910 Thromb Haemost 2002; 87: 910 –7 © 2002 Schattauer GmbH, Stuttgart Tirofiban Blocks Platelet Adhesion to Fibrin with Minimal Perturbation of GpIIb/IIIa Structure Roy R. Hantgan 1 , Mary C. Stahle 1 , W. Gray Jerome 2 , Chandrasekaran Nagaswami 3 , John W. Weisel 3 Departments of 1 Biochemistry and 2 Pathology , Wake Forest University School of Medicine, Winston-Salem, NC and 3 Department of Cell and Developmental Biology , University of Pennsylvania School of Medicine, Philadelphia, PA, USA Keywords GpIIb/IIIa, integrin  IIb  3 , integrin antagonist, fibrin, structure Summary A biophysical approach tested the hypothesis that tirofiban, like eptifibatide, perturbs GpIIb/IIIa structure. Tirofiban bound tightly to platelet GpIIb/IIIa (EC 50 ~ 24 nmol/L) and effectively inhibited platelet aggregation (IC 50 ~ 37 nmol/L) but blocked platelet adhesion to clotted fibrin only at much higher doses (IC 50 ~ 580 nmol/L). Electrophoretic analyses demonstrated that tirofiban protected GpIIb/IIIa from SDS- induced subunit dissociation. However, saturating tirofiban concentra- tions had little or no effect on GpIIb/IIIa secondary or tertiary structure, as determined by circular dichroic spectroscopy, dynamic light scat- tering, and sedimentation velocity measurements performed with purified receptors in octyl glucoside. Moderate dose-dependent effects on GpIIb/IIIa quaternary structure were detected by sedimentation equilibrium. Transmission electron microscopy showed minimal tiro- fiban-induced receptor activation or oligomerization. Thus, even at the increased concentrations needed to block platelet:fibrin adhesive inter- actions, tirofiban exhibited only limited effects on GpIIb/IIIa con- formation and clustering. Our results provide new insights into the mechanisms and potential prothrombotic complications of integrin antagonists. Introduction Tirofiban (Aggrastat) is a potent, selective Glycoprotein IIb/IIIa (GpIIb/IIIa) inhibitor (1, 2) whose effectiveness in treating acute coro- nary syndromes has been established in three large-scale clinical trials: RESTORE, PRISM, and PRISM-PLUS (3-5). However, tirofiban and the other integrin antagonists abciximab and eptifibatide have small in- creased risks of hemorrhage and thrombocytopenia (4-7). Their possi- ble prothrombotic effects, especially if administered without heparin, have also raised concern (8). In fact, the observation of increased mor- tality halted the tirofiban-only arm of the PRISM-PLUS trial (4). The issue of thrombotic complications associated with GpIIb/IIIa inhibitors has recently received increased attention, as a pooled analysis of the data emerging from four clinical trials of a new generation of orally active GpIIb/IIIa antagonists has demonstrated a highly significant 37% excess mortality (9). The extent to which receptor activation contributes to the prothrom- botic effects of GpIIb/IIIa inhibitors remains controversial. Formation of neo-antigenic sites, termed Ligand-Induced Binding Sites (LIBS) (10, 11), has been demonstrated for tirofiban, eptifibatide, and abcixi- mab, suggesting an integrin-antagonist class-effect (1, 12, 13). While Peter et al. (14) reported that abciximab and the RGD analog fradifiban induced fibrinogen binding and platelet aggregation, a recent report challenges this conclusion and attributes the apparent receptor-acti- vating effects of integrin antagonists to artifactual thrombin genera- tion (15). Given this dilemma, there is a critical need for a better understanding of the molecular mechanisms by which integrin antagonists function. We have developed an integrated biophysical and electron microscopy strategy to detect and to describe the effects of low-molecular weight integrin antagonists on the conformation and oligomerization state of the purified GpIIb/IIIa complex (16, 17). Our unique approach uses spectroscopic “nanometersticks” to avoid the steric hindrance that limits the resolution of immunoprobes. We have recently shown that eptifibatide, at near-stoichiometric concentrations, shifts a conforma- tional equilibrium toward an “open” receptor with increased frictional drag, a distinct subunit separation, and enhanced self-association, or clustering (17). Here, we will demonstrate that while increased tirofi- ban concentrations are needed to block the strong multisite interactions that stabilize a platelet:fibrin thrombus, these concentrations had mini- mal effects on the secondary and tertiary structure of the GpIIb/IIIa complex and induced only moderate GpIIb/IIIa receptor clustering. Materials and Methods Reagents AGGRASTAT (tirofiban hydrochloride, L-tyrosine-N-(butylsulfonly)-O- [-4-4(piperidinebutyl)] monohydrochloride) was provided (Merck, West Point, PA) as a dry powder and stored in the dark at room temperature for up to 18 months with no loss in activity. Tyrosine was obtained from Sigma Chemical Co. (St. Louis, MO). Tirofiban and tyrosine concentrations were determined spectrally using experimentally determined molar extinction coefficients ( 275 ) of 1099 ± 122 L/mol-cm and 1126 ± 106 L/mol-cm, respectively. Highly puri- fied human fibrinogen (free of plasminogen and Factor XIII) was purchased from American Diagnostica (Greenwich, CT) and highly purified human -thrombin from Sigma Chemical Co. (St. Louis, MO). Platelet Isolation and Characterization Procedures Blood was obtained by venipuncture from healthy, adult volunteer donors into 1/10 vol of sodium citrate (110 mmol/L) anticoagulant. The Clinical Re- search Practices Committee of Wake Forest University School of Medicine fully examined and approved these procedures. Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were isolated by differential centrifugation. Gel-fil- tered platelets were isolated from PRP in HEPES-Tyrode’s-Albumin buffer Correspondence to: Roy R. Hantgan, PhD, Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, Win- ston-Salem, NC 27157-1019, USA – Tel.: 336-716-4675; Fax: 336-716-7671; E-mail: rhantgan@wfubmc.edu For personal or educational use only. No other uses without permission. All rights reserved. Downloaded from www.thrombosis-online.com on 2018-04-27 | ID: 1001066444 | IP: 54.70.40.11