Research article
Eptifibatide-induced thrombocytopenia and
thrombosis in humans require FcγRIIa and
the integrin β3 cytoplasmic domain
Cunji Gao,1 Brian Boylan,1 Dan Bougie,1 Joan C. Gill,2 Jessica Birenbaum,1 Debra K. Newman,1,3,4
Richard H. Aster,1,5 and Peter J. Newman1,4,6,7
1Blood
Research Institute, BloodCenter of Wisconsin, Milwaukee, Wisconsin, USA. 2Department of Pediatrics, 3Department of Microbiology,
of Pharmacology, 5Department of Medicine, 6Department of Cellular Biology, and 7Cardiovascular Research Center,
Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
4Department
Thrombocytopenia and thrombosis following treatment with the integrin αIIbβ3 antagonist eptifibatide are
rare complications caused by patient antibodies specific for ligand-occupied αIIbβ3. Whether such antibodies induce platelet clearance by simple opsonization, by inducing mild platelet activation, or both is poorly
understood. To gain insight into the mechanism by which eptifibatide-dependent antibodies initiate platelet
clearance, we incubated normal human platelets with patient serum containing an αIIbβ3-specific, eptifibatide-dependent antibody. We observed that in the presence of eptifibatide, patient IgG induced platelet secretion and aggregation as well as tyrosine phosphorylation of the integrin β3 cytoplasmic domain, the platelet
FcγRIIa Fc receptor, the protein-tyrosine kinase Syk, and phospholipase Cγ2. Each activation event was inhibited by preincubation of the platelets with Fab fragments of the FcγRIIa-specific mAb IV.3 or with the Src family kinase inhibitor PP2. Patient serum plus eptifibatide did not, however, activate platelets from a patient with
a variant form of Glanzmann thrombasthenia that expressed normal levels of FcγRIIa and the αIIbβ3 complex
but lacked most of the β3 cytoplasmic domain. Taken together, these data suggest a novel mechanism whereby
eptifibatide-dependent antibodies engage the integrin β3 subunit such that FcγRIIa and its downstream signaling components become activated, resulting in thrombocytopenia and a predisposition to thrombosis.
Introduction
The integrin αIIbβ3 (also known as glycoprotein IIb-IIIa [GPIIb-IIIa])
is a member of the integrin family of cell adhesion receptors and is
essential for normal hemostasis (1). Following platelet activation,
the αIIbβ3 complex undergoes a dramatic conformational change
that allows the adhesive protein fibrinogen to bind, forming a bridge
between platelets that mediates platelet-platelet interactions and
thrombus formation. Inappropriate activation of αIIbβ3 contributes
substantially to cardiovascular disease (2) — a leading cause of death
in the Western world (3). The development of effective fibrinogen
receptor antagonists (FRAs), therefore, has been a major advance in
the management of coronary artery diseases (4, 5).
Eptifibatide (Integrilin), one of several FDA-approved αIIbβ3
inhibitors, is a small, cyclic RGD-like heptapeptide that selectively
inhibits ligand binding to the αIIbβ3 complex and rapidly dissociates from its receptor after cessation of therapy (6, 7). Eptifibatide
has proven in numerous clinical trials to be effective in reducing
the frequency of adverse outcomes in patients with acute coronary
syndromes and secondary complications following percutaneous
transluminal coronary angioplasty (8–11).
Despite their clinical efficacy, administration of all parenteral
fibrinogen receptor antagonists, including eptifibatide, has been
shown to increase the incidence of clinically significant thromConflict of interest: P.J. Newman is a consultant for Novo Nordisk and a member of
the Scientific Advisory Board of the New York Blood Center.
Nonstandard abbreviations used: ddAb, drug-dependent antibody; ITAM, immunoreceptor tyrosine-based activation motif; LIBS, ligand-induced binding site; SFK,
Src family kinase.
Citation for this article: J. Clin. Invest. 119:504–511 (2009). doi:10.1172/JCI36745.
