Binding of a fibrinogen mimetic stabilizesintegrin �IIb�3’s open conformation
ROY R. HANTGAN,1 MATTIA ROCCO,2 CHANDRASEKARAN NAGASWAMI,3 AND
JOHN W. WEISEL3
1Department of Biochemistry, Wake Forest University School of Medicine, Winston-Salem, North Carolina27157, USA2U.O. Biologia Strutturale, Istituto Nazionale per la Ricerca sul Cancro (IST), c/o CBA, Genova, Italy I-161323Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia, Pennsylvania19104, USA
(RECEIVED JANUARY 22, 2001; FINAL REVISION MAY 6, 2001; ACCEPTED MAY 16, 2001)
Abstract
The platelet integrin �IIb�3 is representative of a class of heterodimeric receptors that upon activation bindextracellular macromolecular ligands and form signaling clusters. This study examined how occupancy of�IIb�3’s fibrinogen binding site affected the receptor’s solution structure and stability. Eptifibatide, anintegrin antagonist developed to treat cardiovascular disease, served as a high-affinity, monovalent modelligand with fibrinogen-like selectivity for �IIb�3. Eptifibatide binding promptly and reversibly perturbedthe conformation of the �IIb�3 complex. Ligand-specific decreases in its diffusion and sedimentationcoefficient were observed at near-stoichiometric eptifibatide concentrations, in contrast to the receptor-perturbing effects of RGD ligands that we previously observed only at a 70-fold molar excess. Eptifibatidepromoted �IIb�3 dimerization 10-fold more effectively than less selective RGD ligands, as determined bysedimentation equilibrium. Eptifibatide-bound integrin receptors displayed an ectodomain separation andenhanced assembly of dimers and larger oligomers linked through their stalk regions, as seen by transmis-sion electron microscopy. Ligation with eptifibatide protected �IIb�3 from SDS-induced subunit dissocia-tion, an effect on electrophoretic mobility not seen with RGD ligands. Despite its distinct cleft, the openconformer resisted guanidine unfolding as effectively as the ligand-free integrin. Thus, we provide the firstdemonstration that binding a monovalent ligand to �IIb�3’s extracellular fibrinogen-recognition site sta-bilizes the receptor’s open conformation and enhances self-association through its distant transmembraneand/or cytoplasmic domains. By showing how eptifibatide and RGD peptides, ligands with distinct bindingsites, each affects �IIb�3’s conformation, our findings provide new mechanistic insights into ligand-linkedintegrin activation, clustering and signaling.
The �IIb�3 integrin is the prototypical member of a familyof integral membrane proteins that maintain communicationand contact between a cell’s interior and its external envi-
ronment (Critchley et al. 1999; Giancotti and Ruoslahti1999; Hynes 1992; Plow et al. 2000). Like many integrins,the 226-kD �IIb�3 complex must be activated before it canfunction as a high-affinity receptor for adhesive proteins(Hughes and Pfaff 1998; Shattil et al. 1998; Liddington andBankston 2000;). Ordinarily, �IIb�3 resides embedded inthe plasma membrane of human blood platelets where po-tentially saturating concentrations of its primary physiologi-cal ligand, the 340-kD protein fibrinogen, surround its
Reprint requests to: Roy R. Hantgan, Ph.D., Department of Biochemis-try, Wake Forest University School of Medicine, Medical Center Boule-vard, Winston-Salem, NC 27157, USA; e-mail: [email protected];fax: 336-716-7671.
Article and publication are at http://www.proteinscience.org/cgi/doi/10.1101/ps.3001.
globular ligand-recognition domain (Phillips et al. 1988;Shattil 1999). However, contacts are only established fol-lowing vascular injury, when internal molecular messengersinitiate conformational changes (Sims et al. 1991) that aretransmitted outward from its cytoplasmic regions, throughits entwined membrane-spanning polypeptides (Du et al.1993), and to its large extracellular domain. These processeslead to a functional integrin capable of fibrinogen binding(Ginsberg et al. 1992).
Like other integrins, �IIb�3 can signal bidirectionally(Dedhar and Hannigan 1996). Ligand binding to its ectodo-main soon links the 15-nm-distant cytoplasmic domain andthe actin cytoskeleton (Hughes and Pfaff 1998; Giancottiand Ruoslahti 1999). Conformational changes in the �IIb�3receptor, including increased self-association or clustering,are considered to be involved in this process of outside-insignaling (Shattil et al. 1998; Giancotti and Ruoslahti, 1999;Hantgan et al. 1999). Although high-resolution structuraldata are still emerging (Leahy 1997; Dickeson and Santoro1998; Emsley et al. 2000), much remains to be learnedabout the molecular bases for integrin activation, ligandbinding, and signal transduction.
�IIb�3’s extracellular domain contains distinct yet inter-acting ligand-binding sites with specificity for fibronectin orfibrinogen (Wippler et al. 1994; Peterson et al. 1998; Hu etal. 1999). Synthetic peptides that contain fibronectin’s Arg-Gly-Asp-Ser (RGDS) integrin-targeting motif (Ruoslahtiand Pierschbacher 1987) can be cross-linked to a narrowsegment of the �3 chain (D’Souza et al. 1988). In contrast,peptides homologous to the critical HHLGGAKQAGDVintegrin-recognition site found at the carboxyl termini offibrinogen’s � chains (Kloczewiak et al. 1989; Farrell et al.1992; Hettasch et al. 1992; Weisel et al. 1992) bind prima-rily to a region on the �IIb subunit (Santoro and Lawing Jr.1987; D’Souza et al. 1990). The observation, by surfaceplasmon resonance, that prebound fibrinogen can be readilydisplaced from �IIb�3 by an RGD ligand provides directevidence that fibrinogen’s binding site is separate from, butallosterically linked to, its RGD site (Hu et al. 1999). Thisconclusion has recently been supported by the demonstra-tion, using human/rat �IIb�3 chimeras, that sequenceswithin �IIb’s third and fourth amino-terminal repeats regu-late the ability of RGDS to block fibrinogen binding (Basaniet al. 2001).
It has been proposed that steric hindrance between a li-gand-recognition region (termed the A-domain [Lee et al.1995a,b) or �-I-like domain (Leitinger and Hogg 2000)] onthe �3 subunit and a putative �-propeller fold on the �IIbsubunit (Huang and Springer 1997; Springer 1997) preventsthese macromolecular ligands from binding to either site inthe receptor’s inactive state (Loftus and Liddington 1997).However, low molecular weight RGD peptides can bind tothe �3 subunit on the resting integrin (D’Souza et al. 1988)and block access by their parent adhesive proteins (Plow et
al. 1985). We have recently shown that RGDX ligands(X � Ser, Trp, Phe) shift a conformational equilibrium to-ward an open integrin, thus providing a mechanistic expla-nation for their effects on �IIb�3’s conformation and ac-tivity (Hantgan et al. 1999). The present study extends thoseobservations by examining the structural consequences ofligation of �IIb�3’s fibrinogen binding site. Because thebinding sites for RGD ligands and fibrinogen are spatiallydistinct yet coupled allosterically (Hu et al. 1999; Basani etal. 2001) we hypothesized that binding a fibrinogen-mi-metic would have a significant impact on �IIb�3’s tertiaryand quaternary structure.
