Simultaneous Adsorption of Fibrinogen and Kininogen at a Silica

The competitive adsorption of fibrinogen and high molecular weight kininogen at different flow rates on silica capillaries has been studied at 37 °C ...
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Langmuir 1998, 14, 3356-3364

Simultaneous Adsorption of Fibrinogen and Kininogen at a Silica/Solution Interface M. T. Leˆ and P. De´jardin* Laboratoire des Mate´ riaux et Proce´ de´ s Membranaires, UMR 5635 (CNRS-UM II-ENSCM), 2 Place Euge` ne Bataillon, 34095 Montpellier Cedex 5, France Received December 9, 1997. In Final Form: March 23, 1998

The competitive adsorption of fibrinogen and high molecular weight kininogen at different flow rates on silica capillaries has been studied at 37 °C in Tris buffer (Tris 0.05 M; NaCl 0.10 M) and at a dilution of 10-2 with respect to the plasma concentrations. By radiolabeling (125I and 131I) each protein differently, it is possible to follow the adsorption of both molecules from the mixture simultaneously. An accumulation of both proteins at the interface followed by a progressive release of fibrinogen was observed. This release was not necessarily associated with an increase of the kininogen interfacial concentration. From an analysis of the initial kinetics of adsorption of kininogen vs wall shear rate, the diffusion coefficient (D ≈ 4.4 × 10-7 cm2 s-1) and the adsorption constant (ka ≈ 2.4 × 10-4 cm s-1) have been derived. The final adsorption kinetics of kininogen in the presence of fibrinogen is more compatible with a site adsorption model than with a random sequential adsorption model and leads to a much smaller adsorption constant, of the order of 10-5 cm s-1. This suggests that kininogen does not interact directly with the bare silica but with fibrinogen molecules, which act as adsorption sites. For the molecular ratio of proteins in solution used here, it was found that the interfacial concentrations of both proteins are independent of shear rates at the maximum of fibrinogen concentration (0.4 µg cm-2 for fibrinogen (Fib) and 0.1 µg cm-2 for kininogen (HK)), although appearing at varying times. These concentrations correspond to a molecular ratio HK/Fib of 0.9, which is close to an equimolecular composition at the interface. However at the beginning of the adsorption process, we measured one molecule of kininogen for 10 molecules of fibrinogen at the surface, in accordance with a theoretical estimation from the solution concentrations and molecular masses.

Introduction Protein adsorption at solid/liquid interfaces1,2 is important in many fields such as biocompatible materials, especially hemocompatible materials3 or diagnostic kits.4 Natural and artificial vessels adsorb proteins from blood. In blood compatibility applications, for example, it is important to know which of the plasma proteins are adsorbed to the blood-contacting surface since the identity of these proteins determines subsequent cell interactions.5 These adsorption phenomena must generally be avoided. Hydrophilization, by the pretreatment of surfaces with polymers having a high polyoxyethylene content for example can inhibit or limit these phenomena. This has been demonstrated in solid-phase diagnostics,4 in hemodialysis hollow fibers,6 and with polymer surfaces.7 Similarly, from studies on model self-assembled organic monolayers on gold surfaces it has been demonstrated that protein adsorption was inhibited on surfaces, that were made hydrophilic with oligomers of ethylene glycol compared to hydrophobic residues.8 An understanding of the mechanisms of molecular transfer from the solution

to the interface and vice versa is thus particularly important. Displacement of preadsorbed fibrinogen on different solid polymers by blood plasma has already been studied.9 The amount displaced was much less for a preadsorption time of 1 h than for 1 min, as a transition from a weakly bound (displaceable) to tightly bound (nondisplaceable) state occurred. Numerous kinetic models have been concerned with changes of conformation of adsorbed molecules.10-13 These exchange aspects were considered 20 years ago by Brash and Samak14 who recognized that if adsorption of proteins appears irreversible by rinsing with the buffer, dynamic exchange between molecules at the interface and in solution in fact takes place. This exchange however is limited because a part of the interfacial population cannot be exchanged. Similar studies were performed with polymers where, in contrast, the whole interfacial population could be exchanged.15 These experiments were done with a constant interfacial concentration mostly at full coverage of the interface, either under static conditions on flat surfaces16 or in flow conditions on the wall of a

