Reversible conformational changes induced in glycoprotein IIb-IIIa by a potent and selective peptidomimetic inhibitor

Abstract

Platelet glycoprotein (GP) IIb-IIIa inhibitors may become useful antithrombotic agents. Ro 4–5054 is a low molecular weight, noncyclic, peptidomimetic inhibitor that is three orders of magnitude more potent than RGDS in inhibiting fibrinogen binding to purified GPIIb-IIIa and in preventing platelet aggregation. Comparisons of RGDS and Ro 4–5054 in cell adhesion assays showed that, in contrast to RGDS, Ro 4–5054 was highly selective GPIIb-IIIa inhibitor. Effects of RGDV and Ro 4– 5054 on the conformation and activation state of GPIIb-IIIa were also examined. RGDV and Ro 4–5054 induced conformational changes in purified inactive GPIIb-IIIa as determined by binding of the monoclonal antibody D3GP3 (D3). These conformational alterations were not reversed after inhibitor removal, as indicated by the continued exposure of the D3 epitope and a newly acquired ability to bind fibrinogen. Similarly, RGDV and Ro 4–5054 induced conformational changes in GPIIb-IIIa on the intact platelet. However, after removal of the inhibitors, exposure of the D3 epitope was fully reversed and the platelets did not aggregate in the absence of agonist. Thus, while RGD(X) peptides and Ro 4–5054 transformed purified inactive GPIIb-IIIa into an irreversibly activated conformer, the effects of these inhibitors were reversible on the intact platelet. This suggests that factors present in the platelet membrane or cytoplasm may regulate in part the ability of the complex to shift between active and inactive conformers.

The Amino-terminal One-third of αIIb Defines the Ligand Recognition Specificity of Integrin αIIbβ3

“…To determine the capacity of the chimeras containing substitutions of the divalent cation repeats to bind small activation-independent ligands specific for ␣ IIb ␤ 3 , we examined the capacity of the ␣ IIb ␤ 3 -selective peptidomimetic Ro 43-5054 (38,51) to increase the binding of mAb anti-LIBS1 by flow cytometry. Since the mAb anti-LIBS1 binds preferentially to the occupied conformation of the receptor (41), increased binding of mAb LIBS1 is evidence of receptor-ligand interaction.…”

Tetrafibricin, a novel non-peptide fibrinogen receptor antagonist, induces conformational changes in glycoprotein IIb/IIIa

“…pl-80 is a useful tool to probe conformational changes in GPIIb/IIIa; however, there are different anti-LIBS antibodies with different epitopes on GPIIb/IIIa [24,31,42]. Moreover, ligand-induced conformational changes in the GPIIb/IIIa complex have been demonstrated from various approaches, such as susceptibility to proteolytic cleavage, decreases in intrinsic fluorescence and sedimentation coefficient, and increases in stroke radius [43,44]. We cannot, therefore, draw a simple conclusion that the conformational changes induced by tetrafibricin were exactly the same as those induced by RGDS.…”

“…The purification of GPIIb/IIIa was performed as described previously [26,27]. Briefly, the Triton X-100 lysate of human platelets from outdated blood was applied to a concanavalin A affinity chromatography column.…”

“…The quantitative assay to examine the expression of the pl-80 epitope on GPIIb/IIIa was performed as described previously [27]. Briefly, the wells of microtitre plates were coated with 2 ,tg/ml of pl-80 (100 ,ul/well) overnight at 4°C in buffer B (150 mM NaCl, 1 mM MgCl2, 20 mM Tris/HCl, pH 7.4).…”

Thrombocytopenia Resulting from Sensitivity to GPIIb-IIIa Inhibitors

“…It is known that binding of RGD peptide and RGD-mimetic drugs to GPIIb-IIIa produces conformational changes in the integrin that are detected by monoclonal antibodies specific for ligand-induced binding sites (LIBS). 66,70,71 It is tempting to speculate that antibodies causing thrombocytopenia in patients treated with ligandmimetic GPIIb-IIIa inhibitors may be specific for LIBS determinants on GPIIb-IIIa. A recent report is consistent with this possibility 62 but requires confirmation.…”