Dataset Information

Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcgammaRIIa and the integrin beta3 cytoplasmic domain.

ABSTRACT: Thrombocytopenia and thrombosis following treatment with the integrin alphaIIbbeta3 antagonist eptifibatide are rare complications caused by patient antibodies specific for ligand-occupied alphaIIbbeta3. 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 alphaIIbbeta3-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 beta3 cytoplasmic domain, the platelet FcgammaRIIa Fc receptor, the protein-tyrosine kinase Syk, and phospholipase Cgamma2. Each activation event was inhibited by preincubation of the platelets with Fab fragments of the FcgammaRIIa-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 FcgammaRIIa and the alphaIIbbeta3 complex but lacked most of the beta3 cytoplasmic domain. Taken together, these data suggest a novel mechanism whereby eptifibatide-dependent antibodies engage the integrin beta3 subunit such that FcgammaRIIa and its downstream signaling components become activated, resulting in thrombocytopenia and a predisposition to thrombosis.

SUBMITTER: Gao C

PROVIDER: S-EPMC2648674 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcgammaRIIa and the integrin beta3 cytoplasmic domain.

Gao Cunji C Boylan Brian B Bougie Dan D Gill Joan C JC Birenbaum Jessica J Newman Debra K DK Aster Richard H RH Newman Peter J PJ

The Journal of clinical investigation 20090209 3

Thrombocytopenia and thrombosis following treatment with the integrin alphaIIbbeta3 antagonist eptifibatide are rare complications caused by patient antibodies specific for ligand-occupied alphaIIbbeta3. 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 patien ...[more]

PMID: 19197137

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Project description:Integrin alpha(IIb)beta(3) plays a pivotal role in hemostasis and thrombosis by mediating adhesive interactions of platelets. Binding of alpha(IIb)beta(3) to its physiological ligands, immobilized fibrinogen and fibrin, induces outside-in signaling in platelets, leading to their adhesion and spreading even without prior stimulation by agonists. Implicit in these phenomena is a requirement for the linkage between integrins' cytoplasmic tails and intracellular proteins. However, the nature of the initiating signal has not been established. In this study, we examined whether binding of alpha(IIb)beta(3) to immobilized fibrin(ogen), per se, triggers interaction of the integrin with cytoplasmic proteins. Using the integrin-binding skelemin fragment as a marker of exposure of residues involved in the clasp between alpha(IIb) and beta(3) cytoplasmic tails, we showed that its binding site in the membrane-proximal beta(3) 715-730 segment is cryptic and becomes exposed as a result of binding of isolated alpha(IIb)beta(3) to immobilized ligands. Furthermore, the skelemin-like protein present in platelets and CHO cells does not associate with alpha(IIb)beta(3) in resting platelets or suspended alpha(IIb)beta(3)-expressing CHO cells but is recruited to integrin during cell adhesion. In addition, not only beta(3) but also the membrane-proximal 989-1000 segment of the alpha(IIb) cytoplasmic tail binds the skelemin fragment. Finally, the same residues, alpha(IIb) Val(990), alpha(IIb) Arg(995), and beta(3) His(722), involved in the formation of the clasp between the tails are also required for skelemin binding. These studies suggest that ligation of alpha(IIb)beta(3) by immobilized ligands during platelet adhesion induces a transmembrane conformation change in the integrin, resulting in unclasping of the complex between the membrane-proximal parts of cytoplasmic tails, thereby unmasking residues involved in binding the skelemin-like protein. Thus, the junction between alpha(IIb) and beta(3) cytoplasmic tails may contain the critical structural information for the initiation of outside-in signaling.

Project description:Using green fluorescent protein (GFP) as an autofluorescent tag, we report the first successful visualization of a beta3 integrin in a living cell. GFP fused in frame to the cytoplasmic tail of either alphaIIb or beta3 allowed normal expression, heterodimerization, processing and surface exposure of alphaIIbGFPbeta3 and alphaIIb(beta3)GFP receptors in Chinese hamster ovary (CHO) cells. Direct microscopic observation of the autofluorescent cells in suspension following antibody-induced alphaIIb(beta3) capping revealed an intense autofluorescent cap corresponding to unlabelled immunoclustered GFP-tagged alphaIIb(beta3). GFP-tagged alphaIIbbeta3 receptors mediated fibrinogen-dependent cell adhesion, were readily detectable in focal adhesions of unstained living cells and triggered p125(FAK) tyrosine phosphorylation similar to wild-type alphaIIb(beta3) (where FAK corresponds to focal adhesion kinase). However, GFP tagged to beta3, but not to alphaIIb, induced spontaneous CHO cell aggregation in the presence of soluble fibrinogen, as well as binding of the fibrinogen mimetic monoclonal antibody PAC1 in the absence of alphaIIb(beta3) receptor activation. Time-lapse imaging of living transfectants revealed a characteristic redistribution of GFP-tagged alphaIIb(beta3) during the early stages of cell attachment and spreading, starting with alphaIIb(beta3) clustering at the rim of the cell contact area, that gradually overlapped with the boundary of the attached cell, and, with the onset of cell spreading, to a reorganization of alphaIIb(beta3) in focal adhesions. Taken together, our results demonstrate that (1) fusion of GFP to the cytoplasmic tail of either alphaIIb or beta3 integrin subunits allows normal cell surface expression of a functional receptor, and (2) structural modification of the beta3 integrin cytoplasmic tail, rather than the alphaIIb subunit, plays a major role in alphaIIb(beta3) affinity modulation. With the successful direct visualization of functional alphaIIb(beta3) receptors in living cells, the generation of autofluorescent integrins in transgenic animals will become possible, allowing new approaches to study the dynamics of integrin functions.

Dataset Information

Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcgammaRIIa and the integrin beta3 cytoplasmic domain.

Publications

Eptifibatide-induced thrombocytopenia and thrombosis in humans require FcgammaRIIa and the integrin beta3 cytoplasmic domain.

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