(1) Andrade, J. D., Ed. Surface and Interfacial Aspects of Biomedical Polymers Vol. 2 Protein Adsorption; Plenum Press: New York, 1985. (2) Brash, J. L., Horbett, T. A., Eds. Proteins at Interfaces II: Fundamentals and Applications; ACS Symposium, No. 602; American Chemical Society: Washington, DC, 1995. (3) Leonard, E. F., Turitto, V. T., Vroman, L., Eds. Blood in Contact with Natural and Artificial Surfaces; Annals of the New York Academy of Sciences, Vol. 516; New York Academy of Science: New York, 1987. (4) Malmsten, M.; Lassen, B.; Holmberg, K.; Thomas, V.; Quash G. J. Colloid Interface Sci. 1996, 177, 70. (5) Collins, W. E.; Mosher, D. F.; Tomasini, B. R.; Cooper, S. L. Ann. N. Y. Acad. Sci. 1987, 516, 291. (6) Yan, F.; De´jardin, P.; Mulvihill, J. N.; Cazenave, J. P.; Crost, T.; Thomas, M.; Pusineri, C. J. Biomater. Sci.: Polym. Ed. 1992, 3, 389. (7) Lee, J. H.; Kopeckova, P.; Kopecek, J.; Andrade, J. D.; Biomaterials 1990, 11, 455. (8) Prime K. L.; Whitesides, G. M. Science 1991, 252, 1164.

(9) Slack, S. M.; Horbett, T. A. J. Colloid Interface Sci. 1989, 133, 148. (10) Lundstro¨m, I.; Elwing, H. J. Colloid Interface Sci. 1990, 136, 68. (11) Soderquist, M. E.; Walton, A. G. J. Colloid Interface Sci. 1980, 75, 386. (12) Sevastianov, V. I.; Belomestnaia, Z. M.; Zimin, N. K. Artif. Organs 1983, 7, 126. (13) Beissinger R. L.; Leonard, E. F. J. Colloid Interface Sci. 1982, 85, 521. (14) Brash J.; Samak, Q. M. J. Colloid Interface Sci. 1978, 65, 495. (15) Pefferkorn, E.; Carroy A.; Varoqui, R. J. Polym. Sci., Polym. Phys. Ed. 1985, 23, 1997. (16) Douglas, J. F.; Johnson H. E.; Granick, S. Science 1993, 262, 2010. (17) Huetz, P.; Ball, V.; Voegel, J. C.; Schaaf, P. Langmuir 1995, 11, 3145.

S0743-7463(97)01351-6 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/23/1998

Simultaneous Adsorption of Fibrinogen and Kininogen

capillary,17 on beads of latex,18 and recently on titanium oxide particles.19 Experiments under flow conditions have been reported on the in situ observation of the displacement of adsorbed molecules while the total interfacial concentration is still increasing.20 In these previous investigations the homogeneous exchange of fibrinogen using a simple model has been analyzed. From this model the kinetic constant of conformational changes could be estimated, together with the desorption and exchange constants. Competitive protein adsorption in blood and plasma2,9,21-30 and phenomena referred to as the Vroman effect have been reported previously. Cornelius et al. observed such effects for IgG in plasma,31 and Vroman has provided evidence that in this medium there is a continuous change of composition at the surface with more abundant proteins being replaced sequentially by less abundant ones.23 Vroman effects have been observed on many surfaces, and in general displacement decreases as hydrophobicity increases, presumably reflecting stronger binding on the more hydrophobic materials.25 Some surfaces do not exhibit the Vroman effect. Sulfonated polyurethanes, for example, adsorb large amounts of fibrinogen from plasma without subsequent displacement.32 It has been observed by both Vroman et al.21 and Brash et al.26 that in plasma the contact phase coagulation factor, high molecular weight kininogen (HK), plays a major role in displacing adsorbed fibrinogen, since plasma deficient in this protein is much less active. This is particularly true on hydrophilic surfaces such as glass. A domain rich in histidine and lysine residues has been identified as being responsible for binding to anionic surfaces.33 It has been shown in a recent work that the Vroman effect seen in plasma has its counterpart in simpler systems34 such as fibrinogen/HK mixtures in the presence of glass. The analysis was essentially focused on the final interfacial concentrations whose ratio was a linear function of the dilution, according to several possible mechanisms.35 Here we are studying the kinetics of the (18) Ball, V.; Huetz, P.; Elaissari, A.; Cazenave, J. P.; Voegel, J. C.; Schaaf, P. Proc. Natl. Acad. Sci. 1994, 91, 7330. (19) Bentaleb, A.; Ball, V.; Haı¨kel, Y.; Voegel, J. C.; Schaaf, P. Langmuir 1997, 13, 729. (20) De´jardin, P.; Le, M. T. Recent Research Developments in Polymer Science: Polymers and Surfaces, a versatile combination; Hommel, H., Ed.; Research Signpost: Trivandrum, India, in press. (21) Vroman, L.; Adams, A. L.; Fischer, G.; Munoz, P. Blood 1980, 55, 156. (22) Adams, A. L.; Fischer, G. C.; Munoz, P. C.; Vroman, L. J. Biomed. Mater. Res. 1986, 18, 643. (23) Vroman, L.; Adams, A. L. J. Colloid Interface Sci. 1986, 111, 391. (24) Brash, J. L.; ten Hove, P. Thrombos. Haemostas. 1984, 51, 326. (25) Wojciechowski, P.; ten Hove, P.; Brash, J. L. J. Colloid Interface Sci. 1986, 111, 455. (26) Brash, J. L.; Scott, C. F.; ten Hove, P.; Wojciechowski, P. W.; Colman, R. W. Blood 1988, 71, 932. (27) Horbett, T. A. Thrombos. Haemostas. 1984, 51, 174. (28) Slack, S. M.; Horbett, T. A. J. Colloid Interface Sci. 1988, 124, 535. (29) Breemhaar, W.; Brinkman, E.; Ellens, D. J.; Beugeling, T.; Bantjes, A. Biomaterials 1984, 5, 269. (30) Slack S. M.; Horbett, T. A. J. Colloid Interface Sci. 1989, 133, 148. (31) Cornelius, R. M.; Wojciechowski, P. W.; Brash, J. L. J. Colloid Interface Sci. 1992, 150, 121. (32) Santerre, J. P.; ten Hove, P.; Vanderkamp, N. H.; Brash, J. L. J. Biomed. Mater. Res. 1992, 26, 39. (33) Colman, R. W. Pure Appl. Chem. 1994, 66, 27. (34) De´jardin, P.; ten Hove, P.; Yu, X. J.; Brash, J. L. Langmuir 1995, 11, 4001. (35) De´jardin, P.; Le, M. T. Langmuir 1995, 11, 4008. (36) Le, M. T.; Mulvihill, J. N.; Cazenave, J. P.; De´jardin, P. In Proteins at Interfaces II: Fundamentals and Applications; ACS Symposium 602; Brash, J. L., Horbett, T. A., Eds.; American Chemical Society: Washington, DC, 1995; pp 129-137.