Functional and biophysical studies have been reportedwith the fibrinogen-derived peptide HHLGGAKQAGDV(Parise et al. 1987; Hawiger et al. 1989; Hawiger 1995; Erbet al. 1997). However, the millimolar concentrations re-quired to affect �IIb�3 conformation (Parise et al. 1987;Erb et al. 1997) suggest this flexible synthetic peptide(Donahue et al. 1994; Ware et al. 1999) may not accuratelymodel the integrin recognition site on the �-domain of thenative fibrinogen molecule (Weisel et al. 1992). In thisstudy, eptifibatide (see below) served as a high-affinity li-gand mimetic with fibrinogen-like selectivity for �IIb�3(Phillips and Scarborough 1997). We have followed an in-tegrated biophysical, ultrastructural, and molecular model-ing approach to investigate the relationship between occu-pancy of �IIb�3’s fibrinogen binding site and the receptor’ssolution structure and stability.
Eptifibatide (N6-(aminoiminomethyl)-N2-(3-mercapto-1-oxopropyl - L - lysylglycyl - L - � - aspartyl - L - tryptophanyl -L-prolyl-cysteinamide, cyclic (1–6)-disulfide) is the genericname for Integrilin (COR Therapeutics), a recently ap-proved pharmaceutical integrin antagonist used to treat car-diovascular disease by blocking platelet:fibrinogen adhesiveinteractions (Phillips and Scarborough 1997; Goa and Noble1999). Eptifibatide’s design was initially based on the Lys-Gly-Asp (KGD) site on barbourin, a 73-residue snakevenom peptide that binds tightly to the �IIb�3 integrin butnot to �v�3 (Scarborough et al. 1991). Eptifibatide wasdesigned to retain similar selectivity for �IIb�3 by incor-porating a lysine derivative, homoarginine, into its struc-ture. A recent molecular mechanics study indicates that akey tryptophan present at equivalent positions in both bar-bourin and eptifibatide also contributes to their fibrinogen-like selectivity for �IIb�3 (Minoux et al. 2000). The netresult is a conformationally constrained heptapeptide that atlow micromolar concentrations blocks fibrinogen binding tothe �IIb�3 receptor but not the �v�3 integrin (Scarboroughet al. 1993a,b; Phillips and Scarborough 1997; Goa andNoble 1999).
We will show that near-stoichiometric eptifibatide con-centrations trigger a multistep process in �IIb�3 that startswith a conformational change in its extracellular domainand leads to integrin self-association, mediated at least in
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part through its distant transmembrane and/or cytoplasmicregions. We will also show that eptifibatide binding stabi-lizes noncovalent interactions between the �IIb and �3 sub-units, an effect not observed with RGDX ligands. By show-ing how both eptifibatide and RGDX peptides perturb�IIb�3’s structure, our findings provide a new structuralframework for understanding the dynamic processes of in-tegrin affinity regulation and ligand-induced signal trans-duction, as well as new insights into the mechanism of aclass of cardiovascular-disease drugs known as integrin an-tagonists.
Results
Effects of eptifibatide on �IIb�3 solution conformation
Molecular models of eptifibatide and the control cyclic pep-tide used in this study are shown in Figure 1. Despite theoverall chemical and structural similarity of these confor-mationally constrained peptides, they differed markedly intheir biological activity and, as will be shown in the sectionsthat follow, in their effects on �IIb�3 structure. The bioac-tivity of each peptide (dissolved in buffered saline) wasmeasured in a platelet aggregation assay and the data ana-lyzed by nonlinear regression to determine the peptide con-centration required to reduce the initial rate of aggregationto 50% of its control value, denoted the IC50 (Hantgan et al.1992). Eptifibatide exhibited an IC50 � 0.24 ± 0.06 �M,whereas control peptide had no significant effect on plateletaggregation at concentrations up to 100 �M.
Light-scattering measurementsThe effects of eptifibatide on �IIb�3’s biophysical pa-
rameters were examined to determine how fibrinogen re-ceptor occupancy effected the integrin’s conformation. Dy-
namic and static light-scattering data collected from dilutesolutions of �IIb�3 (2.7–3.8 �M) yielded D20,w
� 2.74 ± 0.05 F and Mw � 226 ± 29 K, respectively, con-sistent with the receptor’s asymmetric structure (Hantgan etal. 1993, 1999). Addition of eptifibatide (3 �M) to achievea 10% molar excess over �IIb�3 decreased D20,w by ∼10%in <15 min, with no significant increase in the weight-av-erage molecular weight. As shown in Figure 2 (solid tri-angles), similar decreases in D20,w (0.904 ± 0.009 timescontrol, P < 0.001, n � 4) were obtained at eptifibatideconcentrations ranging from 3–140 �M. In contrast, addi-tion of control cyclic peptide (open triangles) caused little orno change (0.978 ± 0.022 times control, n � 2). Each datapoint is an average of 6–8 values obtained within 90 min. ofpeptide addition. The solid line was calculated from hydro-dynamic theory for multisubunit particles (De La Torre andBloomfield 1981; Spotorno et al. 1997), utilizing bead mod-els of the closed and open forms of the integrin, whichpredict a 7% change in the frictional coefficient upon liga-tion (Hantgan et al. 1999), coupled with our ligand-linkedisomerization and oligomerization model. An eptifibatidebinding constant of 0.2 �M and an integrin association con-stant of 3 ✕ 104 M−1, which predicts ∼5% dimer formation,were employed in this simulation.
Sedimentation velocity measurementsThe effects of eptifibatide on �IIb�3’s solution structure
were also examined by sedimentation velocity analyses.Sedimentation velocity determinations were performedwith the �IIb�3 integrin alone, in the presence of eptifi-batide and control cyclic peptide. Analyses with SVEDBERGsoftware yielded a weight-average sedimentation coeffi-cient, s20, w � 8.35 ± 0.15 S (n � 5) for �IIb�3 alone (1.6–4.0 �M) and a similar value, 8.31 ± 0.04 S (n � 2), with
Fig. 1. Molecular models of eptifibatide and control cyclic peptide. (A) Eptifibatide, (N6-(aminoiminomethyl)-N2-(3-mercapto-1-oxopropyl-L-lysylglycyl-L-�-aspartyl-L-tryptophanyl-L-prolyl-cysteinamide, cyclic (1–6)-disulfide). (B) Inactive control peptide, cy-clo-L-cysteinyl-L-lysyl-D-alanyl-L-aspartyl-L-tryptophanyl-L-prolyl-L-cystinyl-amide (CKADWPC). Both models were prepared andminimum-energy configurations obtained with ALCHEMY and SYBYL molecular graphics/analysis software (Tripos, Inc.).
