J.A. Peterson, D.W. Bougie, T.J. Gaulke, G.P. Visentin, R.H. Aster
Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53201, USA
Abstract


Drug-induced immune thrombocytopenia is caused by an unusual class of Ab that recognizes epitopes on platelet membrane glycoproteins (GP) only when drug is present in soluble form.
Little is known about how soluble drugs promote high affinity binding of drug-dependent Abs (DDAbs) to specific GP targets, in part because the epitopes recognized have not been precisely defined.
We studied three quinine-induced DDAbs that react with non-reduced GPIIIa in Western blotting and are blocked by the GPIIIa-specific monoclonal Ab (mAb) AP3, suggesting that they are specific for a linear peptide sequence in GPIIIa.
To identify target epitopes, we used the fact that these Abs do not recognize rat GPIIIa, although it is 80% identical to human GPIIIa.
In studies of rat/human hybrid molecules, we found that the three DDAbs and mAb AP3 require human sequence only at residues 50-98, a region in which human GPIIIa differs from rat by only 9 amino acids (AA 50, 62, 63, 66, 67, 71, 77, 78 and 80).
Conversion of AA 50, 62, and 63 in rat GPIIIa to the corresponding human residues was sufficient to create the epitope required for AP3 binding. However, the three DDAbs also required human residues at positions 66 and 67.
A reciprocal effect (loss of binding) was seen when the same AA in human GPIIIa were converted to the corresponding rat residues.
Our findings indicate that the three DDAbs require five AA within GPIIIa residues 50-67 for quinine-dependent recognition of their target.
This region of GPIIIa may be a common motif recognized by DDAbs induced by quinine.
Characterization of its tertiary structure may enable construction of a model to explain how drugs promote binding of DDAb to a specific target and cause platelet destruction.