Langmuir, Vol. 14, No. 12, 1998 3357

processes and moreover of both proteins simultaneously. It may be noted that pure fused silica has been used in the present work; whereas in a preceding study with plasma36 the interaction with glass has been investigated. Finally, it should be emphasized that the interpretation of experimental data obtained from exchange experiments can be complicated because transport processes are involved. This is the case particularly under static conditions, and a critical analysis37 has appeared recently concerning the interpretation of polymer exchange data by stretched exponential functions whose exponent is not constant due to the readsorption process. Moreover under static conditions, especially at low concentrations where exchange is readily observed, the depletion due to adsorption can be high and can vary with protein type, leading to changes in relative bulk concentrations. Therefore, a study under flow conditions would seem easier to interpret as there is always an arrival of fresh solution in a welldefined velocity field. For such complex processes of adsorption at interfaces, especially from blood or plasma, the transport phenomena governed by hydrodynamics are important, especially in practical devices. This has been demonstrated recently by a study on the spatial arrangement of proteins at interfaces in complex pattern flows using dyed beads.38 In this work, we report and analyze the simultaneous adsorption kinetics of fibrinogen and kininogen on silica capillaries from flowing solutions in Tris saline buffer at 37 °C and different wall shear rates (50-760 s-1). Solution concentrations, 30 and 0.80 µg mL-1 for fibrinogen and kininogen, respectively, correspond to 10-2 plasma dilution. This enables the investigation of the transient adsorption of fibrinogen, over several hours with an acceptable time resolution, in a cost-effective way. Materials and Methods Silica Capillaries. High-quality fused silica capillaries of diameter 530 µm and length 100 m (SGE, Australia) were purchased from Perichrom (France). An average length of 10 m was treated with dilute sulfochromic acid (1/10) at 50 °C followed by a mixture of 30% (w/w) aqueous H2O2 and 25% (w/w) aqueous NH3 with water (respective volume ratios 25 /5/70) at 80 °C under flow conditions for 1 h. This cleaning procedure was completed by thorough rinsing with deionized water (SuperQ, Millipore) at 20 °C for 2 h and a low flow of 10-2 M Tris buffer overnight. This procedure makes the surface very hydrophilic with a high density of SiO- groups.39 The capillary was then cut into 24 cm long sections, and the streaming potential ∆Es was measured under varying pressure drops ∆P to deduce the ζ potential of the interface from the slope dEs/dP.40 Maximum values of about -80 mV (Tris 10-2 M; pH 7.4; T ) 23 °C) were stable only over 1-2 days and the subsequent decrease in ζ potential with time was not accurately reproducible. One or two fibers were assembled in a polystyrene pipet of internal diameter 3 mm by injecting an epoxy type glue at its extremities, which were then cut before determining the streaming potential of the fiber bundle. It was often noted that the ζ potential was reduced (-77 ( 2 mV) after assembly. The flow rate was measured to determine whether Poiseuille’s law could be derived from the pressure drop data and to verify that no fiber was blocked. Proteins. Human fibrinogen (Sigma, Type F4883, lot number 53H9304) and single chain human kininogen (Enzyme Research Lab., supplier Kordia, The Netherlands) were used as received without additional purification. Fibrinogen was dissolved in tris(37) Wang, Y.; Rajagopalan, R.; Mattice, W. L. Macromolecules 1995, 28, 7058. (38) Mandrusov, E.; Vroman, L.; Leonard, E. F. J. Biomater. Sci., Polym. Ed. 1996, 8, 1. (39) Iler, R. K. The chemistry of Silica; Wiley-Interscience Pub.: New York, 1979. (40) Zembala, M.; De´jardin, P. Colloids Surf. B 1994, 3, 119.