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control cyclic peptide (10 and 100 �M). In contrast, sig-nificantly smaller sedimentation coefficients were obtainedin the presence of eptifibatide (10 and 100 �M), namely,7.88 ± 0.12 S (n � 6, P � 0.001 vs. integrin alone). Asshown in Figure 2, analysis of the complete dataset indi-cated that eptifibatide (10 and 100 �M, open symbols) re-duced �IIb�3’s sedimentation coefficient to 0.947 ± 0.017times the control value. In contrast, this ratio was0.986 ± 0.022 (n � 2) with control cyclic peptide (10 and100 �M; open symbols). The dashed line, which closelyfollows the experimental data obtained with eptifibatide,was again calculated from hydrodynamic theory for mul-tisubunit particles (De La Torre and Bloomfield 1981; Spo-torno et al. 1997) coupled with our ligand-linked isomer-ization model (Hantgan et al. 1999). The difference in mag-nitude between the ligand-induced changes observed bysedimentation velocity and dynamic light scattering aremost likely due to the increased sensitivity of the lattertechnique to small quantities of oligomers (Hantgan et al.1999).
Evidence for reversible conformational changes
Because sedimentation velocity provided a more precisemeasure of eptifibatide-induced conformational changes,
this technique was used to test for reversibility. Figure 3depicts analyses of sedimentation velocity profiles obtainedwith DCDT+ software; results are presented as g(s*) pro-files (i.e., weight-average distributions of sedimenting spe-cies [Stafford III 1992]). Data were collected with �IIb�3alone (open symbols and dashed line), in the presence of 10�M eptifibatide (solid symbols and solid line) and with asample incubated with 10 �M eptifibatide for 60 min andthen separated free of ligand by rapid size exclusion chro-matography (Hantgan et al. 1999; dark gray symbols anddashed-dot line). Note the shift in the peak of the g(s*)profile from 8.71 S to 8.20 S induced by eptifibatide. Thiseffect was substantially reversed following ligand removal,as these data can now be described by a single species at8.51 S. Averaging results obtained in this and a replicatetransient ligand exposure experiment indicated 79 ± 9% ofeptifibatide’s effects on �IIb�3’s sedimentation coefficientwere reversed following ligand removal.
Effects of eptifibatide on �IIb�3 self-associationSedimentation equilibrium measurements with �IIb�3
alone (3.2–3.8 �M) and in the presence of eptifibatide (3–106 �M) tested the hypothesis that fibrinogen receptor oc-cupancy promotes integrin self-association. The data wereanalyzed initially in terms of a single ideal species model toobtain a set of weight-average molecular weight parameters.In the absence of ligands, �IIb�3 exhibited Mw � 221 ± 5K (Fig. 4), a value in excellent agreement with our previousresults (Hantgan et al. 1999). Addition of a stoichiometric
Fig. 3. Effects of transient exposure to ligand on the distribution of sedi-menting species observed with �IIb�3. Sedimentation velocity data for�IIb�3 alone (open circles and dashed line) and in the presence of eptifi-batide (10 �M, solid circles and solid line) were analyzed with DCDT+time derivative software (J. Philo) to obtain sedimentation coefficient dis-tribution functions, g(s*) vs. s* (Stafford III 1992). Solid lines were ob-tained by fitting the resultant distribution functions to a single ideal species.In addition, a separate �IIb�3 sample (dark gray circles and dashed-dotline) was isolated free of eptifibatide following a 60-min incubation at23°C prior to the onset of the sedimentation velocity run. Note how theshift observed in the presence of eptifibatide was substantially reversedfollowing ligand removal.
Fig. 2. Fractional change in �IIb�3 integrin’s translational diffusion andsedimentation coefficients as a function of ligand concentration. Diffusioncoefficients were determined by dynamic light-scattering measurements ofthe (90 °) intensity autocorrelation function of �IIb�3 (in the presence andabsence of ligand). Data were analyzed by the method of cumulants, fol-lowing correction for solvent contributions. Error bars denote the standarddeviation of replicate measurements (n � 6–8) performed with eachsample. Solid triangles, eptifibatide; open triangles, control cyclic peptide.Sedimentation velocity data were analyzed with SVEDBERG to obtainweight-average sedimentation coefficients as a function of ligand concen-tration. Solid circles, eptifibatide; open circles, control cyclic peptide. Be-cause the errors were typically <0.005 S, the error bars fall within thesymbols. The solid and dashed lines were obtained from simulations of thechanges in D20,w calculated for bead models utilizing hydrodynamic theoryfor multisubunit particles (De La Torre and Bloomfield 1981; Spotorno etal. 1997) and a ligand-linked isomerization and oligomerization model(Hantgan et al. 1999). In both cases, the ligand dissociation constantKL � 0.2 �M. The solid line was obtained using an occupied receptorself-association constant Ka � 0.03 L/�M, whereas Ka � 0 for the dashedline (to simulate isomerization without oligomerization).
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concentration of eptifibatide caused a sharp increase in Mw
to 301 ± 8 K; similar values were obtained over the range3–106 �M eptifibatide (Fig. 4, solid symbols). In contrast,adding control cyclic peptide (10 and 100 �M) yielded nosignificant changes in Mw (Fig. 4, open symbols)
Next, the complete dataset obtained with eptifibatide wasanalyzed globally to obtain an estimate of the associationconstant for dimerization of the liganded receptor, yieldingKa � 5.9 ± 0.9 ✕ 104 M−1. Computation of the predictedchange in Mw using this value of Ka in a ligand-linkedisomerization and oligomerization model (Hantgan et al.1999) yielded the solid line shown in Figure 4, whichclosely follows the experimentally determined molecularweight values. This simulation corresponds to an equilib-rium mixture of �IIb�3 at 3 �M containing some 22%dimers and 5% trimers. We note that this oligomer fractionexceeds those observed either by dynamic light scattering orsedimentation velocity (Fig. 2), suggesting that additional�IIb�3 self-association took place in the 48 h required tocomplete the sedimentation equilibrium experiments. How-ever, because comparable weight-average molecularweights were obtained at two rotor speeds, the oligomeriza-tion process appears reversible (Johnson et al. 1981).
