echistatin and roxifiban

This investigation addressed the paradox that disintegrins and small RGD-ligands readily bind to the resting alphaIIbbeta3 integrin, while macromolecules with similar integrin recognition motifs require an activated, or primed, receptor. Three structurally similar pharmaceutical integrin antagonists (eptifibatide, tirofiban, and roxifiban) were each incubated with resting alphaIIbbeta3; after drug wash-out, the receptor's ability to recognize PAC-1, an activation-dependent IgM with an RYD integrin-targeting site was measured. Their promotion of PAC-1:alphaIIbbeta3 binding (solid phase assay), eptifibatide > tirofiban > roxifiban, correlated with their ability to shift the receptor to an open conformer, as measured by analytical ultracentrifugation. Surface plasmon resonance (SPR) demonstrated that PAC-1 bound rapidly (k(on) approximately 5 x 10(5) l/mol-s, 25 degrees C) and tightly (Kd approximately 1 nM) to eptifibatide-primed integrins, captured on a biosensor using an IgG specific for alphaIIb's cytoplasmic domain. Varying the interval between integrin capture and antagonist dissociation indicated that transiently primed alphaIIbbeta3 retains the ability to rapidly bind PAC-1 from 2-90 min, although the dissociation rate increased at later times, indicative of a weakening of the complex. Fluorescence anisotropy (fluorophore-tagged analogue exchange assay) demonstrated that eptifibatide dissociates rapidly from alphaIIbbeta3 (half-time <2 min), consistent with the priming window determined by SPR. van't Hoff analysis of alphaIIbbeta3:PAC-1's temperature-dependent Kd indicated entropy/enthalpy compensation, similar to (resting) integrin binding to the disintegrin echistatin. Eyring analysis of k(on) yielded DeltaG degrees approximately 10 kcal/mol for PAC-1 binding to primed alphaIIbbeta3, 3 kcal/mol lower than that of echistatin. These observations suggest that priming lowers the transition-state energy barrier, enabling rapid macromolecular ligand binding to activated integrins. Recognizing the limitations in extrapolating from laboratory to pathophysiological conditions, we propose that similar priming mechanisms may contribute to the unexpected platelet-activating effects of pharmaceutical integrin antagonists.

Topics: Amidines; Dual Specificity Phosphatase 2; Eptifibatide; Integrins; Intercellular Signaling Peptides and Proteins; Isoxazoles; Models, Molecular; Peptides; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Thermodynamics; Tirofiban; Tyrosine

Drugs that block platelet-platelet and platelet-fibrin interactions via the alpha(IIb)beta(3) (glycoprotein IIb/IIIa) receptor are used daily in patients undergoing percutaneous coronary interventions. Along with expected increases in spontaneous bleeding, clinical trials have revealed a surprising increase in thrombosis when these drugs are used without other anticoagulants. A better understanding of their mechanisms can minimize these risks.. This study tested the hypothesis that interventions designed to block fibrinogen binding inevitably leave the alpha(IIb)beta(3) receptor in an activated state. It compared the effects on platelet function and alpha(IIb)beta(3) conformation of the orally active compounds orbofiban and roxifiban, the i.v. agents eptifibatide and tirofiban, and echistatin, an arginine-glycine-aspartate (RGD) disintegrin.. The integrin antagonist concentrations required to saturate platelets and to block platelet-platelet and platelet-fibrin interactions were determined by flow cytometry, aggregometry, and clot-based adhesion assays, respectively. Analytical ultracentrifugation measured each antagonist's effects on the solution structure of alpha(IIb)beta(3). Fluorescence anisotropy provided equilibrium and kinetic data for integrin:antagonist interactions.. Both orally active drugs bound more tightly and inhibited platelet aggregation and adhesion to fibrin more effectively than echistatin. Analytical ultracentrifugation yielded this order for perturbing alpha(IIb)beta(3) conformation (priming) and promoting oligomerization (clustering): echistatin > eptifibatide > orbofiban > tirofiban > roxifiban. Roxifiban was also most effective at disrupting the rapidly forming/slowly dissociating alpha(IIb)beta(3):echistatin complex.. Our results suggest that the same molecular mechanisms that enable glycoprotein IIb/IIIa inhibitors to bind tightly to the alpha(IIb)beta(3) receptor and block fibrinogen binding contribute to their ability to perturb the resting integrin's conformation, thus limiting the safety and efficacy of both oral and i.v. integrin antagonists.

Topics: Administration, Oral; Alanine; Amidines; Binding, Competitive; Blood Platelets; Dimerization; Dose-Response Relationship, Drug; Eptifibatide; Female; Fibrinogen; Humans; In Vitro Techniques; Injections, Intravenous; Intercellular Signaling Peptides and Proteins; Isoxazoles; Kinetics; Male; Models, Molecular; Peptides; Platelet Activation; Platelet Aggregation Inhibitors; Platelet Function Tests; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Binding; Protein Conformation; Pyrrolidines; Reference Values; Structure-Activity Relationship; Tirofiban; Tyrosine