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Leˆ and De´ jardin isotope of high energy in the lower window. Analysis of the raw data by internal calibration to obtain the adsorption kinetics was performed as previously.6,41 Briefly, when switching from flow of solution to a flow of buffer there is a drop in recorded activity that is proportional to the amount of protein in solution (Av ≈ πR2Cb), where R is the capillary radius and Cb is the solution concentration, while the increase of activity dA/dt when solution is flowing is proportional to the increase of interfacial concentration Γ: dA/dt ≈ 2πR dΓ/dt. Hence dΓ/dt ) (R/2)CbAv-1 (dA/dt). If internal calibration was too imprecise, an external calibration was performed by cutting the capillary and counting with another detector (Riastar, Canberra).

Results and Discussion

Figure 1. Experimental setup (top) and details of radiation screening around the sample and the detector (bottom). (hydroxymethyl)aminomethane buffer 0.05 M, NaCl 0.10 M, pH 7.35 at 37 °C under gentle stirring, and then filtered with MillexHV 0.45 µm (Millipore) membranes to eliminate any aggregates that may possibly be present. The concentration of the solutions was determined by UV absorbance at 280 nm (Fib ) 1.55 mg-1 cm2; HK ) 0.70 mg-1 cm2). Iodination was performed with the Iodogen technique. For fibrinogen, the removal of free iodide was achieved by flowing the solution through an anion-exchange resin (Accell Plus QMA, Waters-Millipore) that had previously been conditioned in the same buffer as the solution. The procedure for kininogen involved dialysis with a membrane (Molecular weight cutoff 10000 g/mol), against a flowing suspension of resin bearing quaternary ammonium (AG1-1X4, Biorad) in the dialysate compartment. Invariance of the adsorption results with different mixtures of labeled/unlabeled protein solutions was verified. Samples of solutions were analyzed by gel electrophoresis (PhastSystem Pharmacia Biotech) and Comassie Blue staining. According to the supplier the content of plasmin was 0.002 unit/mg solid in fibrinogen; plasminogen was not detectable. Measurement of Adsorbance. To measure simultaneously the adsorbance of both molecules, a two labeling method was used. The solutions were mixtures of 131I-fibrinogen (30 µg/ mL) and125I-kininogen (0.8 µg/mL) with a ratio of concentrations equal to that existing in human plasma with a dilution of 10-2. Appropriate radiological protection was ensured in the manipulation of 131I because of its high-energy γ-radiation. It was verified that labeling did not modify the adsorption process by using mixtures of labeled and nonlabeled solutions of proteins. The experimental setup is illustrated in Figure 1. The solutions were sucked from a reservoir instead of being pushed from syringes to avoid possible exchange processes, for instance, on the walls of tubing where the ratio of area to volume is quite high. An adjustable hydrodynamic resistance was used to obtain a level difference ∆H ≈ 50 cm, much higher than the level variations in the collecting beaker and the reservoir. This ensures a constant wall shear rate in the range 50-760 s-1. The adsorption at 15 cm from the entry of the bundle of capillaries was measured. The bundle was positioned inside a slit of a γ-radiation detector (Quartz et Silice, France). The spectrum of energy was recorded with time. For each spectrum the window of energy characteristic of each isotope was integrated: low energy for 125I and high energy for 131I. Corrections must be carried out for contributions of the

