THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 9, Issue of March 25, pp. 6 8 0 0 - 6 8 0 8 , 1993 Printed in U.S.A.

Two-step Binding Mechanism of Fibrinogen to aIIbD3 Integrin Reconstituted into Planar Lipid Bilayers*

(Received for publication, September 4, 1992)

Beate Muller$, Hans-Gunter ZerwesQ, Kirsten TangemannS, Jiirg PeterQ, and Jurgen Engel$1[ From the $Department of Biophysical Chemistry, Biocenter of the University of Basel and the $Sandoz Pharma AG, Preclinical Research, CH-4056 Basel, Switzerland

The platelet integrin aIIb83 binds to fibrinogen and thus mediates platelet aggregation after stimulation. This integrin was isolated from human platelets and reconstituted into lipid vesicles. As judged by electron microscopy the integrin incorporated adequately only into 1,2-dimyristoylglycero-3-phosphocholine/l,2-di- myristoylphosphatidylglycerol vesicles after removal of the detergent by adsorption to Bio-Beads. These vesicles were then used to generate planar lipid bilay- ers. The binding of fluorochrome labeled fibrinogen or the peptide ligand Gly-Arg-Gly-Asp-Ser-Pro-Cys (GRGDSPC) was monitored by total internal reflection fluorescence microscopy and a solid phase binding as- say. Analysis of the kinetics revealed fast reversible formation of a fibrinogenlintegrin precomplex (KD = 60 nM) followed by formation of a stable irreversible complex. This transition was monitored by measuring the fraction of precomplex which could be dissociated by addition of excess Gly-Arg-Gly-Asp-Ser (GRGDS). For the peptide, the KO was 1200 nM, and the rates of association and dissociation were faster than the time resolution of the method. Similar KD values were found by inhibition of fibrinogen binding to aIIb83 in the immobilized receptor assay. Since the binding of fi- brinogen was irreversible, KD values were dependent on the time period between fibrinogen incubation and peptide addition. These and results by other authors point to the biological importance of the biphasic bind- ing process of fibrinogen to its receptor on platelets.

Integrins are important and widely distributed cellular receptors which can mediate interactions of cells both with the extracellular matrix (ECM)’ and with other cells. Each integrin molecule is composed of two subunits, a and (3, which are noncovalently connected in their large NH2-terminal ex-

* This work was supported by Swiss National Science Foundation Grant 31-32251.91. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 To whom correspondence should be addressed Biozentrum, Bio- physikalische Chemie, Klingelbergstrasse 70, CH-4056 Basel, Swit- zerland. Fax: 41-61-267-2189.

The abbreviations used are: ECM, extracellular matrix; POPC, l-palmitoyl-2-oleoylglycero-3-phosphocholine; POPS: l-palmitoyl-2- oleoylphosphatidylserine; POPG, 1-palmitoyl-2-oleoyl-phosphatidyl- DL-glyCerOl; DMPC, 1,2-dimyristoylglycero-3-phosphocholine; DMPG, 1,2-dimyristoylphosphatidylglycerol; FITC, fluorescein-5- isothiocyanate; TRITC, tetramethylrhodamine-5-(and-6)-isothio- cyanate; T488, tetramethylrhodamine-5-(and-6)-iodoacetamide; TIRFM, total internal reflection fluorescence microscopy; BSA, bo- vine serum albumin; ELISA, enzyme-linked immunosorbent assay; HPLC, high performance liquid chromatography.

tracellular domains (Hynes, 1992). Both of these consist of globular units joined to 14-nm-long arms. Toward the COOH terminus membrane-spanning and short cytosolic domains follow (Carrell, 1985; Kelly et al., 1987; Nermut et al., 1988). The ligand binding regions in the extracellular domains (Hynes, 1992) are well separated from the membrane by the two arms (Parise and Phillips, 1985).

A large number of ECM proteins has been found to interact with integrins with different a- and (3-subunits. For some integrins ligand specificity appears to be high, whereas for others several apparently unrelated ECM proteins have been identified as ligands (Hynes, 1992). The integrin aIIbp3 (MI = 235,000) which was used in the present study is one of the most promiscuous integrins (Phillips et al., 1991). It is the platelet fibrinogen receptor but also recognizes fibronectin, von Willebrand factor, vitronectin, and thrombospondin (Hynes, 1992). An Arg-Gly-Asp (RGD) sequence is recognized by this receptor in all ligands, and, in addition, a Lys-Glu- Ala-Gly-Asp-Val (KQAGDV) sequence is recognized in fibrin- ogen (Kloczewiak et al., 1984; Cheresh et al., 1989). Conse- quently, synthetic peptides containing the RGD sequence bind strongly to aIIbP3 and inhibit the interaction with ECM proteins.

Knowledge of quantitative binding constants for aIIb(33 and its protein and peptide ligands is of great interest because of the great importance of this integrin in platelet aggregation, a process involved in wound healing, thrombosis, and hemo- stasis (Marguerie et al., 1980; Kieffer and Phillips, 1990). Most binding data have been obtained with solid phase assays in which the purified integrin was usually immobilized on a plastic surface. Several authors (Parise and Phillips, 1985; Pytela et al., 1985; Conforti et al., 1990) have tried to avoid artefacts potentially caused by coating to plastic by incorpo- ration of integrins into vesicles, which were then used in different biochemical binding assays. In other studies binding data were derived from platelet attachment assays (Marguerie et al., 1980; Cheresh et al., 1989). This introduces the possi- bility of interference by other cellular receptors or cell surface components.

