Location of the Quinine-Dependent Epitope on Platelet Glycoprotein IlIa

Abstract

Quinine-dependent (Q) IgG antibodies (Q.Ab) in drug-induced immune thrombocytopenia are heterogeneous and bind to different platelet surface glycoproteins (GP), namely GPIb, IX, lIb, IlIa and an unidentified 57-kDa membrane proteins. Although both the Q-dependent epitope on GPIIIa and the P1^A1 antigen require intact disulphide bonds for their expression, they are distinct because Q.Ab bind to GPIIIa lacking P1^A1. Epitopes for both antigens were examined on Western blots of either intact washed human platelets or purified GPIIIa. When intact platelets were digested with trypsin and washed and solubilised prior to electrophoresis, membrane-associated fragments of GPIIIa of 78 kDa were found to be reactive with both antibodies. In addition, 60- and 68-kDa fragments bound anti-P1^A1 but not Q.Ab. Similar digestion with chymotrypsin produced only 60-kDa fragments containing both epitopes. Digestion of purified GPIIIa with chymotrypsin produced 60-kDa peptides reactive with Q.Ab and anti-P1^A1 in immunoblotting studies. Similar digestion with elastase produced 58-kDa fragments also containing the epitopes for both antibodies. Longer digestion times or sequential digestion with different enzymes did not reveal extra fragments. However, immunoprecipitation of trypsin-digested ¹²⁵I-labelled GPIIIa with affinity-purified Q.Ab produced a 17-kDa fragment containing the Q-dependent epitope.