Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic immune thrombocytopenic purpura

Abstract

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to antiplatelet autoantibodies, many of which are directed against platelet membrane glycoprotein (GP) IIb-IIIa or GPIb-IX. In a recent study, we described plasma autoantibodies from 13 selected ITP patients, which required the presence of the putative GPIIIa cytoplasmic region for antibody binding. Since this region may not be available for antibody binding under physiologic conditions, we evaluated the frequency of binding to this or other regions of GPIIb- IIIa by platelet-associated and plasma autoantibody from a group of chronic ITP patients. We studied platelet-associated autoantibodies in 27 patients and plasma antibodies in 21 patients; in 15 patients, both were studied. To determine if autoantibodies were directed to the cytoplasmic portion of GPIIIa or to another portion of the GPIIb-IIIa molecule, antibody eluted from patient platelets or plasma antibody was tested in an antigen capture assay for binding to GPIIb-IIIa obtained from Chinese hamster ovary (CHO) cells transfected with GPIIb and either intact GPIIIa or GPIIIa lacking the carboxy terminal 35 residues. Of the 21 plasma autoantibodies tested, 13 bound primarily to the carboxy terminus of GPIIIa and eight to other epitopes. Conversely, all 26 platelet-associated autoantibodies, including eight of the 13 with anti-carboxy terminus antibodies, bound to epitopes in other regions of GPIIb-IIIa. Comparison of the degree of antibody adsorption by intact or lysed platelets indicated that epitopes on the c-terminal region of GPIIIa are relatively inaccessible on the surface of intact washed platelets when compared with other epitopes. We conclude that the importance of plasma autoantibodies in chronic ITP patients should be interpreted cautiously, since their specificity may differ from that of antibodies bound to the platelet. Whether antibodies against the c- terminus of GPIIIa are of pathogenetic importance remains to be determined, although patients with these antibodies have particularly severe disease.

Quantitation of platelet‐specific autoantibodies in platelet eluates of ITP patients measured by a novel ELISA using the purified glycoprotein complexes GPIIb/IIIa and GPIb/IX as antigens

“…The data using P-GP-ELISA suggest that free Ab differ from Ab bound to the Plt in specificity and/or affinity, i.e. plasma Ab are not just the unbound remainder of Plt-associated Ab, which is consistent with previous reports (McMillan et al, 1987;Fujisawa et al, 1992). Therefore it has to be emphasized that the importance of circulating Ab in ITP patients should be interpreted very cautiously.…”

“…In an intact Plt, epitopes on the cytoplasmic domain of membrane proteins are inaccessible. Nevertheless, such epitopes may be recognized by plasma Ab (Fujisawa et al, 1992), possibly provoked as a secondary phenomenon of Plt destruction. Since in P-GP-ELISA, Pltglycoprotein-binding Ab were eluted from intact Plt, Ab directed against cytoplasmic domains should not be present.…”

International study to compare antigen‐specific methods used for the measurement of antiplatelet autoantibodies

“…These findings support the view that measurement of plasma autoantibodies against platelet glycoproteins is inferior to methods for platelet-associated autoantibodies in AITP. In addition, it has been shown that the specificity of plasma antibodies may differ from that of antibodies bound to platelets and that some of them may bind to cytoplasmic epitopes (Fujisawa et al, 1991(Fujisawa et al, , 1992. This suggests that measurement of plasma autoantibodies may be misleading in the evaluation of AITP.…”

“…These autoantibodies are detected in the majority of AITP patients as platelet-associated antibodies, whereas plasma autoantibodies are noted in a smaller number of patients Berchtold & Wenger, 1993). This difference may be important, since the specificity of antibodies bound to the platelet may differ from that of plasma antibodies and some of the latter are directed against antigens on the cytoplasmic portion of GPIIIa (Fujisawa et al, 1991(Fujisawa et al, , 1992. been developed Kiefel et al, 1987).…”

Anti‐platelet antibodies in patients with systemic lupus erythematosus and the primary antiphospholipid antibody syndrome: their relationship with the observed thrombocytopenia

“…In comparison, chronic idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease caused by circulating antibodies that react with the target antigen on the platelet membrane, with the consequence that the platelets are recognized and eliminated by the host's immune system. Specific tests that characterize the target of such anti-platelet antibodies have shown that many are directed against surface glycoproteins such as GP IIb-IIIa (Woods et al, 1984b;Fujisawa et al, 1992) and GP Ib-IX (Woods et al, 1984a;Kiefel et al, 1991). Other glycoproteins implicated in isolated cases include GP Ia-IIa (Deckmyn et al, 1990(Deckmyn et al, , 1994, GP IV (Beer et al, 1993) and GP V (Mayer & Beardsley, 1996).…”