In the present work we reconstituted aIIba3 into vesicles and planar lipid bilayers. The method of total internal reflec- tion fluorescence microscopy (TIRFM) was used to monitor binding. This physical method had been used earlier to ex- amine the binding of laminin to planar bilayers (Kalb and Engel, 1991), of an antibody to a lipid hapten (Kalb et al., 1990), and of antibody to F, receptors (Poglitsch and Thomp- son, 1990). This technique yields quantitative data and allows the measurement of the kinetic time course of the binding of fibrinogen to aIIbp3. The data obtained with this method were compared with results from solid phase binding assays, in which aIIb(33 was immobilized on plastic. In both systems the binding of the peptide GRGDS was reversible. However,

6800

Two-step Binding of Fibrinogen to aIIbp3 6801

the high molecular weight ligand fibrinogen bound irreversibly to the integrin. A similar phenomenon has been found also for the 0lvp3 fibrinogen interaction (Orlando and Cheresh, 1991; Felding-Habermann et al., 1992). Progressive stabiliza- tion of the complex was also observed in studies of the interaction of fibrinogen with platelets (Marguerie et al., 1980). We propose a mechanism for the integrin fibrinogen interaction in which fast weak binding with measurable KD is followed by slow strong complex formation.

MATERIALS AND METHODS

Reagents-Outdated human platelets were obtained from the local blood bank. Triton X-100 and fibrinogen were obtained from Sigma. It was verified by an enzymatic assay with a chromogenic substrate by C. Tapparelli, Sandoz Pharma AG, Basel, Switzerland, that the fibrinogen did not contain thrombin (detection limit lower than M of the fibrinogen content). The lipids DMPC, DMPG, POPC, and POPS were obtained from Avanti Polar Lipids (Alabaster, AL). Fluorescence labels TRITC, FITC, and T488 were from Molecular Probes (Eugene, OR). Octyl-6-D-glucopyranoside was from Bachem Feinchemikalien (Bubendorf, Switzerland). The peptides Arg-Gly- Glu-Ser (RGES) and Gly-Arg-Gly-Asp-Ser (GRGDS) were purchased from Novabiochem (Laufelfingen, Switzerland) and Sephadex G-25 M and Sephacryl S-300 HR were obtained from Pharmacia LKB Biotechnology (Uppsala, Sweden). Linbro 7X-PF was from ICN Flow Biochemicals (Irvine, Scotland) and the Bio-Beads SM-2 were from Bio-Rad. Quartz slides were purchased from Heraeus (Zurich, Swit- zerland), and the biotinylation kit was obtained from Amersham Corp. (Buckinghamshire, Great Britain). All other reagents and buff- ers were from Merck (Darmstadt, Germany), Fluka (Buchs, Switzer- land), or Sigma. Gly-Arg-Gly-Asp-Ser-Pro-Cys (GRGDSPC) was synthesized with an automatic peptide synthesizer by P. Jeno, De- partment of Biochemistry, Biocenter, Basel, Switzerland, and purified by HPLC. The monoclonal anti-av antibody LM 142 was a kind gift of Dr. D. A. Cheresh.

Purification of aZZb(33-aIIbfi3 was purified from outdated platelets (method based on personal communication by J. J. Calvete). Briefly, washed human platelets were disrupted by sonication, and the partic- ulate fraction was pelleted at 120,000 X g for 30 min. The pellet was extracted with 4% Triton X-100, 50 mM Tris-HC1, pH 7.3, 1 mM CaCl,, 1 mM MgC1, plus protease inhibitors (phenylmethanesulfonyl fluoride, aprotinin, leupeptin). The soluble extract was applied to a DEAE-Sephacel column equilibrated in the same buffer but contain- ing 0.2% Triton X-100. aIIb83 was eluted from the column with 0.5 M NaCl in the equilibration buffer, and the eluate was further purified by two rounds of gel permeation chromatography on Sephacryl S- 300. Some preparations were passed over a column with monoclonal antibodies against fibrinogen to remove copurifying fibrinogen. For some experiments, Triton X-100 was exchanged for octyl-@-D-gluco- pyranoside by chromatography on DEAE-Sephacel. The presence of avp3 in the preparation was tested by a monoclonal anti av-antibody (LM 142) which was described by Coller et al. (1991). We could not detect avp3 by an ELISA. This was in agreement with previous findings of a 500-fold excess of aIIbp3 over avp3 in platelets (Coller et al., 1991).

Reconstitution-Vacuum-dried lipids were solubilized in 0.1% Tri- ton X-100 or 25 mM octyl-p-D-glucopyranoside in 20 mM Tris-HC1, pH 7.4, 50 mM NaC1, 0.5 mM CaCl,. After the lipid (870 nmol) had dissolved completely, integrin solubilized in Triton X-100 or octyl-fl- D-glucopyranoside was added. The total volume of this mixture was 1 ml, and the molar lipid to protein ratio was varied from 900 to 2000. Octyl-0-D-glucopyranoside was removed by continuous dialysis in a dialysis chamber at a temperature which was about 3 "C higher than the phase transition temperature of the lipids used. Alternatively Triton X-100 was removed by two to three additions of about 70 mg of Bio-Beads SM-2 which were washed before use with methanol and water as described by Holloway (1973). Vesicles were separated from free protein by loading on top of a stepwise sucrose gradient (2 M, 1 M, 0.6 M, 0.4 M in 20 mM Tris-HC1, pH 7.4, 50 mM NaCI, 0.5 mM CaC12) and centrifugation at 275,000 X g at 4 'C in an ultracentrifuge for 24 h. The visible vesicle band obtained at about 1 M sucrose solution was collected and dialyzed against buffer.

Lipid and Protein Determination-Lipid determination was per- formed with the phosphate assay (Bottcher et al., 1961), and protein concentrations were determined by the Fohn-Ciocalteau reagent ac- cording to Peterson (1977).