The kinetics of protein adsorption from binary solutions of fibrinogen (30 µg/mL) and kininogen (0.8 µg/mL) at different flow rates are illustrated in Figure 2. Several characteristics can be noted: (i) An extremum in fibrinogen interfacial concentration vs time is evident as previously mentioned for various supports in the literature. This extremum occurs earlier when the flow rate is increased, as transport at short times affects the adsorption process. (ii) There is an increase of the rate of adsorption of fibrinogen before the extremum, more obvious at the lowest shear rates. (iii) Decrease of fibrinogen adsorbance is not necessarily associated with an increase in kininogen interfacial concentration in the presence of the solution. (iv) The fibrinogen continues to leave the interface, even in the presence of buffer only, while the kininogen interfacial concentration is kept constant. (v) The adsorption kinetics of kininogen is monotonic and depends on shear rate, with initially a large linear increase before attaining a plateau value. The experimental results are in good accord with a mechanism whereby the release of fibrinogen from the interface occurs, at constant kininogen interfacial concentration, through a mutual rearrangement of the proteins in the interfacial layer. This suggests that the release mechanism might be still the main one as the amount of fibrinogen begins to decrease when kininogen concentration reaches its maximal value. Among other possible models, this is consistent with the scheme recently proposed where an interfacial complex involving both molecules is assumed.35 This complex is then reversed from a surface contact via fibrinogen to a contact via kininogen before the expulsion of the fibrinogen toward the bulk of the solution. Whatever the exact mechanism of expulsion of fibrinogen from the interface, it does not occur exclusively through a direct exchange between both types of molecules. There is an accumulation of the two proteins at the interface before fibrinogen leaves significantly the interfacial layer. The following analysis is divided in three parts. In the first part, we focus on the kinetics of adsorption of fibrinogen, especially the coordinates of the extremum, the increase of the rate of adsorption before this extremum and the decreasing function of the interfacial concentration with time after the extremum, in the presence and absence of solution. We will analyze in a second part the molecular ratio of both proteins at the interface vs time. Finally we will discuss the kinetics of adsorption of kininogen. 1. Fibrinogen. The molecular ratio of kininogen to fibrinogen at the extremum of the interfacial concentration of fibrinogen (ΓFib) for the different wall shear rates is shown in Figure 3. At the equivalent plasma dilution of (41) Boumaza, F.; De´jardin, P.; Yan, F.; Bauduin, F.; Holl, Y. Biophys. Chem. 1992, 42, 87.

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Figure 2. Adsorption kinetics of fibrinogen (O) and kininogen (4) from binary solutions at different shear rates (s-1): (a) 50; (b) 100; (c) 500; (d) 760. Closed symbols indicate for solution, Open symbols indicate for rinsing with buffer. Straight lines correspond to the initial variation of kininogen adsorption. (e) All fibrinogen adsorption kinetics together. (f) All kininogen adsorption kinetics together.

10-2 used here, it is almost constant with a mean value of 0.9. Therefore for a molecular ratio HK/Fib ) 1/12 in solution the decrease of ΓFib begins when the interfacial layer has an almost equimolecular composition of fibrinogen and kininogen. In fact the individual concentrations at those extrema are independent of shear rate, with values of 0.1 and 0.4 µg cm-2 for kininogen and fibrinogen, respectively, although these concentrations are reached

at times tmax which are dependent on flow rate. This can be related to a transport-controlled process for the adsorption of kininogen until a high fraction of the plateau value due to its especially low bulk concentration (see section 3). By analogy with the transport-controlled processes that lead to adsorption rate varying with wall shear rate γ to the power 1/3, we observed a linear variation of tmax-1 with γ1/3 (Figure 4) according to tmax-1 ) -8.2 ×

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Leˆ and De´ jardin

Figure 3. Top graph: Interfacial concentrations of fibrinogen (closed symbol) and kininogen (open symbol) vs wall shear rate when fibrinogen interfacial concentration is maximum. Bottom graph: Molecular ratio of these concentrations vs wall shear rate. Figure 5. Schematic representation of possible elementary mechanisms of conformational changes of kininogen and fibrinogen with respect to the surface. Protein-protein interactions can be modified by interaction of individual proteins with the surface. With fibrinogen aligned along the y direction, and kininogen above it (step 1): the rotation around the y axis does not provide any new space available on the surface while a rotation around the x axis will create space for molecules of the solution (step 2), without release of material from the interface (step 3, right).

Figure 4. Inverse of the time when fibrinogen interfacial concentration is maximum vs wall shear rate at power 1/3.