Electron microscopy analyses of the effects ofeptifibatide on �IIb�3 structureThe preceding biophysical analyses indicated that eptifi-
batide binding to �IIb�3 causes a change to a more openconformation and increases integrin self-association. Theseconclusions are supported by electron microscopy exami-nation of integrin:eptifibatide complexes. Complexes of�IIb�3 were diluted in 0.05 M ammonium formate buffer at
pH 7.4, 30 mM octyl glucoside, 30% glycerol to a finalconcentration of 20–25 �g/ml, sprayed onto freshly cleavedmica and shadowed with tungsten. Glycerol is necessary toprevent drying artifacts, but its concentration was loweredconsiderably from previous experiments to minimize its ef-fects on �IIb�3 oligomerization (Hantgan et al. 1999). Theresultant �IIb�3 preparations displayed mostly single par-ticles (>85%) with a globular head about 8 ✕ 12 nm andtwo tails about 15 nm long projecting from one side andusually joined distally (Fig. 5A), features we and othershave observed previously for unligated integrins (Carrell etal. 1985; Weisel et al. 1992; Hantgan et al. 1999).
Eptifibatide at a final concentration of 10 �M was incu-bated with �IIb�3 at a concentration of 350 �g/mL for 0.5h prior to shadowing. Examination of the complexes byelectron microscopy after incubation with eptifibatide re-vealed that there was often a separation of the heads inmany complexes (Fig. 5C). In other words, two smaller,separate nodules were observed instead of a large, singlenodule. On average, about 55% of integrin complexes ap-peared to be made up of two separated nodules (n � 400).
In interpreting these results, some cautionary notes are inorder. These molecular features are near the resolution ofthis technique, so that very good preparations were neces-sary to observe the separation, and it could have beenmissed in some cases. Also, the separation was more diffi-cult to detect in oligomers of �IIb�3, which were commonin these preparations, as noted below. Finally, the separationmight not be visible in complexes that lie on the mica sur-face in certain orientations. For all of these reasons, theestimate of molecules showing a conformational change isan underestimate. As a control, 10 �M of a cyclic controlpeptide was used (Fig. 5B). Here, the images were essen-tially the same as those from experiments without any ad-ditions (Fig. 5A). Some complexes, however, appeared tohave separated heads in all preparations. With no peptide,about 10% had separated heads; whereas with cyclic controlpeptide, 9% did.
An increase in self-association of �IIb�3 particles wasalso observed in the presence of eptifibatide. Because the�IIb�3 complexes aggregate in the absence of detergent, 30mM octyl glucoside was used in all experiments, includingduring dilution prior to spraying. Nevertheless, some oligo-mers, generally dimers with tail-to-tail interactions, werealways present. With no peptide, 14% of complexes werepresent as dimers. The amount of self-association increasedstrikingly in the presence of eptifibatide but not in the pres-ence of the control peptide, in which only 15% were seen asdimers. In the presence of eptifibatide, the percentage oftotal �IIb�3 particles present in aggregates was 65%. Ofthese, 87% were dimers, 8% were trimers and 5% werepresent as larger particles. Dimers were oriented 180° apartwith the distal ends of their tails (transmembrane or cyto-plasmic domains) interacting (Fig. 5D); whereas larger
Fig. 4. Effects of eptifibatide on �IIb�3 molecular weight distributiondetermined by sedimentation equilibrium measurements. Sedimentationequilibrium data obtained with �IIb�3 as a function of the concentration ofeptifibatide (solid symbols) or control cyclic peptide (open symbols) wereanalyzed with WinNONLIN (Johnson et al. 1981) to obtain the weight-average molecular weight of the free and ligand-bound receptor. Additionalanalyses were performed with data from the �IIb�3:eptifibatide samples toobtain the receptor’s self-association constant, Ka � 5.9 ✕ 104 L/mole.The solid line was obtained with an isomerization and oligomerizationmodel (Hantgan et al. 1999), as described in the Fig. 2 legend, with KL �
0.2 �M and this value of Ka.
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1618 Protein Science, vol. 10
oligomers were arranged as rosettes (Weisel et al. 1992).Approximately 48% (n � 400) of the oligomers exhibitedseparated nodules in their head regions, although this ismost likely an underestimate, as noted earlier. In addition,the visualization of the open form is somewhat more diffi-cult in the aggregates, so the determination of the percent-age of integrins in the open conformation is more accuratefrom the individual particles.
Interpretation of the images in Figure 5 may be aided byour integrin models, based both on electron microscopy andhydrodynamic data, that will be presented in the Discussion.
Effects of eptifibatide on �IIb�3 stability
Subunit associationBecause the ligated form of �IIb�3 exhibited a subunit
separation visible by electron microscopy, we were sur-prised to find this new conformer was resistant to SDS-induced subunit dissociation. This observation was made inthe course of routine electrophoretic analyses of �IIb�3
samples obtained at the beginning and end of the precedingbiophysical characterizations. As shown in Figure 6A, lanes1 and 2, control integrin samples incubated in 1% SDS atroom temperature showed two prominent bands at molecu-lar masses of 134 ± 2 kD and 100 ± 2 kD, corresponding tothe �IIb and �3 subunits, respectively. In contrast, samplesincubated for as little as 2 h with 3 �M eptifibatide showeda major new band at an apparent molecular mass of 180 ± 2kD (Fig. 6A, lanes 3, 4). The ∼180-kD species was consis-tently observed in �IIb�3 samples incubated with eptifi-batide (3–140 �M/2–80 h) and accounted for 67 ± 18%(n � 22) of the average staining intensity of the �IIb and�3 subunits. The ∼180-kD band was not observed in �IIb�3samples incubated with control cyclic peptide (e.g., Fig. 6A,lanes 5, 6). Western blotting demonstrated that the ∼180-kDband observed with eptifibatide-containing samples exhib-ited immunoreactivity with antibodies specific for both the�IIb and �3 subunits (data not shown).
Like the conformational change detected by sedimenta-tion velocity, the effects of eptifibatide on �IIb�3’s elec-
Fig. 5. A Gallery of electron microscope images of rotary shadowed �IIb�3 complexes, alone and in the presence of eptifibatide orcontrol cyclic peptide. (A) Examples of �IIb�3 images obtained in the absence of peptides. Individual �IIb�3 complexes arecharacterized by a globular head with two long tails that are most often joined at their tips. (B) Examples of �IIb�3 images obtainedin the presence of control cyclic peptide (10 uM). Here the molecular features of the �IIb�3 complexes closely resemble those seenwithout peptide (as in panel A). (C) Examples of �IIb�3 complexes obtained in the presence of eptifibatide (10 uM). In most cases,a separation of the globular head into two distinct nodules can be seen. (D) Examples of �IIb�3 dimers observed in the presence ofeptifibatide (10 uM). Dimers joined tail to tail were the most common structure seen, as detailed in the text. Bar � 50 nm. Interpre-tation of these electron micrographs may be aided by schematics of the closed, open, and oligomeric integrins in Fig. 8.
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trophoretic mobility were reversible following ligand re-moval. For example, Figure 6B shows analyses of samplesobtained during the course of the sedimentation velocityexperiments depicted in Figure 3. The ∼180-kD species wasnot seen with �IIb�3 alone (Fig. 6B, lanes 1, 2) but ac-counted for 66 ± 3% of the staining intensity in an �IIb�3sample incubated for 1–8 h with 10 �M eptifibatide (Fig.6B, lanes 3, 4). Whereas a similar level (65%) was observed30 min after ligand removal (lane 5), by 6 h, the bandintensity had decreased to 24% (lane 6). In a replicate ex-periment, the ∼180-kD band decreased from 78% to 31%over a 7-h period.