Electron Microscopy-For negative staining vesicle suspensions were diluted 10-fold and 10 pl of the dilution was put on a glow- discharged collodium and carbon grid. After about 4 min, 5 ~1 of 0.2 g/ml sodium phosphotungstate solution, pH 7, was added for 0.5 min. After removal of the first stain, incubation was repeated with 10 pl of the same solution for about 5 min.

Fluorescence Labeling of Proteins and Peptide-Fibrinogen and aIIbp3 were labeled with TRITC and FITC, respectively (Kalb et al., 1990). The amount of bound fluorescein was determined by measuring the extinction at 493 nm (t = 153.8 cm2/mg). The amount of bound rhodamine was determined by measuring the extinction at 555 nm ( e = 151.5 cm2/mg). The protein concentration was determined as described above. The degree of labeling was between one and three fluorescein molecules bound per integrin molecule and between four and six rhodamine molecules bound per fibrinogen molecule. To label GRGDSPC, the peptide was incubated with the rhodamine label T488 in a 1:l molar ratio for 2 h at room temperature (buffer: 0.1 M NH,HC03). The mixture was separated by HPLC on a LiChrosorb RP18 column (Merck) with a continuous gradient of 0.1% trifluoro- acetic acid and 80% acetonitrile, 0.09% trifluoroacetic acid. The separation of free label, labeled peptide, and nonlabeled peptide could be monitored at 560 and 214 nm. The concentration of the labeled peptide was determined by amino acid analysis (P. Jeno, Department of Biochemistry, Biocenter, Basel, Switzerland). Fluorescently labeled fibrinogen and peptide as well as biotinylated fibrinogen had the same binding affinity to integrin aIIbp3 as the unlabeled ligands. This was shown in immobilized receptor assays and platelet aggregation assays. The data for rhodamine-labeled peptides are shown in Fig. 5.

Formation of Supported Planar Bilayers-Quartz slides were cleaned with a 20% solution of Linbro 7X-PF in deionized water by boiling for 20 min, followed by immediate sonication in a bath sonicator for 30 min. They were then rinsed with deionized water, washed with methanol, and dried for 1 h at 150 "C. Immediately before use, the quartz slides were further cleaned in an argon plasma cleaner (Harriet Corp., Ossining, NY) for 10 min. The clean quartz slide was then assembled in the TIRFM cell. Direct fusion of the vesicles on the quartz slide has been described previously by Brian and McConnell (1984). The cell was filled with buffer (20 mM Tris- HC1, pH 7.4,150 mM NaC1,l mM CaCl,) and mounted on the stage of the laser fluorescence microscope. Vesicle suspension was injected leading to formation of bilayers only upon stirring. Excess vesicles were removed by washing.

TZRFM-The details of the equipment have been described earlier (Kalb et al., 1990). Bilayers were formed as mentioned above, and increasing amounts of labeled ligand were injected while stirring continuously. In some cases the bilayer was preincubated with 1 mg/ ml BSA to prevent unspecific binding. The laser beam of an argon laser was directed onto the sample through a quartz prism and immersion oil at an angle of 72 "C so that an evanescent field was created at the quartz-buffer interface. The penetration depth of the laser beam into the solution was in the range of 100 nm. Therefore only those labeled ligands were excited which were close to the quartz- buffer interface and usually bound to the receptor or the membrane. After addition of labeled peptide or protein, the time course of the fluorescence intensity was recorded. Final data were averaged from the fluorescence intensity of 20 different spots on the membrane. All experiments were performed in measuring buffer: 20 mM Tris-HC1, pH 7.4,150 mM NaCl, 1 mM CaC12, 1 mM MgCl,, 1 mM MnC12. The temperature in the sample cell was kept constant at 24 "C with a Peltier element. For competition experiments unlabeled peptide or fibrinogen was added. For dissociation of bound ligand, EDTA, pep- tide, or fibrinogen was added or alternatively the cell was washed with buffer.

Immobilized Receptor Assay-Fibrinogen was biotinylated with a biotinylation kit following the suggestions of the manufacturer. All steps of the assay were carried out in measuring buffer. After the plates had been coated with integrin (200 ng/well) for 2 h at 37 "C, free protein binding sites on the plastic were blocked by 5% BSA for 2 h. Biotinylated fibrinogen was allowed to bind to immobilized receptor at 37 "C for the desired time in the presence or absence of inhibitors. Unbound fibrinogen was removed by three washing cycles (200 pl each) after which 100 pl of a 1:lOOO or 1:500 dilution of biotinylated horseradish peroxidase-streptavidin complex was added to the wells and incubated for 1 h at room temperature. The excess reagent was washed away, and 100 pl of premixed peroxidase substrate solution was added. The color reaction was stopped with 100 pl 2% oxalic acid, and the plates were read in an ELISA reader at 414 nm.

Calculation of the Ratio of the Fluorescence Intensity of Bound and

6802 Two-step Binding of Fibrinogen to aIIbp3

Unbound Ligands-The ratio of the contribution of bound ( J b ) and unbound (J.) ligands to the total fluorescence intensity was calculated according to the equation proposed by Harrick (1967).

(Eq. 1)

where d is the penetration depth (about 100 nm) of the evanescent wave which equals the distance at which the intensity drops to l/e of its initial value. The distance of the bound ligand from the interface z = 25 nm was estimated from the dimension of the integrin and the bilayer.

