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.

Determination of Kinetic Constants for the Interaction Between the Platelet Glycoprotein IIb-IIIa and Fibrinogen by Means of Surface Plasmon Resonance

“…The second population of immobilized glycoprotein IIb-IIIa is formed following contact with fibrinogen or RGDV. It has been demonstrated earlier that the binding of RGDS or fibrinogen to glycoprotein IIb-IIIa induces conformational changes within the receptor (Du et al, 1991 ;Kouns et al, 1992) that result in the exposure of neo-epitopes called LIBS. It is, therefore, likely that these ligand induced conformational changes cause a slowing down of the association as well as of the dissociation process.…”

Prevention of thrombosis and rethrombosis and enhancement of the thrombolytic actions of recombinant tissue‐type plasminogen activator in the canine heart by DMP728, a glycoprotein IIb/IIIa antagonist

“…Furthermore, blockade of the GPIIb/IIIa receptor with monoclonal antibodies has proved to be antiaggregatory in experimental (Mickelson et al, 1990a,b;Bates et al, 1991;Rote et al, 1994a) and clinical studies (Coller, 1990;Gold et al, 1990;Kleiman et al, 1993). More recently, peptidomimetic and non-peptide RGD mimetics have been developed in an effort to achieve greater antithrombotic potency and selectivity at the site of the GPIIb/IIIa receptor (Kouns et al, 1992;Peerlinck et al, 1993;Rote et al, 1993;Roux et al, 1993). The present study provides experimental findings with DMP728, a non-peptidomimetic antagonist of the platelet GPIIb/IIIa integrin receptor.…”

Stimulation of platelet glycoprotein IIb‐IIIa (αIIbβ3‐integrin) functional activity by a monoclonal antibody to the N‐terminal region of glycoprotein IIIa

“…Ligand binding induces additional conformational changes which cause exposure of ligand-induced binding sites (LIBS) neoepitopes [4][5][6]. Similar transformations of GPIIb-Ilia are stimulated via occupation of the receptor by RGD peptides [4][5][6] or peptidomimetics [7] which bind to a resting form of the complex. Alterations in GPIIb-IIIa are also induced by platelet adhesion [8].…”

“…These antibodies are directed against epitopes within *Corresponding author. Fax: (7) (95) 414 6699 or 415 2962.…”