Conformational change in fibrinogen induced by adsorption to a surface

Conformational Change in Fibrinogen Induced by Adsorption to a Surface JEANNETTE SORIA, 1 CLAUDINE SORIA, MASSOUD MIRSHAHI, CLAUDE BOUCHEIX, ANDRIS, AURENGO, JEAN-YVES PERROT, ALAIN BERNADOU, MEYER SAMAMA AND CLAUDE ROSENFELD Services des Professeurs Bernadou, Fabiani, Sarnama, H~tel-Dieu, Paris 4~; Services des Professeurs Caen, Rousselet, Hrpital Lariboisi~re, Paris 10~; INSERM U 253, Villejuif; and D£partement de biophysique et biomath~mathiques (Pr Grimy), CHU Piti~-Salp£tridre, Paris 13~, France Received May 30, 1984; accepted February 19, 1985 It is of great interest to know whether proteins adsorbed on a solid surface are conformationally altered, particularly for the problem of blood-foreign surface interaction. With this aim in mind, we tested the reactivities of immobilized or soluble fibrinogen against monoclonal antibodies obtained by mouse immunization with fibrin derivatives. Using an immunoenzymological assay with a monoclonal antibody that revealed an epitope present in the D domain of the molecule but not exposed on soluble undegraded fibrinogen, we found that the binding of fibrinogen to polystyrene rendered the epitope accessible to the monoclonal antibody. We speculate with these results that a conformational alteration of fibrinogen occurred by adsorption to the solid phase. Such results may have important applications in the testing of artificial surfaces that are designed to come in contact with the circulation. © 1985 Academic Press, Inc.

INTRODUCTION

associated in complexes such as the D - D - E complex (6). The results concerning the possible changes of conformation of fibrinogen during its adsorption are conflicting (7, 8), Investigations of fibrinogen mapping have led us to the production of a monoclonal antibody which reacts with polystyrene-adsorbed fibrinogen or to the D fragment either in solution or adsorbed to polystyrene, but which does not interact with soluble nondegraded fibrinogen. This antibody has been proposed for the investigation of the conformational changes of adsorbed fibrinogen.

The adsorption of blood proteins on artificial materials is the first step in a sequence of events which may lead to either biocompatibility or thrombosis. The role of the protein layer composition and of the conformation of the adsorbed proteins in blood material interactions still remains controversial. The case of fibrinogen (Fg) has been widely investigated since it is known to selectively adsorb from plasma onto artificial materials (1, 2) and since platelets adhere to fibrinogen-coated substrates (3-5). Fibrinogen is trinodular, composed of two lateral D domains and one central E domain MATERIAL AND METHODS linked together by coils. The complete degREAGENTS radation of fibrinogen by plasmin leads to the release of two D fragments and one E The following reagents were used: purified fragment. When fibrinogen is converted into human fibrinogen (Kabi); Tween 20, bofibrin, the plasmin degradation products are vine albumin, and orthophenylenediamine (Sigma); Peroxidase-labeled goat Ig antimouse l To whom all correspondence should be addressed: Laboratoire de Recherches (Sainte-Marie), Hrtel-Dieu Ig (Nordic); and 96 well disposable polysty75681, Paris Cedex 04, France. rene microplates for Elisa, Dyna Washer, and 204 0021-9797/85 $3.00 Copyright © 1985 by Academic Press, Inc. All a'ights of reproduction in any form reserved.