The ∼180-kD band was absent following disulfide bondreduction (data not shown), raising the possibility that itcould represent a transient cystine-linked integrin con-former. However, we also determined that the intensity ofthe ∼180-kD band decreased markedly following incubationof integrin + eptifibatide samples in 1% SDS at elevatedtemperatures in the absence of reducing agent. For example,
Fig. 6C (insert, lane 1] shows a strongly staining ∼180-kDband in an �IIb�3:eptifibatide sample that was incubated inSDS for 1h at 23°C. In contrast, the band is not seen in asimilar sample incubated for 1h at 40°C (lane 2). As shownin Fig. 6C, the intensity of the ∼180-kD species decreasedwith increasing temperature over the range 23°C–40°C, ex-hibiting a melting point of ∼30°C. Additional studies (datanot shown) showed that preincubation of integrin sampleswith excess fluorescein maleimide prior to addition of ep-tifibatide had no effect on the intensity of the ∼180-kDband. These observations argue against a thiol exchangemechanism and indicate that ligation with eptifibatide pro-vides partial protection against SDS-induced subunit disso-ciation through noncovalent stabilization of the �IIb�3complex.
Global stability
The influence of eptifibatide on �IIb�3 conformation andstability were examined in more detail by a series of UV-absorbance and intrinsic fluorescence measurements.�IIb�3 exhibited a broad absorbance band with a maximumextinction coefficient at 277.5 nm (Fig. 7A, solid line), bothalone and in the presence of 10 �M eptifibatide. Fluores-cence intensity measurements (�ex 278 nm) of �IIb�3 aloneexhibited an emission maximum (�em) at 341 nm (Fig. 7B,solid line). These observations indicate that in the absenceof ligands, �IIb�3 exhibited a fluorescence emission spec-trum dominated by its 24 tryptophan residues, although 55tyrosines are also present (Fitzgerald et al. 1987; Poncz etal. 1987;). Addition of 10 �M eptifibatide caused an ∼19%decrease in fluorescence intensity with �em now at 340 nm(not shown). However, a similar decrease in intensity(∼24%) was observed in the presence of 10 �M controlcyclic peptide, suggesting that an inner filter effect due to∼20% increased absorbance at the excitation wavelength inthe presence of these tryptophan-containing peptides wasresponsible for the decreased fluorescence (Cantor andSchimmel 1980).
In contrast, addition of 4 M guanidinium chloride(GdnCl) shifted �IIb�3’s absorbance maximum to 276 nm(Fig. 7A, dashed line); the resultant difference spectrum wasmaximum at 286 nm (∼5% increased extinction coefficient),and an isosbestic point at 277.5 nm was observed. Underthese denaturing conditions, �IIb�3 exhibited a red-shiftedfluorescence emission maximum at 344 nm and a 31% de-crease in intensity (Fig. 7B, dashed line).
These observations formed the basis for monitoring un-folding of the �IIb�3 complex in the presence/absence ofeptifibatide. In particular, fluorescence emission data werenormalized by the absorbance at 278 nm to provide an index(F*) of the concentration-corrected fluorescence emissionspectra. These data were analyzed as follows to obtain U,the fraction unfolded, as a function of the [GdnCl]:
Fig. 6. Electrophoretic analyses of the effects of eptifibatide on �IIb�3subunit structure. (A) Samples of �IIb�3 obtained before/after sedimenta-tion velocity measurements (as described in Fig. 3’s legend) were dena-tured with sodium dodecyl sulfate, subjected to polyacrylamide gel elec-trophoresis, and then stained with Coomassie Brilliant Blue to visualize thepolypeptides. Lanes 1, 2: �IIb�3; 3, 4: +10 �M eptifibatide; 5, 6: +10 �Mcontrol cyclic peptide. Note the characteristic �IIb & �3 doublet (molecu-lar masses ∼134 kD and ∼100 kD) in lanes 1, 2 and 5,6, as well as theadditional ∼180-kD band seen with the eptifibatide sample. Molecular-mass markers are shown to the left of the gel photograph. (B) Samples of�IIb�3 obtained in the transient-exposure-to-ligand experiment (describedin Fig. 3’s legend) were subjected to electrophoretic analyses, as describedabove. Note the appearance of the ∼180-kD species in �IIb�3 samplescontaining eptifibatide (3, 4) and the decrease in this band’s intensity ∼6 hafter ligand removal (compare lanes 5 and 6). (C) Temperature-dependentchanges in the relative staining intensity of the ∼180-kD band observedwith integrin + eptifibatide samples. Samples were incubated at the indi-cated temperature for 1 h in the presence of 1% SDS (but no reducingagent) prior to electrophoretic analysis and Coomassie Brilliant Blue stain-ing. (Inset) Integrin + eptifibatide samples incubated in 1% SDS at 23°C(lane 1) and 40°C (lane 2) prior to electrophoretic analysis.
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1620 Protein Science, vol. 10
U = F*max − F*GdnCl�F*max − Fmin (1)
where the subscripts max and min refer to the normalizedfluorescence emission obtained at 0 and 4 M GdnCl, re-spectively, whereas the term F*GdnCl defines the emission ata particular denaturant concentration.
Fig. 7C shows GdnCl denaturation profiles obtained with�IIb�3 (0.6–1.4 �M) alone (solid symbols) and in the pres-ence of excess eptifibatide (open symbols, 3.4 & 10.1 �M).The solid line was obtained by fitting the combined data (±eptifibatide) to a cooperative unfolding model with a mid-point [GdnCl]mid at 1.8 ± 0.18 M GdnCl (where b is anempirical index of cooperativity):
U = 1�1 + exp − ��GdnCl� − �GdnCl�mid�b� (2)
Fitting each dataset separately yielded unfolding midpointsat 1.69 ± 0.35 and 1.85 ± 0.22 M GdnCl in the presence/absence of eptifibatide, respectively. These observations in-dicate that eptifibatide binding does not destabilize the�IIb�3 complex, despite the major rearrangements it in-duces in integrin conformation.
Discussion
This work is the first to show that occupancy of the fibrino-gen-binding site on the �IIb�3 integrin with a monovalentligand initiates a multistep process involving structuralchanges in the receptor’s extracellular domain. Thesechanges are transmitted over distances up to 15 nm to en-hance integrin clustering through its transmembrane and/orcytoplasmic domains. We have shown by laser light scat-tering, analytical ultracentrifugation, and electron micros-copy that near-stoichiometric concentrations of eptifibatide,a cyclized heptapeptide with fibrinogen-like selectivity for�IIb�3, shift a conformational equilibrium toward an openintegrin. Despite its distinct cleft, its cooperative gua-nidinium chloride-induced unfolding profile shows this ep-tifibatide-bound conformer retains the structural stability ofthe ligand-free integrin. Electrophoretic analyses showedthat eptifibatide binding actually protects the �IIb�3 com-plex from SDS-induced subunit dissociation, an effect notseen with RGDX peptides (Hantgan et al. 1999). Both sedi-mentation equilibrium measurements and electron micros-copy observations show that ligation with eptifibatide en-hances the formation of integrin clusters more effectivelythan �3-targeted RGD ligands (Hantgan et al. 1999).