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bocytopenia (9, 10, 12–17). Though ligands that bind αIIbβ3 are
capable of directly inducing both integrin and platelet activation
(18–22), the acute thrombocytopenia that is infrequently observed
after administration of eptifibatide is thought to be most commonly caused by the binding of either preexisting or neoantigeninduced drug-dependent antibodies (ddAbs) that bind to the
αIIbβ3 complex in the presence of the drug (23). A recent case
study suggests that thrombosis might also be an additional rare
complication of eptifibatide therapy (24); however, whether this is
antibody mediated has not been investigated.
Though the mechanism by which eptifibatide-dependent antibodies clear platelets from circulation has not been well examined,
understanding the activating properties of other αIIbβ3-specific
antibodies may provide relevant insights. For example, although
the vast majority of murine mAbs that target the αIIbβ3 complex
have no effect on platelet activation, several are potent stimulators. Anti-αIIbβ3-specific platelet-activating antibodies appear to
fall into 2 broad categories. One class of mAbs, known as ligandinduced binding site (LIBS) antibodies, recognize conformational
epitopes that are exposed upon integrin activation, ligand binding,
or denaturation and activate platelets by stabilizing the open, or
active, conformation of the integrin, enabling the binding of multivalent ligands such as fibrinogen (25–27). Antibody-mediated
fibrinogen binding not only serves to bridge adjacent platelets but
also initiates a broad series of reactions, collectively termed “outside-in” signaling, that augment a wide range of platelet activation
responses, including shape change, granule secretion, and generation of cell-surface procoagulant activity (1).
The other class of activating αIIbβ3-specific murine mAbs all
appear to bind in such a way as to present their Fc regions, either
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Figure 1
Serum IgG from a patient who developed severe thrombocytopenia and thrombosis following eptifibatide treatment induces platelet aggregation and granule secretion.
(A) Washed human platelets were preincubated with
eptifibatide (Ept) in a lumi-aggregometer for 3 minutes
before addition of either patient or normal human serum.
Collagen-related peptide (CRP) was added to the normal
human platelets at the end of the experiment to demonstrate their ability to aggregate and secrete their granules. Note the scale on the y axis, indicating the blunted
aggregation response in the presence of the drug. Granule secretion induced by patient antibodies, in contrast,
was 100% of that induced by CRP, indicating the strong
degree of platelet activation induced by the eptifibatidedependent antibodies. (B) Patient serum was depleted
of IgG using protein G Sepharose beads to reduce IgG
content (SDS gel inset) and added to platelets in the
presence of eptifibatide. Note that patient serum has lost
its ability to activate platelets.
in cis (intraplatelet) or in trans (interplatelet) (28, 29) to the platelet
Fc receptor FcγRIIa — a 40-kDa integral membrane protein (30)
comprising 2 extracellular Ig-like domains, a single-pass transmembrane domain, and a 76-amino-acid cytoplasmic tail (31,
32) containing 2 YxxL sequences that together constitute a single
immunoreceptor tyrosine-based activation motif (ITAM) (33, 34).
Human platelets express, on average, approximately 3,000–5,000
copies of FcγRIIa per cell (35), and when the extracellular domains
of these receptors become engaged or crosslinked, associated Src
family kinases (SFKs) phosphorylate the ITAM tyrosines within the
cytoplasmic domain (36), creating a docking site for the tandem
Src homology 2 (SH2) domains of the protein-tyrosine kinase Syk
(34, 37). Recruitment of Syk to the phosphorylated ITAMs at the
inner face of the plasma membrane leads to its activation and subsequent assembly in lipid rafts of a multiprotein signaling complex
consisting of the adaptor molecules Cbl (38) and LAT (39–41), the
SFK Lyn (39, 42), PI3K (43–45), Tec family kinases Btk and Tec (46),
and PLCγ2 (39, 45). Once activated, PLCγ2, via its lipase activity,
generates lipid products that support a multitude of cellular activation responses, including integrin activation as well as platelet
secretion and aggregation. Downregulation of FcγRIIa signaling
appears to be accomplished through the activity of low-molecularweight protein tyrosine phosphatase (LMW-PTP), which dephosphorylates the ITAM tyrosines of FcγRIIa (41). PECAM-1 has also
been implicated in suppressing FcγRIIa function (47), though the
mechanism by which this occurs has not yet been defined.
