pre-existing antibodies. The specificity of
neoantigen exposure on the GP IIb/IIIa receptor to the ligand structure
is likely an explanation for different DDAbs found with different drugs
in human plasma samples. Similar effects of subtle changes in the
structure of drugs on neoepitopes exposure were described previously
for sulfonamide-dependent15 and quinine-dependent
antibodies.38 An analysis of plasma samples from patients
who experienced thrombocytopenia during clinical trials with FRAs would
help to determine if the DDAbs are always implicated in drug-induced
thrombocytopenia in humans.
Structural differences, and thus likely differences in the composition
of the epitope between A3 and the compounds that induced thrombocytopenia, could explain why this compound had no effect on
platelet count in either chimpanzee A-264 or rhesus monkey 94-R021. One
could also speculate that the affinity of the drug for the receptor
could have an important role, because both compounds that induced
thrombocytopenia in chimpanzee A-264 have high affinity for GP IIb/IIIa
with a half time for dissociation from the receptor of 20 and 45 minutes for A1-L and A2-L, respectively. The FRA B, which induced
thrombocytopenia in rhesus 94-R021, has a lower affinity for the
receptor than A1-L and A2-L, and the half time for dissociation from
the receptor is also much shorter.29 Interestingly, the
recovery of the platelet count after the administration of B to rhesus
94-R021 was much faster than it was in both cases of thrombocytopenia
in chimpanzee A-264, with higher affinity compounds. The affinity of A3
for resting platelets is low29 (Kd, 640 nmol/L). Because
receptor occupancy by the drug is required for binding of DDAbs to
platelets (as demonstrated in this report with A2-L-dependent
antibodies), the kinetics of drug binding that control the kinetics of
antibody binding may be critical. This is supported by the correlation
between the time course of thrombocytopenia and the rate of the
clearance of the FRAs that induced thrombocytopenia in chimpanzee A-264
and rhesus monkey 94-R021. In other words, the platelet count recovers
in the chimpanzee after several days, whereas in the rhesus monkey, it
recovers after several hours.
It is generally understood that in immune thrombocytopenia the
platelet-antiplatelet antibody complex is trapped via the Fc receptors in the reticuloendothelial system of the liver and
spleen.12,36 Based on our results, we hypothesize that the
mechanism by which the GP IIb/IIIa antagonists induce the antibody
binding to platelets requires binding of the antagonists to the
receptors. The binding to the receptors results in the conformational
change and an exposure of new epitopes on the surface of the GP
IIIb/IIIa. Antibodies in the plasma that bind to platelets only in the
presence of the drug recognize these neo-epitopes, as demonstrated by
flow cytometric measurements. Platelet-drug-antibody complex is then
captured via the Fc receptors in the reticuloendothelial systems of
the liver and the spleen. The removal of the antibodies and return of
the platelets to the circulation or eventual destruction of sequestered
platelets is likely controlled by the off-rate of antibodies, which
depends on the off-rate and Kd of the drug.
Highly integrin GP IIb/IIIa-selective FRAs, which induced
thrombocytopenia in both primates, suggest a critical role of GP IIb/IIIa in the mechanism of thrombocytopenia. DDAb binding to primate
platelets was abolished by incubating platelets either with EDTA at
37°C or with a rabbit antihuman GP IIb/IIIa polyclonal antibody that
blocks platelet aggregation and binding of fibrinogen to platelets.
These results, together with the identification of DDAbs using an ELISA
and coated purified GP IIb/IIIa, confirmed that DDAbs bind to epitopes
on GP IIb/IIIa. Flow cytometric measurements of DDAbs using platelets
from a patient with type I Glanzmann's thrombasthenia further
confirmed that the GP IIb/IIIa receptor is the target of DDAbs found in
chimpanzee A-264 and rhesus monkey 94-R021 plasma. Structural analysis
of the epitopes on GP IIb/IIIa by flow cytometric and plasmon resonance
measurements of competition between peptides derived from sequences of
GP IIb and GPIIIa and DDAbs are in progress. It is interesting that
autoantibodies to epitopes on GPIIb/IIIa have been reported in a low
percentage of otherwise healthy subjects in EDTA-dependent
thrombocytopenia.39
In summary, these results provide evidence to suggest that the
described drug-induced thrombocytopenia in chimpanzee A-264 and rhesus
monkey 94-R021 is caused by circulating IgG type DDAbs. In the presence
of specific FRA(s), these antibodies react with GP IIb/IIIa on all
primate platelets tested. The DDAbs were found in plasma samples
collected before the first exposure of the primates to any FRA. We
further showed that the incidence of DDAbs for two FRAs that induced
thrombocytopenia in chimpanzee A-264 was approximately 1% in 1,032 human plasma samples. It remains to be seen whether the apparent
correlation between the frequency of plasma samples positive for DDAbs
and the incidence of thrombocytopenia in clinical trials is just
coincidence, thus implicating alternate mechanisms in drug-induced
thrombocytopenia, or if the thrombocytopenia is purely immune mediated.
| |
ACKNOWLEDGMENT |
The authors greatly appreciate the significant contributions of Benny
C. Askew, Melissa S. Egbertson, and John H. Hutchinson for design and
synthesis of the FRAs used in these experiments; Maria T. Stranieri,
Gary R. Sitko, Joan D. Glass, and Raymond F. Stupienski for the careful
performance of numerous experiments with conscious nonhuman primates;
and Charles Chang and Lee Gaul for the screening of multiple human
plasma samples for the presence of DDAbs. We also thank Daniel M. Bollag for critical reading of the manuscript.
| |
FOOTNOTES |
Submitted June 1, 1998; accepted March 12, 1999.
B.B. and J.J.C. contributed equally to this report.
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 Bohumil Bednar, PhD,
Department of Pharmacology, Merck Research Laboratories, WP26-265,
Sumneytown Pike, West Point, PA 19486; e-mail:
bohumil_bednar{at}merck.com.
| |
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