For the calculation of the integrin surface concentration rb = 1.5 10”’ nmol nm-* in the bilayer, it was assumed that one lipid molecule covers an area of 0.57 nm2 (Buldt et al., 1979) and that the protein- lipid ratio in the bilayer remains the same as in the vesicles. Assuming that integrin orientation is random, only half of the integrin molecules are accessible for the ligand. The volume of the measuring cell was 1.1 ml, and the quartz slide surface was 6.9 cm2. At the lowest total fibrinogen concentration (10 nM), the molar ratio of fibrinogen to integrin was thus larger than 10.

From the concentration of labeled GRGDSPC peptide and its fluorescence intensity in the unbound state, the specific fluorescence per fluorophore was determined. From this value and the integrin surface concentration, the ratio of ligand to total receptor was cal- culated at maximum binding. For estimates of the amounts of active receptor equimolar complexes with peptide and fibrinogen were as- sumed. Consequently bifunctionality of fibrinogen would increase the fraction of active receptor by a factor of two.

Kinetics of the Two-step Mechanism-An analytical solution was developed for the two-step mechanism

in which L stands for the ligand (fibrinogen), R for the receptor (integrin), I for the intermediate complex, and C for the irreversibly formed final complex. Small letters stand for time-dependent concen- trations, and the sum of the concentrations of the two-step complexes was defined as s = i + c. The measured normalized fluorescence or ELISA signal is s/r, with s/r, = 1 for full binding, and r, stands for the total receptor concentration. It follows with

F = & + k-, + k‘ and G = k’k2 (Eq. 3)

and

with

F 2

H , = - - + E - G ) 112

and

(Eq. 7)

(Es. 8)

Since &was always more than 10-fold larger than r,, it follows that 1 = & and k’ = kl& is a pseudo first order rate constant. For fitting to the experimental data, a fitting-program (Goldberg, 1989) and an IBM computer were used.

Calculation of KO for True Binding Isotherms-The concentration of the complex was always much smaller than the total ligand con- centration (1 = &). Therefore KD = & at half of the maximum signal.

RESULTS

Integrin Can Be Reconstituted Correctly into Homogeneous Vesicles”cuIIb~3 was isolated from human platelets by puri- fying Triton X-100-solubilized membrane proteins by ion

exchange and gel permeation chromatography. We then tried to incorporate this pure integrin into lipid vesicles using a previously described dialysis method with octyl-8-D-glucopyr- anoside as detergent (Pytela et al., 1985). Separation of non- incorporated integrin was attempted by sucrose gradient cen- trifugation. lntegrin incorporation was determined by protein and lipid quantitation and in addition by electron microscopy. All lipid compositions employed seemed to yield good incor- poration according to protein and lipid determinations (Table IA). However, electron microscopy revealed proper incorpo- ration only into POPC/POPS vesicles. In DMPC and DMPC/ DMPG vesicles integrin adhered very strongly as large aggre- gates on the vesicle surface. Similarly, incorporation could not be detected by electron microscopy into POPC, POPC/ POPG, and egg phosphatidylethanolamine vesicles (data not shown). POPC/POPS vesicles showed a large size heteroge- neity probably due to fusion of negatively charged vesicles in the presence of Ca2+ and were for this reason not useful in our assay.

More successful incorporations were achieved with Triton X-100 as detergent and by removing it by adsorption to Bio- Beads SM-2. With this method integrin did not incorporate into POPC/POPS vesicles, but high yields were obtained in DMPC and DMPC/DMPG vesicles (Table IB). DMPC/ DMPG vesicles showed a homogeneous size distribution (100- 200 nm) and excellent incorporation as judged by electron microscopy (Fig. 1). This incorporation method is also suitable for other integrins as tested with alp1 from chick gizzard (data not shown).

DMPCIDMPG Vesicles form Bilayers on a Quartz Slide by Direct Fusion-Fluorescently labeled aIIbp3 with two FITC molecules/integrin was incorporated into DMPC/DMPG (50/ 50, mol/mol) vesicles. These were injected into the TIRFM cell for bilayer formation as monitored by space-restricted excitation by the evanescent field at the quartz-buffer inter- face (Fig. 2). The bilayer was stable and could be damaged neither by washing the cell intensively with buffer nor by increasing the stirring velocity.

Binding of Fibrinogen to aIIbB3 in Bilayers-Fibrinogen was labeled with TRITC to contain between 4 and 6 rhoda- mine residues/molecule. Vesicles with incorporated integrin were used for bilayer formation on the quartz cover of the TIRFM cell. After removal of excess vesicles, BSA was added to prevent nonspecific binding. Upon addition of labeled fibrinogen, the time course of the increase of fluorescence was monitored at different fibrinogen concentrations (Fig. 3).

TABLE I Reconstitution of (uZIbD3 into lipid vesicles

Different lipids and (uIIbp3 were dissolved in 25 mM octyl-0-D- glucopyranoside containing buffer (A) which was removed by contin- uous dialysis for 24 h or in 10 mg/ml Triton X-100 containing buffer (B). Triton X-100 was removed by Bio-Beads. Nonincorporated in- tegrin was removed by sucrose gradient density centrifugation. Di- alyzed vesicles were characterized by electron microscopy, and pro- teinllipid ratios were determined by protein and phosphate assays.

Initial Protein/lipid incE&:zion Lipid protein/lipid ratio after

ratio reconstitution (molar) (molar) microscoDv)

electron

A POPC/POPS, 25/75 1/900 l/2000 DMPC

Partial 1/900 1/5700 No

DMPCDMPG, 50/50 1/900 1/2100 No

POPC/POPS, 75/25 1/800 1/1700 No DMPC 11800 1/2200 Yes DMPC/DMPG. 50/50 1/800 1/1500 Yes

B

From estimates of the specific fluorescence intensity of the ligand and the concentration of the integrin, it was determined that more than 50% of the total integrin in the bilayer did bind fibrinogen. The kinetics were clearly multiphasic and were fitted with theoretical curves calculated for a biphasic mechanism (Equation 2 under “Materials and Methods”) with kl = 4.4 lo‘ M” s-’, = 2.2. s“’, and kp = 1.2. s-’ as fitting parameters. Consequently KO = k-l/kl = 50 nM.