Despite accumulating evidence that thrombocytopenia associated with administration of fibrinogen receptor antagonists is
immune in nature, the underlying mechanism by which this class
of ddAbs cause platelet clearance and, less frequently, thromboThe Journal of Clinical Investigation
sis, remains obscure. The purpose of the present investigation,
therefore, was to gain further insight into etiology of eptifibatideinduced thrombocytopenia and thrombosis. Here, we provide the
first evidence to our knowledge that these antibodies behave like
the class II murine anti-platelet mAbs described above, in that their
Fab regions interact with the αIIbβ3 complex, while the Fc region
of the antibody activates FcγRIIa on the same cell. We also report
the unexpected observation that the cytoplasmic domains of both
integrin β3 and FcγRIIa are required for drug-induced antibodymediated platelet activation to occur and propose what we believe
to be a novel mechanism for thrombocytopenia and thrombosis
following administration of fibrinogen receptor antagonists.
Results
An eptifibatide-dependent antibody that induces platelet secretion and
aggregation. To gain further insight into the mechanism of eptifibatide-induced, antibody-mediated thrombocytopenia and
thrombosis, we incubated normal human platelets with serum
from a patient who had developed an eptifibatide-dependent
antibody 2–3 days following administration of eptifibatide, and
we simultaneously measured platelet aggregation and granule
secretion in a lumi-aggregometer. As shown in Figure 1A, whereas
normal human serum in the presence of eptifibatide had no effect
on platelet activation, patient serum plus eptifibatide induced
marked platelet aggregation that was blunted, as expected, due to
the presence of the fibrinogen receptor antagonist eptifibatide. In
contrast, the degree of granule secretion induced by patient serum
plus eptifibatide approached that induced by strong agonists
such as collagen or collagen-related peptide (CRP; black tracing,
Figure 1A), demonstrating the potential for such antibodies to
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Figure 2
Requisite role for the FcγRIIa ITAM/Syk/PLCγ2 activation pathway in eptifibatide-dependent, antibody-induced platelet activation. (A) Fab fragments from normal mouse IgG or from the
blocking anti-FcγRIIa mAb IV.3 were incubated with human
platelets prior to addition of eptifibatide and patient serum. Note
that blocking FcγRIIa with IV.3 Fabs totally abrogates both platelet aggregation and secretion induced by the patient IgG antibody. (B) Platelets undergoing eptifibatide-induced, antibodydependent platelet aggregation in A were lysed directly in the
aggregometer cuvette by adding 2× lysis buffer, as described in
Methods. Levels of FcγRIIa, Syk, and PLCγ2 antigens (Ag) and
of their tyrosine-phosphorylated counterparts (PY) were detected
by Western blotting using antigen- or phosphotyrosine-specific
antibodies, as indicated. Note that the tyrosine phosphorylation
of all 3 signaling components induced by the binding of patient
antibody is almost completely blocked by preincubation of platelets with IV.3, but not control, Fabs.
fully activate platelets. Platelet activation was found to be caused
by antiplatelet antibodies in the patient serum, as removal of IgG
using Protein G Sepharose beads completely abrogated the ability
of the serum to activate platelets (Figure 1B). Addition of the ADP
scavenger apyrase had no effect (data not shown), indicating that
released ADP is not required for eptifibatide-dependent antibodyinduced platelet aggregation.
Role of platelet Fc receptor signaling in eptifibatide-induced, antibodymediated platelet activation. A small subset of murine mAbs specific
for αIIbβ3 bind to platelets in such a way topographically as to present their Fc region to the platelet Fc receptor FcγRIIa and activate
platelets (28, 29). Though there are as yet no accounts of human
drug-dependent anti-αIIbβ3 antibodies that activate platelets via
FcγRIIa, Pedicord et al. reported several years ago the production of
a murine mAb that bound αIIbβ3 only in the presence of the oral
αIIbβ3 antagonist roxifiban and activated platelets via interactions
of its Fc region with FcγRIIa (48). To determine whether FcγRIIa
might be similarly involved in platelet activation by human eptifibatide-dependent ddAbs, we repeated the experiment described in
Figure 1, but with platelets that had been pretreated with Fab fragments of the FcγRIIa blocking antibody IV.3. As shown in Figure
2A, IV.3 Fabs completely blocked both granule secretion and aggregation induced by the eptifibatide-dependent antibody.