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CONFORMATION OF ADSORBED FIBRINOGEN

205

Elisa Reader (Dynatech). Fragment D and fragment E were obtained by degradation of fibrinogen by plasmin, then separated by ion exchange chromatography on DEAE cellulose according to Nilehn (9). Fibrin split products were obtained according to Gaffney by partial digestion (50%) of a plasminogen-enriched clot immersed in streptokinase (SK) (10).

protein attachment was carried out by adding to each well 100 ul of the coating solution at 2 ~tg/ml in 4 M urea pH 7.5. For both sets of plates, the coating was carried out by incubation overnight at 37°C in a humid atmosphere in order to avoid evaporation of the protein solution used for coating. Then, unadsorbed antigen was removed by washing the wells 4 times with 0.15 M NaC1 containPRODUCTION OF MONOCLONAL ing 0.05% Tween. The screening was carried ANTIBODIES (MAbs) out by incubating plates coated with fibrinogen, fibrin degradation products, fragment 1. Immunization D, or fragment E for 2 h at 37°C with 100 BALB/C mice were injected twice with #1 of each culture fluid supernatant diluted 100 ~zg crosslinked fibrin fragments, first in- to one-half strength. After four washes, the traperitoneally, and then intravenously 1 plates were incubated for another 2 h at 37 oc with 1/ 1000-diluted peroxidase-labeled month later. goat Ig antimouse Ig and washed again 4 times. The peroxidase bound to each well 2. Production of Hybridomas was then determined by adding orthophenyHybridomas were obtained according to lenediamine and hydrogen peroxide as deFazekas de St. Groth and Sheidegger (11). scribed by Wolters et al. (13). After exactly Three days later after the second injection, 3 rain the reaction was stopped with sulfuric immune mouse spleen cells were fused with acid and the adsorbance measured at 492 NS1 myeloma cells. The hybridoma cells nm using an automatic Elisa spectrophotomwere cultured in hypoxanthine-aminoptereter. In order to avoid nonspecific adsorption ine-thymidine medium at 37°C with 7% (14), the 1/2-dilution of supernatant culture COz in a humid atmosphere. fluid was performed in a phosphate buffer containing 0.1% Tween 20 and 0.1% bovine 3. Screening albumin. The other dilutions were carried Two weeks later, the culture fluid super- out in the same buffer containing 0.05% natants were tested for reactivity against im- Tween 20 and 0.1% bovine albumin. Two mobilized fibrinogen, fibrin degradation controls were utilized: the first consisted of products, fragment D, and fragment E. In phosphate-Tween 20 buffer instead of superthis assay, 96 well polystyrene microtiter natant culture fluid; for the second we incuplates for Elisa were coated with fibrinogen, bated diluted supernatant culture fluid in fibrin degradation products, fragment D, or uncoated plates. fragment E. Coating of polystyrene was per4. Cloning formed using either nontreated plates when the proteins to be coated are in solution in Hybridoma cultures were selected by phosphate buffer, or glutaraldehyde activated screening and cloned using limiting dilution polystyrene plates as described by Klasen et in the presence of a feeder layer of spleen al. (12) when proteins to be coated are in cells. solution in 4 M urea. For nontreated plates, passive sensitization was achieved by adding COMPETITIVE INHIBITION BINDING 200 tsl of the coating solution to 1 ~tg/ml in The capacity of soluble fibrinogen or its 0.1 M phosphate buffer (pH 7.4) to each well. For glutaraldehyde-activated plates, degradation products to inhibit the binding Journal of Colloid and Interface Science,

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of these MAbs to fibrinogen-coated polystyrene plates (or plates coated with fibrinogen degradation products) was then determined. For this assay, the optimal dilution of hybridoma culture fluids to be used was determined by first testing serial dilutions of the culture supernatants on fibrinogen-coated plates. The dilution chosen was that which gave 80% of maximal binding. A two-step procedure was carded out. As for the screening test, all dilutions were carried out in phosphate buffer containing 0.05% Tween 20 and 0.1% bovine albumin. In the first step, 500 #1 of serial dilutions of fibrinogen (3 X 10"8 M to 4.5 X 10-l° M) were preincubated with 500 ~tl of the diluted culture fluid supernatant for 18 h at 4°C and then centrifuged. In the second step, 100 #1 of the centrifuged supernatant from each preincubated sample were added to fibrinogen-coated plates. After 2 h incubation at 37°C, the plates were washed 4 times to remove unbound material. The quantity of mouse Ig bound to the plates was determined as described above by the reaction of peroxidase-labeled IgG antimouse Ig. The dose-response curve was established by plotting the bound immunoreactive enzyme (expressed as optical density (0.D.)) versus increasing amounts of soluble fibrinogen added (log scale). Inhibition studies were also performed using a solution of fibrinogen degradation products instead of fibrinogen. Additional experiments were carded out with fragment D- or fragment Ecoated plates. The same experiments concerning the screening and the competitive binding assay were also performed in the absence of albumin. RESULTS AND DISCUSSION SCREENING TEST