10-5 + 6.04 × 10-5 γ1/3, with t in s and γ in s-1. The transport to the interface is important in the adsorption of kininogen over 1-2 h in the range studied, while for fibrinogen it is for a few minutes only until an interfacial concentration of 0.15-0.20 µg cm-2 is attained, as previously observed for adsorption from simple solutions onto glass tubes.41 However the rate of adsorption of fibrinogen is still dependent on the flow rate after the first regime, although the order of magnitude of the rate of adsorption is unrelated to a control by the transport. We suggest that this dependency might originate from the presence of kininogen which interferes with the adsorption of fibrinogen while its own adsorption process is still mainly controlled by the transport. Nevertheless, the coupling between the adsorption processes of the two proteins cannot be interpreted by a simple monolayer occupation of the surface by proteins with fixed conformations: as flow rate is increased, kininogen occupys more rapidly the surface and at a given time would leave less space

available for fibrinogen and we would expect within such a model a decreased rate of adsorption of fibrinogen, which is opposite to the observation. Before the maximum, the acceleration in the accumulation of fibrinogen to the interface can be linked to an increased adsorption term or to a decreased desorption term in the presence of kininogen or to both mechanisms simultaneously. It seems unusual that the same molecule would catalyze the adsorption of another protein before expelling it from the interface. Such a process should involve intricate steps of reorientation of the molecules at the interface. We can consider two possibilities, one for each term: (i) a decrease in the desorption process with the free space fraction on the surface becoming small enough that a part of kininogen adsorbs on the top of a fibrinogen layer and slows down its desorption or its exchange with new molecules from the bulk; then a rearrangement would occur where the main contact with the surface would shift from fibrinogen to kininogen and the fibrinogen molecules would be expelled from the composite interfacial layer; and (ii) an increase in the adsorption term with kininogen-induced newly available space by favoring a side-on to end-on process for the adsorbed fibrinogen molecules. Possible elementary steps are illustrated in Figure 5. As proteins generally have globular structures, it is likely that the main mechanisms involved in the displacement processes are rotations and translations at the interface. Reptation processes however seem more likely with coiled polymers and perhaps with the dangling chains of the proteins. The release of

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Figure 6. Representation of the decreasing fibrinogen interfacial concentration from the maximal value Γmax as ln(Γ/Γmax) vs time. Full line: linear fit of data in the presence of solution. Dotted line: Linear fit of data by rinsing with buffer. Table 1. Fit of Data of Fibrinogen Adsorption When the Interfacial Concentration Is Decreasing (Figure 6) within an Exponential Model Γ ) Γextremum exp[-k(t textremum)]

γ (s-1)

k (10-4 s-1) solution t > textremum

50 100 500 760

0.75 ( 0.02 0.70 ( 0.01 1.0 ( 0.01 1.1 ( 0.02

k (10-4 s-1) solution; t > 90 min

k (10-4 s-1) rinsing with buffer

0.95 ( 0.03 0.90 ( 0.02

0.25 ( 0.02 0.22 ( 0.04 0.77 ( 0.01 0.84 ( 0.05

fibrinogen could also have been induced by the spreading of kininogen molecules on the surface. Such an acceleration was observed by Baszkin and Boissonnade42 for the system albumin (0.2 mg/mL)/fibrinogen (0.035 mg /mL) on polyethylene. In this study a maximum of fibrinogen interfacial concentration occurred after about 5 h, a delay that can be attributed to a stronger interaction of fibrinogen indicating higher barriers to overcome with a hydrophobic surface. The decrease of interfacial fibrinogen concentration is represented in Figure 6 as ln(Γ/Γmax) vs time, where Γmax is the interfacial concentration at the extremum. There is a perceptible tendency for the release rate to increase with shear rate, whose mean value is 0.90 × 10-4 s-1. By considering the rinsing steps, it appears that fibrinogen continues to leave the interface with a three times smaller decreasing rate at the highest interfacial concentrations, which exist at the lowest shear rates. The change in release rate is only 10-20% at the lowest interfacial concentrations, under the highest wall shear rates (Table 1). This observation suggests that a participation of the molecules of the solution occurs when the interfacial concentration is high, through a homogeneous exchange process reducing the average residence time of molecules at the interface; such a participation would not be so crucial at low coverage by fibrinogen. On the same support, we observed in the presence of a diluted plasma pool with similar concentrations of fibrinogen and HK a higher decreasing rate of interfacial concentration of fibrinogen and a smaller maximal value due to the participation of other proteins for surface coverage. In plasma also, this decrease was not stopped by replacing the solution by the (42) Baszkin, A.; Boissonnade, M. M. In Proteins at Interfaces II: Fundamentals and Applications; ACS Symposium 602, Brash, J. L., Horbett, T. A., Eds.; American Chemical Society: Washington, DC, 1995; p 209.