We obtained further evidence that FcγRIIa mediates platelet activation by the patient antibody by examining specific elements of
the FcγRIIa signal transduction pathway (36, 37, 45). As shown
in Figure 2B, addition of patient serum in the presence, but not
absence, of eptifibatide resulted in phosphorylation of FcγRIIa
ITAM tyrosines, the protein-tyrosine kinase Syk, and its downstream effector PLCγ2. These biochemical activation events were
all blocked in platelets that had been preincubated with IV.3 Fabs,
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again demonstrating the requisite role for FcγRIIa in eptifibatidedependent, antibody binding–induced platelet activation.
Phosphorylation of FcγRIIa ITAMs by Src family protein-tyrosine kinases is thought to be a early proximal event following
engagement of the FcγRIIa extracellular domain (36), enabling the
recruitment and activation of Syk to the inner face of the plasma
membrane (34, 37, 43). To confirm that binding of eptifibatidedependent patient antibodies activated the FcγRIIa/Syk/PLCγ2
pathway in an SFK-dependent manner, we pretreated platelets
with the pan-Src inhibitor PP2 before adding eptifibatide and
patient serum. As shown in Figure 3, PP2, but not its nonfunctional control analog PP3, completely blocked granule secretion,
platelet aggregation, and signaling events downstream of FcγRIIa
engagement induced by the eptifibatide-dependent antibody, confirming a requisite role for one or more SFKs in this process.
The integrin β3 cytoplasmic domain is required for ddAb-mediated
platelet activation. As illustrated schematically in Figure 4A, eptifibatide-dependent antibodies engage the extracellular domains of
the αIIbβ3 complexes via their Fab regions and FcγRIIa via their Fc
regions. As a consequence, the potential exists for SFKs associated
with the cytoplasmic domains of either the integrin β3 subunit
(49–52) or FcγRIIa (39, 42) to function as ITAM kinases. To determine whether GPIIIa-associated SFKs might be required for initiating signaling downstream of antibody engagement, we examined
the ability of eptifibatide-dependent antibodies to activate platelets that express normal levels (Figure 4B) of a truncated, mutant
form of the integrin β3 subunit cytoplasmic domain that lacks
SFK binding sequences (Supplemental Figure 1, A and B; supplemental material available online with this article; doi:10.1172/
JCI36745DS1). As was observed in a previously reported Glanzmann thrombasthenic patient harboring a D724 mutation (53),
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from normal platelets treated with eptifibatide but had no effect
on untreated platelets. Platelet activation induced by each of these
3 samples was completely blocked by mAb IV.3, suggesting a common mechanism of platelet activation — i.e., antibody-mediated
bridging of the αIIbβ3 complex and FcγRIIa (data not shown).
Figure 3
Role of SFKs in eptifibatide-dependent, antibody-induced platelet activation. Washed human platelets were pretreated with 25 μM pan-Src
inhibitor PP2 or its nonreactive analog PP3 for 5 minutes at 37°C prior
to addition of eptifibatide plus patient serum. (A) PP2 blocks antibody
binding–induced platelet aggregation and secretion. (B) Dependency
of SFKs on antibody binding–initiated activation of the primary components of the FcγRIIa signaling pathway.
the extracellular domain of this variant αIIbβ3 complex retains
its normal structure and function, as evidenced by its ability to
bind fibrinogen in the presence of Mn2+ (Supplemental Figure 1C)
and bind the LIBS-specific mAb D3 (26) in an RGD-dependent
manner (data not shown). FcγRIIa was also expressed at normal
levels on these Glanzmann thrombasthenic platelets (Figure 4B)
and able, upon engagement, to mediate robust granule secretion (Supplemental Figure 2) and platelet aggregation (data not
shown), demonstrating that the FcγRIIa/SFK/Syk/PLCγ2 signal
transduction pathway was fully operable in the platelets from this
variant thrombasthenic patient. As shown in Figure 4C, however,
despite having normal levels of αIIbβ3 on the surface and functional FcγRIIa-mediated signaling, platelets from the D724 variant
thrombasthenic patient did not become activated by eptifibatidedependent antibodies. These data demonstrate that functional
FcγRIIa-associated SFKs in themselves are not sufficient to initiate
eptifibatide-dependent, antibody binding–induced platelet activation. Rather, the αIIbβ3 cytoplasmic domain, and likely its associated SFKs, are also required.