Figure 8 presents a ligand-linked conformational changeand oligomerization scheme (solid arrows) that, whencoupled with hydrodynamic modeling, quantitatively de-scribes the results we have obtained with eptifibatide, aswell as those we previously reported with RGDX peptides(Hantgan et al. 1999). By showing that eptifibatide andRGD ligands induce similar conformational changes in�IIb�3’s ectodomain, our findings reinforce the conceptthat the receptor’s fibrinogen binding site and its RGD siteare allosterically linked (Hu et al. 1999; Basani et al. 2001).However, as will be explored in detail below, these ligandsexhibited important differences in their effects on �IIb/�3subunit interactions and integrin oligomerization.
Like its RGDX-induced counterparts, the new conformer
Fig. 7. Unfolding of the �II�3 complex monitored by changes in intrinsicfluorescence intensity. (A) Because these samples exhibited an isosbesticwavelength at 277.5 nm, an excitation wavelength of 278 nm (1-nm band-width) was used for fluorescence measurements and the emission signalnormalized by the absorbance of each sample at 277.5 nm to correct forsmall differences in protein concentration. (B) Fluorescence emission spec-tra obtained with �II�3 alone (solid line) and in the presence of 4 Mguanidinium chloride (dashed line). Note how guanidinium chloride de-creased the fluorescence signal by ∼31% and shifted the emission maxi-mum to 344 nm. (C) Guanidinium chloride denaturation profiles obtainedwith �IIb�3 alone (solid symbols) and in the presence of eptifibatide (opensymbols). The fraction unfolded was determined from the changes in nor-malized fluorescence intensity measured as a function of denaturant con-centration. The combined dataset was fit to a cooperative unfolding modelto obtain the solid line, characterized by a transition midpoint at ∼1.8 Mguandinium chloride.
Integrin structure and stability
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we have observed in the presence of eptifibatide exhibits thehallmark of an activated integrin, namely, a distinct subunitseparation in the receptor’s extracellular domain (Loftusand Liddington 1997; Hantgan et al. 1999; Emsley et al.2000). The conformational change alone exposes a cavitylarge enough to accommodate fibrinogen’s � domain (Yeeet al. 1997), the subunit that harbors the key KQAGDVintegrin-targeting sequence on �IIb�3’s primary physi-ological ligand (Kloczewiak et al. 1989; Farrell et al. 1992;Hettasch et al. 1992). In addition to these conformationalchanges, we have found that eptifibatide promotes the for-mation of integrin dimers and trimers. In fact, the self-association constant of ∼6 ✕ 104 M−1 obtained from sedi-mentation equilibrium data indicates eptifibatide was atleast 10-fold more effective at increasing �IIb�3 oligomer-ization than any of the RGDX ligands we had previouslycharacterized (Hantgan et al. 1999). The �IIb�3 clusteringinduced by eptifibatide is especially interesting in light ofthe report that fibrinogen both activated and caused cluster-ing of �IIb�3 in lipid bilayers; whereas the monovalentligands GRGDS and the fibrinogen �-chain dodecapeptidecaused activation only (Erb et al. 1997).
Eptifibatide’s ability to change �IIb�3’s conformationand actively promote its self-association may explain theinterplay between ligand binding to its ectodomain, receptorclustering and the regulation of signal transduction (Miya-moto et al. 1995; Erb et al. 1997; Giancotti and Ruoslahti1999; Humphries 1999). Precedent for long-range propaga-tion of conformational changes comes from studies of sig-naling mechanisms in the aspartate receptor, a member ofthe two-transmembrane-helix receptor family (Koshland1998; Ottemann et al. 1999). These investigators recentlyproposed a piston model that explains how aspartate bindingto the receptor’s periplasmic domain induces a conforma-tional change in a transmembrane helix that is transmitted tothe ∼10 nm distant cytoplasmic domain (Ottemann et al.1999). However, other transmembrane signaling modesthey proposed, including the scissors and seesaw modelsmay be more appropriate for the �IIb�3 integrin, given theconformational changes observed here by electron micros-copy. The scissors model is especially attractive because itprovides a mechanical linkage that can explain bidirectionalintegrin signaling (O’Toole et al. 1994). We propose that bybinding to �IIb�3’s ectodomain, ligand-mimetics such as
Fig. 8. Ligand-linked integrin conformational change and oligomerization model. Illustration of proposed scheme with beads modelsof the closed and open �IIb�3 integrin conformations, as well as integrin dimers (Hantgan et al. 1999). Note the similarity of thesemodels to the electron micrographs in Fig. 6. The �IIb heavy chain is represented by purple beads while the �IIb light chain is shownas blue beads; the �3 subunit is depicted as a string of yellow beads. The white sphere represents an octyl glucoside micelle. The �3chain region to which RGD peptides have been cross-linked (D’Souza et al. 1988) is represented by a red bead; the �IIb region whereKQAGDV peptides have been cross-linked (D’Souza et al. 1990) is represented by a pink bead. Computation of the changes inhydrodynamic parameters (Hantgan et al. 1999) for the scheme indicated by the solid arrows formed the basis of the simulations inFigs. 2 and 4 and helped to understand the shifts in sedimentation velocity in Fig. 3. However, we recognize the possibility thatintermediates interacting through their ectodomains, such as those indicated in the dotted boxes, may also form but may collapse tomore elongated dimeric structures through micelle fusion and/or due to surface interactions during preparation for electron microscopy.This scheme describes the effects of both eptifibatide (this work) and RGD ligands (Hantgan et al. 1999) on �IIb�3 conformation, thusunderscoring the allosteric linkage between their distant binding sites (Hu et al. 1999; Basani et al. 2001).
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1622 Protein Science, vol. 10
eptifibatide (this study) and RGDX peptides (Hantgan et al.1999) perturb the interactions between subunits, by analogy,opening the scissors by pulling on the blades on the recep-tor’s extracellular face, separating its distant transmembraneand/or cytoplasmic regions and making them available foroligomerization. Thus the conformational change they in-duce in �IIb�3’s ectodomain may be similar to that inducedphysiologically by events starting from the cytoplasmic do-mains (inside-out signaling). Likewise, the receptor cluster-ing that results from eptifibatide’s occupancy of the integrin’sfibrinogen-binding site may mimic outside-in signaling.