We tested 720 supernatants and 210 supernatants were found to be reactive with fibrinogen- or fibrin degradation products coated-plates obtained by passive sensitizaJournal of Colloid and Interface Science, Vol. 107,No. 1, September1985

tion. These 210 supernatants were further tested with fragment D and fragment E and the corresponding cells from five of these were cloned and then recloned for further investigation. Two of these MAbs (coded ESB2 and ESB3) revealed epitopes present in the E fragment of the molecule while the 3 others (coded DSB1, DSB2, and DSB3) were directed against an epitope located in the D fragment of the molecule. The screening assay used was shown to be specific since no mouse IgG was detected on polystyrene after incubating diluted supernatant culture fluid in uncoated plates, thus demonstrating that nonspecific adsorption did not occur even onto an uncovered surface. In addition no peroxidase-labeled IgG antimouse IgG was found on fibrinogen-coated plates in the absence of supernatant culture fluid. Furthermore, Tween 20, used at 0.05% final concentration, did not influence the stability of the protein-solid phase bond, since no loss of adsorbed protein plates was found throughout the assay, as demonstrated by Cantarero, Butler, and Osborn (15). In addition, the fact that the antibodies tested at several dilutions did bind to fibrinogen-coated plates to the same extent regardless of the presence or the absence of albumin in the buffer (results not shown) allowed us to conclude that albumin did not mask any epitopes of immobilized fibrinogen involved in this antigen-antibody reaction. IMMUNOENZYMOLOGICAL COMPETITIVE INHIBITION BINDING ASSAY

These five monoclonal Abs against fibrinogen were further examined by characterizing the inhibitory capacity of fibrinogen in solution on the binding of the MAbs to immobilized fibrinogen or fibrinogen degradation products on polystyrene wells. Fibrinogen in solution at low concentration prevented the binding of four of the culture fluid supernatants (ESB2, ESB3, DSB1, DSB3) to plates coated with fibrinogen obtained by passive sensitization. There was a

CONFORMATION OF ADSORBED FIBRINOGEN linear dose response curve between the log of the concentration of fibrinogen and the decrease of Ig binding to fibrinogen-coated wells (expressed as the optical density) (Fig. 1). On the contrary, with the culture supernatant DSB2, the results are quite different, since MAb in the culture fluid did bind to the fibrinogen-coated plates, regardless of the coating procedure used but fibrinogen in solution, up to 3 X 10 -8 M (or 6 X 10 -8 M if fibrinogen concentration is expressed as fragment D equivalent) was unable to prevent t h e binding of the antibody (Fig. 1). On the contrary, fragment D in solution at a very low concentration (up to 1 X 10 -8 M), completely inhibited MAb binding to both fibrinogen- and fragment-D-coated plates (Fig. 2). Similar results were observed when fragment-D-coated plates were used instead of fibrinogen-coated plates (results not shown). Furthermore, in the competitive binding assay, the same quantitative results were found whether the experiment was carried out in the presence or in the absence of albumin (results not shown). Thus, these results allow us to conclude that albumin which represented a large excess relative to antibody did not mask the corresponding epitopes of fibrinogen.