Incidence of platelet-activating eptifibatide-dependent patient antibodies. Antibodies reactive with eptifibatide-coated platelets were
detected, using a well-established, sensitive flow cytometric assay
(23), in the serum of 26 of 42 patients referred to BloodCenter
of Wisconsin’s Diagnostic Platelet and Neutrophil Immunology
Laboratory for suspected eptifibatide-induced thrombocytopenia (23). Serum from 3 of the 26 antibody-positive patients (12%)
induced marked aggregation and granule secretion release of ATP
The Journal of Clinical Investigation
Discussion
Though accumulating evidence supports an immune etiology
for tirofiban- and eptifibatide-induced thrombocytopenia (23),
exactly how tirofiban- or eptifibatide-dependent antibodies
mediate platelet clearance is poorly understood. The observation
that some tirofiban-dependent antibodies are capable of directly
inducing platelet granule secretion (54, 55), together with a recent
report of eptifibatide-induced thrombocytopenia associated with
an increase in circulating procoagulant, platelet-derived microparticles (56), suggested to us that platelet activation might, in some
instances, contribute to the occasional thrombocytopenia and,
rarely, thrombosis (24) that are observed following administration
of these αIIbβ3 ligand–mimetic compounds. The mechanism by
which such antibodies might activate the platelets to which they
are bound, however, is completely unknown.
The first antibody reported to activate human platelets as a consequence of its binding was a murine mAb specific for the tetraspanin
CD9 (57). While most antiplatelet antibodies bind to the platelet
surface without inducing platelet activation, a subset bind to their
target antigens with a topographical orientation that causes them
to elicit strong platelet activation, leading to granule secretion and
platelet aggregation. The range of cell-surface receptors to which
mAbs can bind and activate platelets is large and includes, in addition to CD9, the αIIbβ3 complex (58), CD36 (59), β2-microglobin
(60), class I histocompatibility antigen (61), JAM-A (62), and the
Gas6 receptors Axl, Sky, and Mer (63). With the exception of LIBS
antibodies, which bind to or induce an active conformer of the
αIIbβ3 complex (25–27), and antibodies to kinase domain–containing receptors (63), most of the remaining murine mAbs, including a
recently described murine drug-dependent mAb specific for αIIbβ3
(48), appear to activate platelets by forming inter- or intraplatelet
bridges between their target antigen and the platelet Fc receptor
FcγRIIa (28, 29). Like their murine counterparts, human allo- (64),
auto- (65, 66), and ddAbs induced by currently FDA-approved
fibrinogen receptor antagonists (23, 54–56) have also been implicated in platelet activation. The mechanism by which such human
antiplatelet antibodies activate platelets, however, is not known.
The major finding of the present work is that certain patient antibodies specific for the eptifibatide-bound αIIbβ3 complex activate
platelets by engaging the integrin via their Fab regions and FcγRIIa
via their Fc, regions. While there is no evidence for a direct physical
association between αIIbβ3 and FcγRIIa, and they cannot be coimmunoprecipitated from detergent lysates (P.J. Newman and C. Gao,
unpublished observations), they do appear to be topographically
close to each other on the platelet surface, as evidenced by the finding
that several αIIbβ3-specific mAbs, when prebound, are able to sterically block the binding of the anti-FcγRIIa mAb IV.3 (67, 68). Because
αIIbβ3 complexes are present at relatively high density on the platelet
surface (~40,000–80,000 per platelet; refs. 69, 70), it would seem that
any αIIbβ3-bound antibody whose Fc domain is oriented in such a
way as to engage a single FcγRIIa molecule, even though it is present
at much lower density (3,000–5,000 copies/platelet; ref. 35), would
have the potential to initiate FcγRIIa-mediated signaling. Further
studies are needed to examine the range of antigen/drug/antibody
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Figure 4
Evidence for β3 cytoplasmic domain–associated kinases in initiating eptifibatide antibody-induced platelet activation. (A) Schematic
representation of an eptifibatide-dependent antibody simultaneously engaging both the αIIbβ3 complex and FcγRIIa, resulting
in SFK-mediated phosphorylation of FcγRIIa ITAM tyrosines,
recruitment of Syk, and activation of PLCγ2, ultimately resulting in
platelet aggregation and granule secretion. Note that GPIIIa-associated Fyn and Src are brought into close proximity with FcγRIIaassociated Lyn as a result of antibody-mediated bridging of the
extracellular domains of these 2 receptors. SH2, Src homology 2.