Figure 7. Molecular ratio of kininogen over fibrinogen at the interface vs time at different wall shear rates. From bottom to top: 50; 100; 500; 760 s-1.

buffer36 therefore emphasizing the conformational rearrangement at the interface as the main process to expel molecules from the interface even in the presence of plasma. 2. Molecular Ratio Kininogen/Fibrinogen. The variation of the molecular ratio of the interfacial concentrations with time at different wall shear rates is illustrated in Figure 7. From this plot it is evident that there is a time interval where the ratio remains constant before kininogen becomes predominant, after the extremum in fibrinogen interfacial concentration is reached. This time interval is linked to the approach of the coverage saturation, while both interfacial concentrations are still increasing functions of time. The order of magnitude of the molecular ratio at the very beginning can be estimated using the model of completely transport controlled processes for both proteins

( ) ΓHK ΓFib

mol

)r

( ) ( ) DHK DFib

2/3

=r

MHK MFib

-2/9

) r′

( ) MHK MFib

-11/9

(1)

where r and r′ are the ratios of the solution concentrations (HK over Fib) in mole and mass per unit volume, respectively, D is the diffusion coefficient, and M is the molar mass with D ∼ M-1/3. With a ratio of molar mass of 1/3 and r′ ) 0.8/30 one obtains at initial times an estimation of one molecule of kininogen for 10 molecules of fibrinogen, a value in accord with the order of magnitude observed experimentally. Indeed the diffusion coefficient of kininogen can be estimated from an analysis of the kinetics of adsorption at short times. 3. Adsorption Kinetics of Kininogen. From the large linear variation of the interfacial kininogen concentration, which is observed initially, it is possible to determinate the initial apparent kinetic constant precisely. The inverse of this constant is plotted in Figure 8 versus (x/γ)1/3, where x is the distance from entrance of the fiber (43) De´jardin, P.; Le, M. T.; Wittmer, J.; Johner, A. Langmuir 1994, 10, 3898.

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Leˆ and De´ jardin Table 3. Parameters Γsat and kexp of the Fits for the Adsorption of Kininogen above 80% of the Plateau Value According to the Differential Equation du/dt ) -kexpur with u ) 1 - (Γ/Γsat) γ (s-1)

Γsat

kexp (10-4 s-1)

ka (10-5 cm s-1)

50 100 500 760

0.104 0.165 0.161 0.202

8.9 ( 0.6 6.9 ( 0.3 7.3 ( 0.2 5.2 ( 0.1

1.0 1.0 1.0 1.0

50 100 500 760

0.104 0.165 0.161 0.202

5.1 ( 1.2 4.0 ( 0.5 5.5 ( 0.8 4.1 ( 0.6

0.74 ( 0.10 0.72 ( 0.05 0.87 ( 0.06 0.88 ( 0.10

Ra

1.22 ( 0.08 1.48 ( 0.06 1.40 ( 0.04 1.22 ( 0.02

a With an exponent R fixed to 1 (Langmuir model) and the deduced adsorption constant ka or an exponent adjustable through a leastsquares method.

Figure 8. Inverse of the experimental initial kinetic constant kexp as a function of (x/γ)1/3 where x is the distance from entrance of the fiber and γ the wall shear rate. Intercept gives access to the constant of the interfacial reaction while coefficient of diffusion can be derived from the slope. Points with open symbols were excluded from the linear fit (full line) as the diffusion layer thickness becomes comparable to the radius of the capillary at these low shear rates. Table 2. Determination of the Diffusion Coefficient D of Kininogen and Adsorption Kinetic Constant ka from Equation 2 Applied for the First Five Dataa x (cm)

γ (s-1)

k (10-5 cm s-1)

7.5 7.5 15 15 15

200b 500b 200b 450 760

6.9 ( 0.1 9.0 ( 0.2 6.0 7.4 8.0

15

50 100b

1.6 4.7

eq

ka (10-4 cm s-1)

D (10-7 cm2 s-1)

2a 2b

1.7 ( 0.3 2.4 ( 0.4

4.9 ( 0.6 3.7 ( 0.5

a k is the experimental kinetic constant. k ) C -1 (dΓ/dt) where b Cb is the solution concentration and dΓ/dt is the experimental rate of adsorption. x is the distance from the entrance of the capillary. b Mean value over at least five experiments.

and γ the wall shear rate. Following the interpretation detailed previously,43 this plot can provide the true adsorption kinetic constant ka at the interface and the diffusion coefficient D of the protein (Table 2) according to the approximation valid near the Le´veˆque regime (see Appendix)

k-1 ) kLev-1 + 0.684ka-1

(2a)

or the approximation valid near the control by the interfacial reaction

k-1 ) ka-1 + 0.827kLev-1

(2b)

where kLev ) 0.54(D2 γ/x)1/3. The results according to these two limits are given in Table 2: D ) (3.7 to 4.9) × 10-7 cm2 s-1 and ka ) (1.7 to 2.4) × 10-4 cm s-1 . These values are however not consistent with the corresponding domain of (ka/kLev), as probably the experimental points are lying in the crossover domain between the two regimes. Using the full expression (see Appendix, eqs A1-A4) leads to ka ) 2.4 × 10-4 cm s-1 and D ) 4.4 × 10-7 cm2 s-1, which are values close to the foregoing estimations. For x ) 15 cm and wall shear rates 50, 100, 200, 500, and 760 s-1, the thickness of the Nernst layer yN ) 1.86 (Dx/γ)1/3 can be

