Identification of anti-platelet autoantibodies by western blot in 45 patients with idiopathic thrombocytopenic purpura (ITP).

BACKGROUND

In sera and platelet eluates of ITP patients, antigen specificity was widely studied by means of sensitive methods including immunoprecipitation, monoclonal antibody immobilization, and immunoblot. These studies indicated that GPIIb-IIIa were the main epitopes of ITP autoantibodies.

METHODS

We studied the specificity of antiplatelet autoantibodies in 45 patients with acute and chronic ITP. Patient sera were tested by Western blot on separated platelet proteins in non-reducing conditions; antibody binding was identified using biotinconjugated anti-human IgG and avidin-peroxidase.

RESULTS

Two main nonspecific bands of 200 and 125 kD were visible using normal serum; the first referred to platelet IgG, and the second was due to a naturally occurring antibody towards an internal protein. Twenty-five sera (55%) stained one (n = 11), two (n = 7), three (n = 3) or four (n = 4) specific bands. In patients with chronic ITP there was a prevalence of multiple bands. The relative molecular weights of the recognized antigens were in the range of 140-160, 80-100, 50-70 and 40 kd. The 80-100 epitope was recognized as a membrane protein in only 40% of sera, and it was partially characterized as GPIIIa in 4 patients. The other stained epitopes were absorbed by platelet lysate and then identified as internal proteins.

CONCLUSIONS

This finding might be related to sensitization to antigens exposed by platelets during immune damage, and may pose an important problem in the identification with the immunoblot technique of target antigens responsible for immune sequestration.