To allow comparison with similar evaluations by other authors (see “Discussion”), the signals measured after 2 h were also plotted against fibrinogen concentration (Fig. 4), although it was noticed that even after this time a true equilibrium was not reached. An apparent K D = 30 nM was obtained. The data reveal that fibrinogen bound to incorpo- rated integrin, but not to the lipid bilayer (Fig. 4), nor to the incorporated integrin in the presence of EDTA. In the absence of BSA, fibrinogen bound nonspecifically to the lipid bilayer. The same amount of nonspecific binding was measured when fibrinogen was added to integrin-containing bilayers in the presence of EDTA (data not shown).

To determine the dissociation rate constant, an excess of unlabeled fibrinogen, peptide, or EDTA was added after 2 h of incubation with labeled fibrinogen. We further tried to dissociate bound fibrinogen by washing the cell intensively with buffer. In neither case could fibrinogen be dissociated within 2 h (data not shown), and thus the second step of the binding mechanism was assumed to be practically irreversible.

RGD Peptides Bind to aIIbp3 in Immobilized Receptor As- says with an Apparent KD of 2000 nM-The peptide GRGDSPC was labeled with the rhodamine fluorescent dye T488 which was attached covalently to the cysteine in a 1:l molar ratio. aIIbP3 was coated to plastic wells which were blocked afterwards with BSA. Biotinylated fibrinogen and diff9rent amounts of peptides were added simultaneously to

the plates, and the amount of bound fibrinogen was measured after 2 11. GRGDS, GRGDSPC, and T488-labeled GKGDSPC inhibited fibrinogen binding in the same concentration range. As expected RGES had no effect even at high concentrations (Fig. 5 ) . The KD for the RGD peptides could be calculated on the assumption that the same amount of fibrinogen and peptide had bound to integrin at the half-maximal amplitude of the curve. The K D was estimated to be 2000 nM, but this value is apparent because of the irreversible binding of fibrin- ogen (see “Discussion”).

Peptide GRGDSPC binds to aIIbp3 in bilayers with a KD of 1200 nM-For TIRFM measurements, increasing amounts of labeled GRGDSPC were added to aIIbj33 in bilayers without blocking by BSA (Fig. 6). Already a short time after addition, the fluorescence intensity was constant (Fig. 7), and values obtained after 0.5 h are plotted in Fig. 6. The peptide bound to aIIbp3 in the bilayer but also gave rise to a lower and linear signal in the absence of integrin. The same background signal could be observed if the peptide was added to lipid bilayers with and without aIIbp3 in the presence of EDTA (data not shown). The background signal originates from the fraction J , of unbound peptide in the evanescent wave, which is significant at the high peptide concentrations, but could be ignored for the 100-fold lower protein concentrations in the measurements with fibrinogen. For a peptide concentration of 3000 nM, a Jb/J, ratio of 1 was estimated with Equation 1 (see “Material and Methods”) in satisfactory agreement with the corresponding experimental value of 2 derived from Fig. 6A. The difference between the two curves in Fig. 6A yielded the binding isotherm (Fig. 6B). In accordance with the similar finding for fibrinogen (see above), more than 80% of the integrin was found to bind GRGDSPC peptide. A KD of 1200 nM was estimated from the ligand concentration at which half of the maximum fluorescence intensity was reached.

.. ..II--- ”

Two-step Binding of Fibrinogen to cuIIbp3 6803

FIG. 1. Electron micrograph of aIIb83-containing DMPC/DMPG vesicles. The integrin was reconstituted into DMPC/DMPG (50/50) vesicles by removal of Triton X-100 by Bio-Beads as described under “Material and Meth- ods.” Nonincorporated integrin was re- moved by sucrose gradient density cen- trifugation. Vesicles were negatively stained with sodium phosphotungstate.

6804

FIG. 2. Formation of planar lipid bilayers by fusion of aIIbB3-con- taining DMPCDMPG vesicles to quartz slides. FITC-labeled integrin was incorporated into DMPC/DMPG vesicles (see Fig. 1). Vesicle suspension (50 nmol) was injected into the measur- ing cell (buffer: 20 mM Tris-HCI, pH 7.4, 150 mM NaCl, 0.5 mM CaC12) to let the vesicles fuse onto a quartz slide. Fusion kinetics were followed by the measure- ment of the time course of the fluores- cence intensity (excitation 488 nm) by TIRFM. After 100 min the cell was washed with 50 ml of buffer to remove excess vesicles. No change of fluores- cence was observed after the washing step (arrow).

a

Two-step Binding of Fibrinogen to aIIbP3 400

300

w a s h

200

100

0

400

300

w a s h

200

100

0 0 5 0 100 1 5 0 2 0 0

time (min)

1500 I 1000

500

0 0 1 0 0 2 0 0 300

t ime (min)

TRITC-labeled fibrinogen to aIIbB3 FIG. 3. Association kinetics of

in bilayers by TIRFM. The integrin was incorporated into DMPG/DMPC (60/50, mol/mol) vesicles and planar bi- layers were formed as described in Fig. 2. For blocking of unspecific binding, bilayers were treated with 10 mg/ml BSA in measuring buffer for 1 h. Rhodamine- labeled fibrinogen was injected (0, 10 nM; 0,30 nM; A, 90 nM), and association kinetics were followed by measuring the time course of the fluorescence intensity (excitation 514 nm). The biphasic kinet- ics were fitted to the experimental data (solid curues) by the mechanism (1) and rate constants given in the text. For the experiment with 30 nM fibrinogen the calculated time courses of the interme- diate complex (. - . ) and the final prod- uct (- - - -) are shown.