Cal DAG-GEF is a guanine nucleotide exchange factor for Rap1.
(B) Flow cytometric analysis of αIIbβ3 (detected with mAb AP2)
and FcγRIIa (detected with mAb IV.3) expression on normal versus D724 Glanzmann thrombasthenic (GT) patient platelets analyzed in C. Note normal levels of both. Numbers above each peak
indicate the median fluorescence intensity. Adapted with permission from the American Society of Hematology (82). (C) Failure of
eptifibatide-dependent antibodies to activate FcγRIIa on platelets
expressing a truncated β3 cytoplasmic domain.
combinations that can result in not only opsonization, but also
activation, of platelets and thereby contribute to clinically relevant
thrombocytopenia and occasional thrombosis.
Perhaps the most unanticipated finding of the present work is
the strict requirement for the integrin β3 cytoplasmic domain in
initiating platelet activation induced by eptifibatide-dependent
patient antibodies. Though the molecular components are different, the mechanism of action by which such antibodies are able
to induce platelet secretion and aggregation is not unlike that
underlying cytokine and growth factor receptor signaling, in which
homo- or heterodimeric receptors are brought into close approximation, resulting in transactivation of intrinsic or associated tyrosine kinases. Similarly, when the extracellular domains of 2 or more
FcγRIIa molecules are brought together by IgG immune complexes
(often simulated experimentally by addition of heat-aggregated
IgG or mAb IV.3 plus anti-mouse IgG), homodimerization or multimerization occurs, allowing an SFK-mediated chain reaction to
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begin (illustrated in Supplemental Figure 2), quickly resulting
in robust platelet activation. Likewise, eptifibatide-dependent
antibody-mediated clustering of αIIbβ3, via the Fab domain of
the antibody, with FcγRIIa, via its Fc region (illustrated schematically in Figure 4A), leads to transactivation of integrin- and
Fc receptor–associated protein tyrosine kinases, which function
either directly or indirectly as ITAM kinases to facilitate the
assembly of a signaling complex that initiates platelet activation. Evidence for this model derives from the observation that
addition of the SFK inhibitor PP2 completely abrogates platelet activation by eptifibatide-dependent antibodies (Figure 3)
and that such patient antibodies are unable to activate platelets
that express a mutant integrin lacking most of the cytoplasmic
domain of GPIIIa (Figure 4). Whether other molecular players
are involved in eptifibatide-dependent antibody-induced platelet activation, and whether all activating patient antibodies act
via the same mechanism, is currently under investigation.
The observation that a human eptifibatide-dependent antibody can initiate FcγRIIa-mediated signal transduction leading to granule secretion and residual platelet aggregation in
the presence of the potent αIIbβ3 antagonist eptifibatide (Figures 1–4) strongly suggests that αIIbβ3-independent events are
involved. McGregor et al. showed nearly 20 years ago that platelets from a patient with type I Glanzmann thrombasthenic exhibited residual aggregation and near-normal granule secretion in
response to stimulation with collagen (71) — a strong agonist that
activates platelets via essentially the same ITAM/Syk/PLCγ2 pathway employed by FcγRIIa. In experiments not shown, eptifibatidedependent, antibody-mediated platelet aggregation was induced
in the presence of a 10-fold-higher concentration of eptifibatide
than that employed in Figures 1–4 (i.e., 67.0 versus 6.7 μg/ml) or in
the presence of patient serum plus eptifibatide plus 20 mg/ml of
AP2 — an αIIbβ3 complex–specific mAb that blocks both fibrinogen binding and platelet aggregation (72). Taken together, these
data support the notion that patient antibodies bridging αIIbβ3
and FcγRIIa induce platelet granule secretion and residual aggregation in a αIIbβ3-independent manner. This αIIbβ3-independent pathway of aggregation may be restricted to circumstances
where αIIbβ3 blockade occurs or may even be activated under
such circumstances. It is also possible that αIIbβ3-independent
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aggregation observed here may be restricted to specific individuals. Because RGD-containing ligands such as fibrinogen, vWF, and
fibronectin are prevented from binding to αIIbβ3 in the presence
of eptifibatide, other receptor/ligand pairs are likely to be mediating platelet-platelet interactions. While we have not yet addressed
this issue, CD36/thrombospondin, P-selectin/PSGL-1, and GPIb/
vWF all seem like plausible candidates, since ligands for each of
these receptor/ligand pairs are released from platelet α-granules
following FcγRIIa-mediated platelet activation.