The integrin dimers observed in electron micrographs ap-pear to be joined at their tails, suggesting that transmem-brane and/or cytoplasmic domain interactions may play asignificant role in oligomerization. These concepts are sup-ported by NMR data (Vinogradova et al. 2000) and molecu-lar models (Haas and Plow 1997) that have identified mul-tiple interaction sites on the carboxy-terminal regions of the�IIb and �3 subunits. Furthermore, dimerization of transmem-brane helices in detergent micelles has been carefully docu-mented for glycophorin A using both NMR and small anglex-ray scattering (MacKenzie et al. 1997; Bu and Engelman1999). However, it also is possible that the integrin dimers weobserved were initially stabilized by ectodomain interactionslost following octyl glucoside micelle fusion (VanAken et al.1986; Lorber et al. 1990) and/or surface interactions duringpreparation for electron microscopy. Thus, we present hypo-thetical side-by-side dimeric integrin intermediates within thedotted boxes in Figure 8. Precedent for ligand-induced oligo-mers joined through their extracellular domains comes fromstudies with hematopoietic receptor complexes, such as themultimers that form when the cytokine IL-6 binds to its re-ceptor’s � and � subunits (Wells and deVos 1996).
Eptifibatide is a newly approved cardiovascular-diseasedrug, an integrin antagonist, and our observations may ex-plain key aspects of its pharmaceutical activity (Phillips andScarborough 1997). We propose that eptifibatide perturbs�IIb�3’s conformation and blocks receptor occupancy byits primary physiological ligand, fibrinogen, thus preventingplatelet aggregation (Phillips et al. 1988). Eptifibatide’s ef-fects are specific, as no significant changes in �IIb�3’shydrodynamic or electrophoretic parameters were obtainedwith the biologically inactive control peptide. As illustratedin Figure 1A, eptifibatide displays a cup-shaped configura-tion similar to that described for other high-affinity integrinantagonists (Minoux et al. 1998, 2000). Interestingly, asshown in Figure 1B, this feature, which may be importantfor receptor recognition, is interrupted by a kink in thebiologically inactive control cyclic peptide.
Because eptifibatide also shares key structural elementswith a peptide analog of the fibrinogen � chain site neces-sary for integrin recognition (Hawiger 1995; Suehiro andPlow 1997), our results have special significance for under-standing the relationship between receptor occupancy and
integrin conformation. In particular, cyclo (S,S)KYGCHarDWPC behaves as a high-affinity analog of theKQADGV site on fibrinogen’s � chains (Ware et al. 1999);whereas cyclo (S,S) KYGCRGDWPC mimics fibrinogen’sRGD sequences (Suehiro and Plow 1997; Cierniewski et al.1999). These cyclized ligands bind to distinct regions of the�IIb�3 receptor separated by ∼6–9 nm (Cierniewski et al.1999). Our ligated integrin model (Fig. 8) indicates that theRGD cross-linking site on the �3 chain (D’Souza et al.1988) is now separated by ∼9 nm from the � chain bindingdomain on the �IIb subunit (D’Souza et al. 1990). In theabsence of ligands, our model predicts only a 2-nm subunitseparation (Hantgan et al. 1999).
Despite this cleft, we have found that the open conformeris not a denatured species. In particular, we have shown thatupon eptifibatide binding, �IIb�3 shows increased stabilitytoward SDS-induced subunit separation and a cooperativeguandinium chloride unfolding profile similar to the nativeintegrin. Eptifibatide’s ability to stabilize �IIb�3 subunitinteractions against SDS-induced dissociation seems similarto the recently reported effects of echistatin on other RGD-dependent integrins (Thibault 2000). However, our findingsextend Thibault’s observations by showing that mild heat-ing disrupts the SDS-stable integrin:eptifibatide complexes.Our data also argue against a disulfide-exchange activationmechanism (Yan and Smith 2001), as eptifibatide-inducedcomplexes were observed even in the presence of excessthiol-blocking reagent. Additional evidence for a noncova-lent process comes from our finding that eptifibatide’s sta-bilizing effects on �IIb�3 are readily overcome by gua-nidinium chloride, which denatures proteins without dis-rupting their disulfide bonds (Creighton 1984).
In conclusion, we suggest that the geometric constraintsimposed by insertion of �IIb�3’s stalks into the lipid bilayermay promote clusters of ligand-activated integrins stabi-lized by additional interactions of their ectodomains, suchas those in Figure 8. Such integrin multimers have recentlybeen observed in a 21 Å-resolution structure of the�v�5:adenovirus complex obtained by cryoelectron micros-copy (Chiu et al. 1999). While high-resolution data are stillemerging, our integrated biophysical, electron microscope,and molecular modeling approach has furthered develop-ment of a structural scaffold that can explain the conforma-tional differences between resting and activated integrins, aswell the mechanisms of outside-in signaling (Clark andBrugge 1995; Loftus and Liddington 1997; Hughes and Pfaff1998; Giancotti and Ruoslahti 1999; Leisner et al. 1999).
Materials and methods
Purification and characterization of the �IIb�3 complex
The platelet integrin receptor �IIb�3 was isolated from the deter-gent-solubilized membrane fraction of human blood platelets
Integrin structure and stability
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(American Red Cross) by lentil lectin affinity chromatography andsize exclusion chromatography, as previously described in detail(Hantgan et al. 1993). This procedure yields milligram quantitiesof nearly monodisperse �IIb�3 in a pH 7.4 buffer (HSC-OG)containing 0.13 M NaCl, 0.01 M HEPES, 0.002 M CaCl2, 3 ✕ 10–8
M basic trypsin inhibitor (Aprotinin, Sigma), 10−6 M leupeptin,and 0.03 M n-octyl-�-D-glucopyranoside (octyl glucoside, OG,Sigma). �IIb�3 concentrations were determined by ultraviolet ab-sorbance measurements (LKB Ultrospec) using an extinction co-efficient at 280 m of 1.21 mL mg−1 cm−1, as previously described(Ramsamooj et al. 1990; Hantgan et al. 1993). Samples of �IIb�3obtained prior to and following exposure to ligand-mimetic orcontrol peptides were analyzed by SDS-PAGE using 5%–15%gradient gels (Bio Rad) and followed by protein staining withCoomassie Brilliant Blue R-250, as previously described (Hantganet al. 1999). Selected samples were electrophoretically transferredto PVDF membranes and immunostained with monoclonal anti-bodies specific for either the �IIb or �3 subunits (CD61 and CD41,respectively, Immunotech). Immune complexes were detectedwith an alkaline phosphatase-conjugated goat anti-mouse mono-clonal antibody using a BioRad Enhanced Chemiluminescence kitwith images captured on film for subsequent analyses using SigmaGel software (Jandel Scientific).