FIG. 4. Binding of rhodamine-la- beled fibrinogen to aIIbB3 in planar lipid bilayers by TIRFM. Increasing amounts of rhodamine-labeled fibrino- gen were added and fluorescence inten- sities were recorded 2 h after each addi- tion (H). Unspecific binding to DMPG/ DMPC in the presence of 1 mg/ml BSA is shown in the lower curve (0). The theoretical background value for labeled molecules in solution at 1000 nM fibrin- ogen is about 0.5% of the saturation in- tensity.

1200 a

c loO0- .- c cn

0 2 0 0 4 0 0 6 0 0 8 0 0 1000 fibrinogen (nM)

0' f

0

Two-step Binding of Fibrinogen to aIIbP3

1.6 I

1 . 4 - 1.2 -

1 -

0.8 - 0.6 - 0.4 - 0.2 -

6805

1 0 - 9 1 0- 1 0' 1 0-

peptide (M)

FIG. 5. Inhibition of fibrinogen binding to aIIbg3 by peptides in ELISA-type assays. Purified aIIb03 was coated to mirotiter wells for 2 h at 20 'C. After removal of excess of integrin with measuring buffer wells were blocked with 0.5 mg/ml BSA as detailed under "Material and Methods." Biotinylated fibrinogen and increasing amounts of nonlabeled peptide were added simultaneously. After 2 h of incubation at 37 "C unbound fibrinogen and peptide were removed by washing, and bound fibrinogen was detected by successive addition of the streptavidin-peroxide complex and substrate and measurement of the extinction at 414 nm (see "Materials and Methods"). Each point represents the mean of four independent measurements. Rhodamine-labeled GRGDSPC (0) as well as nonlabeled GRGDSPC (0) and GRGDS (0) inhibited fibrinogen binding identically, whereas the control peptide RGES (A) did not give any inhibition.

Attempts to measure the association and dissociation rates of the labeled peptide GRGDSPC by TIRFM were not suc- cessful, as the dead time of the instrument (about 1 min) was longer than the kinetic processes (Fig. 7). Dissociation was induced by washing with buffer and addition of GRGDS. The experiment clearly demonstrates full reversibility of binding in contrast to the essentially irreversible binding of fibrino- gen. After removal of the peptide by washing, new peptide could be bound. This, in addition to the result of Fig. 2, demonstrates the full stability of the bilayer in the TIRFM experiment.

Inhibition of Fibrinogen Binding to aIIbB3 Is Time-depend- ent-The time course of fibrinogen binding to aIIb@3 was followed by the immobilized receptor assay. Biphasic concen- tration dependencies were observed (Fig. 8). They were qual- itatively very similar to the curves obtained by TIRFM and could be fitted by the same two-step mechanisms with some- what different constants kl = 1. lo6 s-' "', k 1 = 3. s-' and kz = 5. lo" s-' (KD = 27 I"').

The extent of transfer into the stable form C which is predicted by the mechanism was tested by inhibition of fi- brinogen binding by a large excess of GRGDS peptide which was added after different times of incubation (Fig. 9). Only when the peptide was added simultaneously with the fibrino- gen was almost 100% inhibition observed. When the peptide was added after 15, 60, and 120 min, the extent of inhibition decreased dramatically and was only 15-20% after 2 h. Note that even after simultaneous addition the binding of fibrino- gen slowly increased with time, indicating slow but irreversi- ble binding of fibrinogen.

The results shown in Fig. 9 are in full agreement with the predictions of the two-step mechanism. The amount of com- plex formation which cannot any longer be inhibited by addition of peptide at a given time equals the final nondisso- ciable complex C found at this time (Fig. 8).

DISCUSSION

Integrin aIIbB3 is predominant on platelets and plays an important role in thrombosis and hemostasis by binding to

the multifunctional fibrinogen dimer (Cheresh et al., 19891, thus mediating irreversible platelet aggregation. On platelets the receptor has to be activated by ADP (Marguerie et al., 1980) or thrombin (Vu et al., 1991) to induce binding to soluble fibrinogen (Marguerie et al., 1980). After isolation by deter- gents the receptor seems to be in an activated state. We investigated the mechanism of fibrinogen binding to purified aIIb@3 incorporated into lipid bilayers by total internal re- flection fluorescence microscopy, a method which has been used earlier for studies of protein-membrane interactions (Kalb et al., 1990, Kalb and Engel, 1991). Data obtained were compared with fibrinogen binding to aIIb@3 in a solid phase assay.

Integrin crIIb@3 was isolated from human platelets and then incorporated into lipid vesicles. Tests of two different recon- stitution methods, several detergents, and differing lipid com- positions showed that all three parameters were highly critical for reconstitution. Integrin incorporated truly well only into DMPC/DMPG (50/50, mol/mol) vesicles, using Triton X- 100 as detergent and Bio-Beads to remove the latter. Incor- poration and vesicle size were both rather homogeneous, and vesicles did not aggregate due to the negative charge of DMPG. Negatively charged lipids generally seemed to support integrin incorporation. We also obtained integrin incorpora- tion into POPC/POPS (25/75, mol/mol) vesicles after remov- ing octyl-@-D-glycopyranoside by dialysis. However, incorpo- ration was not homogeneous, and the vesicles showed strong size heterogeneity due to vesicle fusion of POPS in the pres- ence of Caz+ and M e . All reconstitutions with other lipid compositions were unsuccessful.

