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
), 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
), 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
). Like their murine counterparts, human allo- (64
), auto- (65
), and ddAbs induced by currently FDA-approved fibrinogen receptor antagonists (23
) 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
). Because αIIbβ3 complexes are present at relatively high density on the platelet surface (~40,000–80,000 per platelet; refs. 69
), 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 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 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 A), 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 ) and that such patient antibodies are unable to activate platelets that express a mutant integrin lacking most of the cytoplasmic domain of GPIIIa (Figure ). 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 –) 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, eptifibatide-dependent, antibody-mediated platelet aggregation was induced in the presence of a 10-fold-higher concentration of eptifibatide than that employed in Figures – (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 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
); (b) that the 2 allelic isoforms (Arg131 versus His131) of FcγRIIa might also contribute to its ability to stimulate platelets (74
); 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.