Finally, given (a) that the density of FcγRIIa can vary by as much
as 2- to 3-fold from individual to individual (35) and that the level
of FcγRIIa expression likely affects platelet responsiveness (73–76);
(b) that the 2 allelic isoforms (Arg131 versus His131) of FcγRIIa
might also contribute to its ability to stimulate platelets (74, 77);
and, as reported herein, (c) the observations of the obligatory
involvement of FcγRIIa in thrombocytopenia and thrombosis in
a large subset of eptifibatide-dependent patient antibodies, further studies appear to be needed to examine whether prescreening
patients for FcγRIIa genotype and/or expression level are warranted
before administration of αIIbβ3 antagonists.
Methods
Patient studies. The index case — a 73-year-old man admitted to a local hospital with partial obstruction of the right coronary artery and a platelet
count of 212,000/μl — was one of 42 patients referred to the BloodCenter
of Wisconsin’s Diagnostic Platelet and Neutrophil Immunology Laboratory who developed thrombocytopenia after being given eptifibatide for
prevention of thrombotic complications following percutaneous transluminal coronary angioplasty (23). The patient was given intravenous infusions of unfractionated porcine heparin and eptifibatide prior to stent
implantation. Approximately 12 hours later, he suffered an acute inferior
wall myocardial infarction. A thrombosed stent in the right coronary artery
was reopened and eptifibatide was restarted with heparin and clopidogrel.
Eighteen hours later, a large hematoma was identified at the site of catheter entry in the right groin, and his platelet count dropped to 25,000/μl.
Heparin and eptifibatide were discontinued. Serological analysis for heparin-dependent antibodies was negative, but eptifibatide-dependent antibodies were found by flow cytometry. Platelet counts remained less than
38,000/μl for the next 3 days. On day 6, a deep venous thrombosis was
identified in the right leg and treated with warfarin and low-molecularweight heparin. The platelet count rose to 244,000/μl on day 7, and the
patient was discharged on day 9 with no further hematologic or cardiac
abnormalities for the next 6 months.
Reagents and antibodies. The synthetic peptides RGDW and CRP (78)
were synthesized by the Protein Chemistry Core Laboratory at the Blood
Research Institute, BloodCenter of Wisconsin. Eptifibatide was obtained
by prescription from a local pharmacy. Luciferase and PGE1 were purchased
from Chrono-Log Corp. and Sigma-Aldrich, respectively. Wortmannin,
PP2, and PP3 were from Calbiochem. The αIIbβ3-specific mAbs AP2 and
AP3 have been previously described (69, 79). The hybridoma cell line secreting the FcγRIIa-specific mAb IV.3 (30, 80) was purchased from ATCC. A
nonblocking FcγRIIa-specific mAb conjugated to FITC was obtained from
BD Biosciences — Pharmingen. Polyclonal antibodies specific for PLCγ2,
phosphotyrosine759 of PLCγ2, and phosphotyrosine525, 526 of Syk were purchased from Cell Signaling Technology. Mouse anti–human Syk and normal human IgG were obtained from Santa Cruz Biotechnology Inc. Anti–
phospho-tyrosine mAb (PY-20) was from Zymed. AP2 and AP3 were labeled
with Cy3 by using a commercial kit from GE Healthcare, while mAb IV.3
was labeled with Alexa Fluor 488 by using a labeling kit from Invitrogen.