OD 492nm 1-

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/z

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A"

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1:5 0:75 0:38 0:~9 0'.10 0'.05008M) Fg in solution

FIG. 1. Inhibition of the binding of 5 monoclonal antibodies (Ab): DSB1, DSB2, DBS3, ESB2, and ESB3 to immobilized fibrinogen induced by fibrinogen in solution. (x axis: molar concentration of fibrinogen in solution (log scale); y axis: optical density (O.D.) representing the quantity of Ab bound to immobilized fibrinogen).

207

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0.5-

a

r~

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m

e

n

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LOgscale 25 112 016 0'3 0'15 0'075(10-8M) Fg or fragrnent D in solution

FIG. 2. Inhibition of the binding of monoclonal antibody DSB2 to immobilizedfibrinogeninduced by fibrinogen or fragment D in solution. (x axis: molar concentration of fibrinogen (or fragment D) in solution; y axis: optical density (O.D.) representing the quantity of Ab bound to immobilized fibrinogen).

For the antibody to react with a protein molecule, the antigenic determinant recognized must be expressed at the surface and the local conformation must allow this binding. Thus, using the DSB2 antibody, it can be assumed that immobilization of fibrinogen yields an accessible epitope which is exposed on fragment D (immobilized or in solution) but poorly exposed in undegraded fibrinogen in solution. Thus DSB2 supernatant culture fluids allow us to detect a conformational change in fibrinogen induced by its absorption onto a solid phase. It can be assumed that surface-induced conformational change of fibrinogen is not related to a fibrinogen polymerization, occurring during the coating. Indeed, the fibrinogen coating to polystyrene was carded out either in the presence of urea or in its absence (urea is known to avoid polymerization) and whatever the coating procedure used, no difference in the binding of the monoclonal antibody to the immobilized fibrinogen could be noted. Such experiments may be of importance in the screening of artificial material used for grafts on extracorporal circulation. Many techniques are available to investigate the conformational changes of plasma proteins during adsorption. Some require specific equipment and utilize the peculiar optic properties of the material of interest. Journal of Colloid and Interface Science, Vol. 107, No. 1, September 1985

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A m o n g such techniques are infrared b o u n d fraction measurements on silica surfaces (16), ellipsometric studies on p l a t i n u m (17) or c h r o m i u m (18), Fourier transform infrared spectroscopy on coated A T R crystals (19), R a m a n spectroscopy on coated fluid prisms (20, 21), or circular dichroism on quartz discs (8). It is noticeable that circular dichroism can be used to study conformational changes o f fibrinogen eluted after adsorption, and can therefore be used whatever the material (7). However, the results o f this m e t h o d conflict with the direct technique (without elution), using quartz discs. Other m e t h o d s required a high specific surface area o f the material, e.g., microcalorimetry (22) or quasielastic light scattering (23). The m e t h o d proposed in this paper is easily performed and can be used for any material whatever its surface and its optical properties. Furthermore, the same technique could be used to investigate conformationat changes o f proteins other than fibrinogen. Moreover, the reported results provide direct evidence that the conformational change o f adsorbed fibrinogen m a y be great enough to reveal an epitope o f the molecule which is masked when fibrinogen is in solution. REFERENCES 1. Morrissey, B. W., Ann. N. Y. Acad. Sci. 283, 50 (1977). 2. Chuang, H. Y. K., King, W. F., and Mason, R. G., J. Lab. Clin. Med. 92, 483 (1978). 3. Adams, G. A., and Feverstein, I. A., Trans. Amer. Soc. Artif. Intern. Organs 27, 90 (1981). 4. Adams, G. A., and Feverstein, I. A., Trans. Amer. Soc. Artif. Intern. Organs 27, 219 (1981). 5. Kim, S. W., Lee, R. G., Oster, H., and Coleman, D., Trans. Amer. Soc. Artif. Intern. Organs 20, 449 (1974). 6. Marder, V. J., Francis, C. W., and Doolittle, R. F.,

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