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.
Thrombocytopenia and thrombosis following treatment with the glycoprotein αIibβ3 antagonist, eptifibatide, are rare complications that are thought to be 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 not well understood. To gain insight into the mechanism by which eptifibatide-dependent antibodies cause platelet clearance, we incubated normal platelets with serum derived from a 73 year old patient who developed profound thrombocytopenia following eptifibatide administration. This patient’s serum was found to contain an αIibβ3-specific eptifibatide-dependent antibody that induced significant platelet secretion and aggregation in the presence, but not in the absence, of eptifibatide. Interestingly, a number of signaling molecules recently shown to function downstream of integrin engagement, including the integrin β3 cytoplasmic domain, the protein-tyrosine kinase Syk, and phospholipase Cγ2 (PLCγ2) each became tyrosine phosphorylated in platelets subjected to patient serum plus eptifibatide. Because the Immunoreceptor Tyrosine-based Activation Motif (ITAM)-bearing platelet Fc receptor, FcγRIIa, plays a prominent role in antibody-induced platelet activation, we examined its role in eptifibatide-induced platelet activation and clearance. We found that (1) FcγRIIa became rapidly and strongly tyrosine phosphorylated following incubation of normal platelets with patient serum+eptifibatide and (2) pre-incubation of platelets with Fab fragments of the FcγRIIa-specific monoclonal antibody, IV.3, completely blocked both phosphorylation of FcγRIIa, Syk, and PLCγ2, as well as platelet secretion and aggregation induced by patient serum+eptifibatide. Because amounts of patient sera are often limiting, a model system comprised of the αIibβ3 complex-specific mAb, AP2, and the ligand mimetic peptide, RGDW was employed, and found to faithfully mimic these reactions. Thus, intact, but not Fab fragments of, AP2 caused tyrosine phosphorylation of FcγRIIa, Syk, PLCγ2, as well as platelet secretion and aggregation, and these reactions could be completely blocked with Fab fragments of mAb IV.3, further implicating FcγRIIa in eptifibatide-induced thrombocytopenia/thrombosis. Finally, to gain further insight into how integrins functionally link to FcγRIIa to activate platelets, we subjected platelets from a patient with a variant form of Glanzmann thrombasthenia whose platelets express normal levels of αIibβ3 and FcγRIIa, but lack most of the β3 cytoplasmic domain, to similar treatment. Neither patient serum+eptifibatide nor AP2+RGDW were able to activate this patient’s platelets, implicating the β3 cytoplasmic domain and its associated tyrosine kinases in eptifibatide-induced platelet activation. Taken together, these data suggest a novel mechanism involving both FcγRIIa and the cytoplasmic domain of integrin β3 in thrombocytopenia and thrombosis following administration of fibrinogen receptor antagonists.
Numerous drugs are known to cause immune thrombocytopenia (TP) mediated by antibodies (abs) that bind to platelets only when the sensitizing drug is present in soluble form. The widely used antibiotic, vancomycin, has been implicated as a cause of TP only in 8 case reports and little is known about antibodies possibly responsible for this complication. We characterized clinical and serologic aspects of TP occurring in 39 patients during treatment with vancomycin. In this group, TP developed after 1–27 days of treatment (median 6 days) and plt nadirs ranged from 1,000 to 60,000 plts/uL (median 14,000 plts/uL). Bleeding occurred in 14 patients and contributed to a fatal outcome in 3. TP persisted for 1–17 days after discontinuing vancomycin (median 7 days). Platelets eventually returned to baseline in all surviving patients. Serum obtained after the onset of TP was studied for vancomycin-dependent, platelet-reactive abs by flow cytometry and by solid phase ELISA using immobilized plt glycoproteins (GP) as targets. Vancomycin-dependent antibodies detected in patients and normal subjects IgG only IgM only IgG + IgM No antibody Total Patients with TP 21 (54%) 5 (13%) 13 (33%) (0)%) 39 Normal individuals 58 (21%) 4 (1%) 1 (0%) 210 (77%) 273 Results of flow cytometric studies are summarized above. All patients had IgG and/or IgM abs that reacted with normal plts in the presence, but not in the absence of vancomycin. The IgG mean fluorescence intensity (MFI) signal in the presence of drug was 1.6 to 32.0 times stronger (mean ratio 5.7) than that obtained in the absence of drug. Vancomycin-dependent IgG abs were also identified in 59 of 273 normal individuals but were much weaker than abs detected in patients (p = 0.004). IgM abs (mean ratio 5.7, range 1.6 – 34) were found in 18 of 39 patients (46%). Weaker IgM abs were found in only 5 of 273 normal subjects (1.8%) (p = 0.001). Two patient abs studied in ELISA reacted preferentially with GPIIb/IIIa. Studies to determine the frequency of abs in patients given vancomycin who do not develop TP are in progress. These findings provide evidence that TP in patients given vancomycin can be caused by drug-dependent abs specific for GPIIb/IIIa that are stimulated by vancomycin exposure. IgG and IgM abs in patients with TP are generally much stronger than drug-dependent, platelet-reactive immunoglobulins found in some normal subjects. The significance of the latter abs is presently unknown. Drug-dependent IgM abs are found almost exclusively in patients with vancomycin-associated TP and may be diagnostic. Patients treated with vancomycin often have life-threatening bacterial sepsis and may have various reasons for developing TP. Serologic testing for drug-dependent, platelet-reactive IgG and IgM abs may provide a means of identifying those in whom vancomycin should be discontinued.
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