Autoantibodies in Patients with ITP Do Not Bind to Patient-Specific Epitopes on Platelet Glycoproteins.

Abstract Idiopathic thrombocytopenic purpura (ITP) remains primarily a diagnosis of exclusion. Evidence indicates that the thrombocytopenia is caused by a shortened platelet lifespan mediated by autoantibodies directed to platelet surface glycoproteins. Methods to detect the autoantibodies vary in their sensitivity and specificity. Earlier tests that measure only the amount of immunoglobulin associated with the platelet were unable to differentiate immune from non-immune thrombocytopenia. Tests that measure glycoprotein-specific autoantibodies directly on the patient platelets are more useful. Measurements of platelet-associated autoantibodies to GPIIbIIIa and GPIbIX using monoclonal-based assays (antigen capture assay) demonstrate a high specificity (>90%) and moderate sensitivity (60%). However, detection of circulating plasma autoantibodies in patients with ITP is relatively insensitive. One proposal is that the autoantibodies bind to restricted conformational epitopes found on patient, but not normal target platelets. To investigate this, we studied samples from patients sent to the McMaster Blood Disorders Clinic for investigation of thrombocytopenia. We identified 22 patients who were consistently positive for autoantibodies in the direct platelet antigen capture assay and used these to study the binding of plasma anti-platelet autoantibodies in the indirect platelet antigen capture assay. Direct testing of the patient platelets demonstrated IgG autoantibodies to GPIIbIIIa (OD 0.34 to 3.21, mean 1.52) and to GP Ib/IX (OD 0.29 to 1.99, mean 0.72). Plasma/serum from the same sample dates was used for indirect testing. Normal target platelets were incubated with 0.5 mL of patient plasma and then tested for bound IgG antibodies to specific glycoproteins. Of 22 patient samples tested, the majority (15/22) were negative in the indirect assay. Only 7 (32%) demonstrated weak binding of plasma antibodies to normal platelets. IgG antibodies to GPIIbIIIa (OD 0.25 to 0.52, mean 0.31) were detected in all 7, and antibodies to GP IbIX (OD 0.31) in one patient sample. These 7 patients had demonstrated high level of autoantibodies on their platelets in the direct assay (OD 0.50 to 3.21, mean 1.90). We then investigated whether the platelet-bound autoantibodies measured in the direct assay using the patient platelets, would recognize epitopes on normal target platelets. We eluted the IgG from the platelets of 9 patients (pH 2.8 citrate buffer) who were positive in the direct antigen capture assay. The eluates were incubated with normal platelets, which were then tested for GPIIbIIIa and GPIbIX specific antibodies. Of the 9 samples, 7 demonstrated efficient rebinding of the IgG to normal target platelets (pre-eluted samples: OD 0.41 to 2.79, mean 1.97; eluted/rebound samples: OD 0.42 to 2.87, mean 1.90). The 2 samples that did not rebind to normal targets had weak binding in the standard direct assay (OD 0.34 and 0.51). These studies investigated and compared plasma and platelet-bound autoantibodies in ITP patients who had high levels of IgG antibodies to GPIIbIIIa or GPIbIX on their platelets. We found that the majority of these patients do not have detectable autoantibody in their plasma, and that only low levels of plasma anti-platelet antibody are detectable in those patients with the highest levels of platelet-bound autoantibodies. The platelet-bound autoantibodies can be eluted from patient platelets and rebound to normal target platelets. These studies indicate that antiplatelet autoantibodies in patients with ITP recognize epitopes expressed on both patient and normal target platelets, and suggest that patient-dependent conformational antigens do not account for the low levels of circulating autoantibody detected in patient plasma.

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