Ligand-mimetic and control peptides
Eptifibatide, (N6– (aminoiminomethyl)-N2– (3-mercapto-1-oxo-propyl - L - lysylglycyl - L - �-aspartyl - L - tryptophanyl - L - prolyl -cysteinamide, cyclic (1–6)-disulfide), was provided by CORTherapeutics (San Francisco, CA), where it has been developed asa pharmaceutical, Integrilin (Phillips and Scarborough 1997). Aninactive control peptide, cyclo-L-cysteinyl-L-lysyl-D-alanyl-L-aspartyl-L-tryptophanyl-L-prolyl-L-cystinyl-amide (CKADWPC),was also provided by COR Therapeutics. Both peptide prepara-tions were dry powders that were dissolved in HSC-OG and storedfor up to 6 months at −70°C. Peptide concentrations were deter-mined by quantitative amino acid composition analyses (Hantganet al. 1992) of samples of stock solutions.
Static and dynamic light-scatteringinstrumentation and data analyses
The molecular weight and translational diffusion coefficient of the�IIb�3 complex were measured with a Brookhaven InstrumentsBI-2030 AT correlator, operated in conjunction with a BI-200 SMlight scattering goniometer/photon counting detector and a SpectraPhysics 127 He-Ne laser (35 mW, equipped with a vertical polar-ization rotator), as previously described (Hantgan et al. 1993,1999).
For molecular weight determinations, right-angle sample scat-tering intensities were corrected for solvent scattering and ex-pressed relative to a benzene standard (Hantgan et al. 1993). Thisinformation, coupled with measurements of protein concentrationby UV-absorbance, was used to calculate the weight-average mo-lecular weight (Mw) of �IIb�3 samples using Rayleigh-Ganstheory (Johnson and Gabriel 1981). For translational diffusion co-efficient determinations, each intensity-normalized photon countautocorrelation function obtained for the �IIb�3 complex was cor-rected for the contributions of octyl glucoside micelles and thenanalyzed by the method of cumulants, as previously described(Hantgan et al. 1993). All translational diffusion coefficients re-ported here have been corrected for solvent viscosity to obtainD20,w values.
Analytical ultracentrifugation:Instrumentation and data analyses
Sedimentation velocity and equilibrium measurements were per-formed in a Beckman Optima XL-A analytical ultracentrifuge(Beckman Instruments) equipped with absorbance optics and anAn60 Ti rotor. Sedimentation velocity data for the �IIb�3 complex(alone and in the presence of ligand-mimetic or control peptide)were obtained in double-sector cells at 20°C at a rotor speed of35,000 rpm, as previously described (Hantgan et al. 1999). Thesedata were analyzed using both SVEDBERG (version 1.04) andDCDT+ (version 6.31) software (J. Philo) to obtain the weight-average sedimentation coefficient (Sw) and distribution of sedi-menting species, g (s*), respectively (Stafford III 1992). All sedi-mentation coefficients reported here have been corrected for sol-vent density and viscosity to obtain S20, w values.
Sedimentation equilibrium data were collected from �IIb�3samples (alone and in the presence of ligand-mimetic or controlpeptide) contained in double-sector cells with buffered octyl glu-coside (and peptides, as required) in the reference sector. Datawere collected at 280 nm at rotor speeds of 6000 and 8000 rpm at20°C, as previously described (Hantgan et al. 1999). The absor-bance versus radial distance data were analyzed by nonlinear re-gression with WinNONLIN3 (Johnson et al. 1981) to obtain theself-association constant for the �IIb�3 complex alone and in thepresence of ligand-mimetic (or control) peptide. WinNONLIN3was provided by David Yphantis and the staff at the NationalAnalytical Ultracentrifugation Facility.
Spectroscopic measurements
UV absorbance measurements of �IIb�3 samples contained in1-cm path length/1 mL volume quartz cuvettes were performed asa function of wavelength on a Milton Roy Spectronic 3000 Arrayspectrophotometer (chosen for its 0.3-nm resolution); single wave-length determinations were performed on an LKB Uvicord II in-strument. Fluorescence emission spectra were obtained with anAMINCO-Bowman Series 2 Luminescence Spectrometer (SLM-AMINCO). Samples were excited at 278 nm (1-nm bandwidth)and the emission spectra recorded from 300–500 nm (1-nm band-width). Samples were contained within quartz microcuvettes (1-cmpathlength, 150 �L filling volume, 4 windows, Hellma Cells, Inc.).All spectral measurements were obtained at 23 ± 1°C and cor-rected for the contributions of octyl glucoside buffer, eptifibatide,and guanidinium chloride, when present in the samples.
Rotary-shadowed specimens for electron microscopy
Rotary-shadowed samples were prepared by spraying a dilute so-lution (final concentration ∼20–25 �g/mL) of molecules in a vola-tile buffer, 0.05 M ammonium formate at pH 7.4, 30 mM octylglucoside, and 30% (v/v) glycerol onto freshly cleaved mica andshadowing with tungsten in a vacuum evaporator (Denton VacuumCo.; Fowler and Erickson 1979; Weisel et al. 1985; Veklich et al.1993). These samples were examined in a Philips CM100 electronmicroscope (FEI Co.) operating at 60 kV and a magnification of53,000✕. Counts of molecules with different conformations ordifferent amounts of oligomers were made from prints of the mi-crographs, using images from many different areas of several dif-ferent preparations to get a random sample.
Molecular modeling
Bead models of the �IIb�3 complex (Hantgan et al. 1999) wereconstructed, visualized, and analyzed using the BEAMS (BEAds
Hantgan et al.
1624 Protein Science, vol. 10
Modeling System) set of computer programs to represent each ofthe �IIb and �3 polypeptide chains of the �IIb�3 complex as anensemble of interconnected spheres (Rocco et al. 1993; Spotornoet al. 1997; see also Byron 2000). Hydrodynamic theory for mul-tisubunit particles (De La Torre and Bloomfield 1981; Spotorno etal. 1997;) was employed to calculate the translational diffusioncoefficient, Dt, the Stokes Radius, Rs, and the sedimentation co-efficient, s, for the models as previously described (Rocco et al.1993; Hantgan et al. 1999). Molecular models of eptifibatide andCKDAWPC were constructed and a minimum energy configura-tion for each obtained (Cohen et al. 1990) using ALCHEMY 2000and SYBYL software (Tripos).
Acknowledgments
Thanks to Drs. R.M. Scarborough, D.R. Phillips, M.M. Kitt, and S.Hollenbach of COR Therapeutics, Inc., for providing eptifibatideand the control cyclic peptide, as well as for helpful discussions;Mary Stahle for her expert technical assistance; and Dr. D.S. Lylesfor his critical reading of the manuscript and to Dr. J.B. Edelsonfor her skilled editing. Support was provided by grantMCB-9728122 from the National Science Foundation (to R.R.H.),grant BI-04-CT96–0662 from the European Community (to M.R.),and grant HL30954 from the National Institutes of Health (toJ.W.W.).
The publication costs of this article were defrayed in part bypayment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 USC section 1734solely to indicate this fact.
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