Correctness of protein incorporation was shown by electron microscopy. In well reconstituted samples single integrins incorporated into the membrane could be clearly seen and frequently their two tails could be distinguished. Comigration of lipid and integrin in a sucrose gradient was not a reliable measure of correct incorporation. Especially during dialysis, nonincorporated integrin formed large protein aggregates which adhered so strongly to the vesicles that they could not

6806 Two-step Binding of Fibrinogen to aIIbS3

b l e d GRGDSPC to aIIbB3 in bilay- FIG. 6. Binding of rhodamine-la-

ers by TIRFM. Bilayers with (rIIb83 in DMPC/DMPG vesicles were formed as described in Fig. 2 but not blocked with BSA. Increasing amounts of TRITC-la- beled peptide were added. Binding iso- therms were determined by measuring the fluorescence intensity for each pep- tide concentration after 30 min. A , the lower curve (W) shows the fluorescence increase for pure DMPGDMPC bilay- ers without integrin. The effect results from peptide in solution (see text). The upper curve (El) shows the fluorescence increase in the presence of (uIIb83 which is the sum of the signal for bound peptide and the background signal of the peptide in solution. B, the difference of the two

of GRGDSPC to aIIb83. The dissocia- curves in A yields the binding isotherm

tion constant KO = 1200 nM was esti- mated by determination of the peptide concentration which gives half of the maximum fluorescence signal (see "Ma- terials and Methods").

Q) 0 C

0 0 10000 20000 30000 40000

peptide (nM)

400 3 +GRGOSPC* + GRGDSPC'

300

200

100

"-~". , . . , . . , - . , . . I - - . 0 3 0 6 0 9 0 120 150 180

time (min)

FIG. 7. Association and dissocia- tion kinetics of peptide binding to aIIb@3 in bilayers by TIRFM. Bilay- ers with aIIb83 in DMPC/DMPG were formed. Rhodamine-labeled peptide GRGDSPC was added at a concentra- tion of 3300 nM and association kinetics were followed by measuring the time course of the fluorescence intensity (0) at an excitation of 514 nm. After reach- ing equilibrium, the cell was washed with buffer (4 ml/min) while monitoring the time course of the fluorescence intensity. A second addition of labeled peptide yielded again the same fluorescence in- tensity. Peptide could be dissociated by addition of a 100-fold excess of GRGDS. Note that labeled peptide which stayed free in solution exhibits a background signal similar to that shown in Fig. 6A. This was abolished by washing with buffer.

Two-step Binding of Fibrinogen to aIIbp3 6807

2.0 I

time (min)

FIG. 8. Association kinetics of fibrinogen binding to aIIb83 determined by the ELISA-type assay. Fibrinogen was added to the immobilized integrin at 30 nM (0) and 90 n M (A) concentration, and the time course of binding was monitored as described under "Materials and Methods." The solid curves were calculated with the two-step mechanism (2) with equal fitting parameters for the two curves (kl = 1.1.10' M" s-', k-l = 3.0. lo-' s-', kz = 5.3 10" s-l). The dashed curues are the corresponding fractions of final complex c (- - - -, 90 nM; . - . , 30 nM), and the bars represent estimates of these fractions obtained from the fraction of nondissociable and therefore not inhibitable material at 30 nM concentration (Fig. 9).

1-8

- A * 0 c A t o

o-2 1 0.0 I I I I I I I I I

0 30 6 0 9 0 120 150 180 210 240 270 t ime (min)

FIG. 9. Displacement of bound fibrinogen by a 300-fold molar excess of GRGDS peptide as monitored by an immobi- lized receptor assay. The fibrinogen concentration was 30 nM, and the binding curve without peptide addition is shown (0). Peptide was added simultaneously with the fibrinogen (0) or after 15 min (I), 60 min (0), and 120 min (A). The degree of inhibition (right ordinate) can be estimated from the variable decrease of maximum binding after addition of the peptide.

be separated on a sucrose gradient. Vesicles with incorporated labeled integrin formed planar

lipid bilayers by fusion to a quartz surface (Brian and Mc- Connell, 1984), and this process was followed by TIRFM. On the assumption that integrin incorporation into vesicles was random, only half of the integrins are expected to be oriented with their extracellular domains toward the solution.

Binding of fluorescence-labeled fibrinogen to aIIbj33 could be monitored by total internal reflection fluorescence micros- copy. In view of the discussion over only partial activation of isolated aIIbj33 in integrins (Du et al., 1991; Kouns et al., 1992), we have determined the binding capacities for fibrino- gen and the RGD-containing peptide. These estimates are not very precise but show that more than 50 or 80% of aIIbB3 bound fibrinogen and the peptide, respectively. We therefore conclude that most, if not all, of our integrin preparation is

activated. This also indicates that possible small impurities of 4 3 , which were not detectable by the immunoassay, do not influence our results. Other integrins were detected in platelets in very small amounts (Hemler et al., 1988), and no fibrinogen binding was demonstrated (Sonnenberg et al., 1988).

The time course of fibrinogen binding followed a biphasic kinetics, which did not reach a plateau value even after several hours. From this we concluded a two-step mechanism, con- sisting of a fast equilibrium reaction followed by a slow practically irreversible second process (see Equation 2). The concentration-dependent kinetics were fitted for the above mechanism with the rate constant kl , k-1, and kl as fitting parameters. We also followed kinetics of fibrinogen binding to aIIbj33 in a solid phase immobilized receptor assay. These experiments were evaluated by the same two-step mechanism. The rate constants were kl = 0.4- 10' (1.1.10') I"' s-', k-1 = 2.2. (3. s-', kz = 1.2. lo" (5.10") s-'. KD was calculated to be 50 (27) nM. Values are from TIRFM meas- urements and values in brackets are the results from immo- bilized receptor assays. The two entirely different methods yield rather similar results. It should be considered that TIRFM measurements were performed at 24 "C and immo- bilized inhibition assays at 37 "C.

