Differential expression of a ligand induced binding site (LIBS) by GPIIb-IIIa ligand recognition peptides and parenteral antagonists

The glycoprotein (GP) IIb-IIIa complex is an attractive anti-platelet targe t for the prevention of thrombotic events associated with coronary artery d isease. Although GPIIb-IIIa antagonists inhibit GPIIb-IIIa binding to its l igands, the interactions have not been fully clarified, particularly with r espect to their ability to induce structural changes in the complex that le ad to exposure of neoantigenic epitopes or ligand-induced binding sites (LI ES). In this study we used the anti-LIES monoclonal antibody (mAb) D3 to fu rther define the activation states of purified active and inactive GPIIb-II Ia. We also compared the data obtained in the purified system to that obser ved with intact human platelets. Active GPIIb-IIIa expressed significantly greater high-affinity D3 LIES sites compared to the inactive form. In addit ion, the ligand recognition peptides RGDS and H12 caused increased expressi on of the D3 epitope, with RODS eliciting a much more potent response. The response of the purified GPIIb-IIIa to these peptides paralleled that obser ved with human platelets. To explore whether the platelet antagonists abcix imab, eptifibatide and tirofiban induced expression of the D3 LIES site, a modified competitive ELISA was developed. Our data indicate that the use of purified GPIIb-IIIa with this ELISA system provides a reproducible approac h for exploring the interactions between GPIIb-IIIa and its antagonists. Wh ereas abciximab caused no detectable increase in the expression of the D3 e pitope on purified GPIIb-IIIa, eptifibatide, tirofiban, RODS, and H12 induc ed differential expression of the high-affinity LIES. Studies with intact p latelets suggested that abciximab blocked the binding of the D3 and LIBS6 m Abs, and that the pre bound anti-LIES D3 sterically hindered abciximab bind ing.