Fab fragments of mAbs IV.3, AP2, and AP3 were produced by using IgG1
The Journal of Clinical Investigation
Fab and F(ab′)2 Preparation Kit (Pierce Biotechnology; Thermo Scientific).
Purified fibrinogen was provided by Michael Mosesson (Blood Research
Institute, BloodCenter of Wisconsin) and labeled with FITC according to
previously described methods (81).
Platelet aggregation and secretion. All studies using human patient samples
were reviewed and approved by the Institutional Review Board of the BloodCenter of Wisconsin, with appropriate informed consent of the participants.
Blood samples were collected into 3.8% sodium citrate, diluted 1:1 with modified Tyrode’s-HEPES buffer, and then allowed to “rest” by incubation at
room temperature for 10 minutes. Platelet-rich plasma (PRP) was prepared
by low-speed centrifugation, washed into modified Tyrode’s-HEPES containing 50 ng/ml PGE1 and 5 mM EDTA, and finally resuspended in Tyrode’sHEPES containing 1 mM CaCl2 to a final concentration of 3.0 × 108/ml.
Platelet aggregation was performed at 37°C in a Chrono-Log whole blood
lumi-aggregometer in the presence of luciferase to simultaneously measure
light transmission and secretion of dense granule–derived ATP. For selected
studies, platelets were obtained, with parent-provided informed consent,
from a two-year-old male child with a variant form of Glanzmann thrombasthenia. DNA sequence analysis revealed a C2268T homozygous mutation
within exon 13 of the patient’s β3 gene that encodes an Arg724Stop mutation in both alleles (Supplemental Figure 1). This results in the expression of
a truncated form of GPIIIa whose cytoplasmic domain contains only 8 of 47
residues. The expression levels of this mutant αIIbβ3 complex on the platelet
surface were normal (see below).
Immunoprecipitation and Western blot analysis. Platelet detergent lysates were
prepared by adding and equal volume of ice-cold 2× lysis buffer (30 mM
HEPES [pH 7.4], 300 mM NaCl, 20 mM EGTA, 0.2 mM MgCl2, 2% Triton
X-100) containing 2× protease and phosphatase inhibitor cocktail (Calbiochem; EMD) directly to the aggregometer cuvette. Syk, phospho-Syk,
PLCγ2, and phospho-PLCγ2 were examined by Western blot analysis of
total platelet lysates, while the phosphorylation state of FcγRIIa was measured after immunoprecipitation with IV.3, followed by capture of immune
complexes using protein G Sepharose beads (Amersham Biosciences; GE
Healthcare). Following SDS-PAGE, immunoprecipitated proteins were
transferred to polyvinylidene fluoride membranes and visualized using an
ECL detection kit (Amersham Biosciences; GE Healthcare).
Flow cytometry. Washed human platelets (50 μl) at a concentration of
2 × 108/ml were incubated with 5 μg/ml of the indicated mAbs for 1 hour at
room temperature. Platelets were washed in 1 ml HEN (0.1 M HEPES, 1 mM
EDTA, 50 mM NaCl, pH 7.4), resuspended in 2 μg/ml PE-conjugated goat
anti-mouse IgG (Jackson ImmunoResearch Laboratories Inc.) for 45 minutes,
and then analyzed on a BD LSR II flow cytometer. In some experiments, the
platelet activation state was evaluated by the addition of 125 μg/ml of FITClabeled fibrinogen in the presence of 1 mM Mn2+ versus 2 mM EDTA.
Acknowledgments
This work was supported by grant HL-44612 from the National
Heart, Lung, and Blood Institute of the NIH.
Received for publication July 10, 2008, and accepted in revised
form December 17, 2008.
Address correspondence to: Peter J. Newman, Blood Research
Institute, BloodCenter of Wisconsin, PO Box 2178, 638 N. 18th
Street, Milwaukee, Wisconsin 53201, USA. Phone: (414) 937-6237;
Fax: (414) 937-6284; E-mail: peter.newman@bcw.edu.
Portions of this work were presented in abstract form at the 49th
Annual Meeting of the American Society of Hematology in Atlanta,
Georgia, USA, December 8–11, 2007.
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