Rough KD values could be obtained from the binding iso- therms measuring the amount of bound fibrinogen after 2 h by TIRFM (KD = 30 nM at 24 "C) and immobilized receptor assay (KO = 7 nM at 37 "C). As equilibrium was not reached, these are only apparent binding constants, which can, how- ever, be compared with the values measured by other authors in a similar way. Apparent KO values of 500 nM have been published for the fibrinogen binding to aIIbB3 on platelets (Marguerie et al., 1980), a KO of 12 nM for the fibrinogen binding to aIIbj33 in isolated platelet membranes (Phillips and Banghan, 1983) and a KD of 12 nM for the fibrinogen binding to immobilized aIIbj33 (Charo et al., 1991).

To strengthen the evidence for the postulated mechanism, dissociation kinetics of the intermediate and the final product were investigated. Fibrinogen binding to aIIbB3 in lipid bilay- ers and in immobilized form became essentially irreversible after 2 h. This was indicated by the lack of dissociation of labeled fibrinogen after addition of unlabeled fibrinogen, GRGDS peptide, or EDTA in large excess or after washing with plain buffer. It was, however, possible to achieve partial dissociation after shorter times of binding of fibrinogen, and this fraction corresponded quantitatively to the fraction of precomplex I predicted by the two-step mechanism.

The two-step mechanism of fibrinogen binding is of large practical importance. It explains why the results of solid phase assays often strongly depend on incubation time and, in particular, on the sequence in which the competing ligands are added. This phenomenon was thoroughly investigated by Orlando and Cheresh (1991) and Felding-Habermann et al. (1992) for the avj33/fibrinogen system. In the present work we found complete inhibition for aIIbj33/fibrinogen/GRGDS only when both ligands were added simultaneously. Even under this condition the extent of inhibition slowly decreased with time, due to a slow increase of the low amount of irreversibly binding fibrinogen. The data clearly demonstrate that binding constants determined in earlier studies for inter- actions of aIIbB3 and avj33 do not reflect the true equilibrium binding situation, but rather a pre-equilibrium of binding. The results may further significantly depend on the experi- mental conditions and on the time of equilibration. Most importantly, inhibition assays will usually strongly depend on the way the experiment is performed. Results may range from

6808 Two-step Binding of Fibrinogen to aIIbP3

no inhibition to full inhibition, depending on the time of preincubation with the protein.

For measurements of binding affinity for small peptides, competition with a large ligand is not needed when the TIRFM method is used. With this method we were able to measure direct binding of labeled GRGDSPC peptide to (rIIb83 in lipid bilayers. Kinetics of peptide binding reached a plateau value after a short time. Full reversibility was indicated by complete dissociation of the labeled peptide after washing of the membrane with plain buffer. This allowed us to measure a binding isotherm and to calculate a KD of 1200 nM at 24 "C. The binding of the peptide was not only revers- ible but also very fast. By use of the TIRFM method with its dead time of about 1 min, we were not able to determine the rates of association or dissociation. To compare the KD of peptide binding to (rIIb83 in a solid phase assay, fibrinogen binding was inhibited simultaneously by increasing amounts of peptide. A KD of 2000 nM at 37 "C was calculated from the ELISA-type inhibition assay. The latter value is again not a true equilibrium value for the reasons outlined above.

What is the cause and biological significance of the very strong binding of fibrinogen in the second step of the mech- anism? Most interestingly, the phenomenon was already ob- served by Marguerie et al. (1980) for binding of fibrinogen to whole ADP-activated platelets. The authors discussed second- ary associations of very high affinity with the lipid bilayer or other components of the platelet membrane. From our results we can state that the almost irreversible binding in the second step is rather an intrinsic property of the fibrinogenlaIIbp3 system. Our data, as well as data by Orlando and Cheresh (1991) on ~ 4 3 , clearly show that irreversible binding also occurs to integrin immobilized on plastic surfaces. On the basis of these different experimental approaches, there can be little doubt that irreversible binding is a reproducible phys- iological process. Aggregation of platelets leads to an irrevers- ible clot and therefore an essentially irreversible fibrinogen/ integrin interaction appears functionally favorable.

The fibrinogen dimer is a multifunctional molecule with probably four or six sites for integrin binding (D'Souza et al., 1988, 1990; Charo et aZ., 1991). The first relatively weak interaction may reflect the initial interaction of one of these sites with a first integrin. This may be followed by interactions with other integrin molecules, leading to essentially irrevers- ible binding. Other possibilities are multifold interactions of the same integrin with different sites on fibrinogen or aggre- gation of fibrinogen, again causing multifold interactions. Aggregation of the ligand, if it occurs at all, must be limited in extent, because it was not apparent by a heterogeneous fluorescence signal, as in the case of laminin aggregation (Kalb and Engel, 1991). Conformational changes between two states of soluble aIIbp3 with different affinities have been

concluded from the opportunity to activate the receptor by monoclonal antibodies (Frelinger et al., 1991). These different possibilities will be tested by additional measurements by the TIRFM method. It will also be of interest to investigate whether irreversible second steps occur also for other types of integrin/ligand interactions in which reversibility of inter- action may be required for biological action.

Acknowledgments-We thank Dr. Edwin Kalb for introduction into the TIRFM method, A. Luthi for initial reconstitution experi- menta, and Dr. Mats Paulsson for helpful discussions.

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