Figure 9. Dotted lines from bottom to top: Adsorption kinetics of kininogen at wall shear rates 50, 100, 500, and 760 s-1 from binary mixtures of fibrinogen and kininogen with the superimposed fit by an exponential model when reaching the plateau. Full line from time zero: adsorption kinetics from a simple solution of kininogen without fibrinogen at wall shear rate 200 s-1. CHK ) 0.8 µg/mL.

then evaluated to be 95, 75, 60, 44, and 38 µm for an infinite radius of curvature and represents a significant fraction of the capillary radius (265 µm), especially at the lowest shear rates; therefore the values at 50 and 100 s-1 were excluded from the linear fit (Figure 8). To take into account the real geometry of the system would require numerical simulations. The analysis of the kinetics when the plateau is almost reached, typically above 80% of the limit, can be performed using different models. The simple Langmuir model

[

∆Γ ) ∆Γ0 exp -

kaCHK (t - t0) Γsat

]

(3)

with ∆Γ ) Γsat - Γ where the desorption term is assumed to be zero (see Figure 2) provides a mean adsorption constant ka of 1.35 × 10-5 cm s-1 (Table 3; Figure 9). This is 20 times smaller than the constant determined at initial times. This slowness in reaching the plateau could be attributed also to an excluded surface effect which would be more sophisticated than the linear expression of the Langmuir model. For instance the random sequential adsorption (RSA) model for spheres leads, near the jamming limit, to the following relationship

(

)

Γ dΓ ≈ kaC 1 dt Γsat

R

(4)

Simultaneous Adsorption of Fibrinogen and Kininogen

with R ) 3. This model predicts that the rate in reaching the final value is greatly reduced.44 To choose the more appropriate model, we performed in addition two fits of data, one with R ) 3 and another with R adjustable. We found that the use of exponent 3 did not improve the fit of data with respect to eq 3, while an adjustable parameter in a least-squares method led to exponents near the unity value, which corresponds to the exponential model (Table 3). Hence the Langmuir model is more likely to apply than the RSA model. The Langmuir model is based on an assumption of sites of adsorption. For the present process we can postulate that these sites are on the adsorbed molecules of fibrinogen, but not on the bare surface, given the differences between the initial (2.4 × 10-4 cm s-1) and final (1.35 × 10-5 cm s-1) kinetic constants. It is remarkable that the kininogen rate of adsorption begins to decrease significantly when fibrinogen concentration reaches a maximum at the interface, indicating a probable saturated surface. With simple solutions of kininogen, in the absence of another protein, the change of slope occurs more abruptly, typically over a few minutes (Figure 9), and very close to the full coverage, due to a continuous large value of the adsorption constant. Conclusion Studies of the mechanisms of simultaneous adsorption of two entities such as fibrinogen and kininogen from binary mixtures in a buffer can be achieved using a twolabeling method. This helps us to understand the process of release of fibrinogen from the interface in the presence of kininogen, a process observed with blood plasma (Vroman effect). This method was used recently in a study on the homogeneous exchange fibrinogen/fibrinogen while the total interfacial concentration was still increasing.20 The results suggest that at 37 °C for the system kininogen/ fibrinogen/hydrophilic silica the rearrangement at the interface was the predominant mechanism to release fibrinogen from the interface and not the direct exchange molecule by molecule, as the decrease of fibrinogen concentration begins at high coverage and continues at a constant kininogen interfacial concentration. If the latter mechanism could still exist in the presence of solution, it cannot be invoked in the rinsing period where the fibrinogen interfacial concentration continues to decrease, while the kininogen interfacial concentration remains constant. Concerning the conformation of both proteins at the interface, the experimental data suggest that kininogen does not interact directly with silica near the extremum of fibrinogen interfacial concentration, when there is high surface coverage. A crude model of complex formation was postulated recently35 and such a motion of molecules normal to the interface was proposed to interpret experimental data of scanning angle reflectometry on an antigen/antibody system.45 When the surface is quite crowded, it is likely that in the conditions of our experiments a part of the kininogen interfacial population first interacts with fibrinogen molecules before a direct contact with the silica surface is made. One of the possible explanations for the increasing rate of adsorption of fibrinogen before the extremum is that new space would become available. It might also arise because of an increased local solution concentration due to upstream desorption. This space would be provided by a kininogencatalyzed side-on to end-on process for fibrinogen before its release from the interface. Such a side-on to end-on (44) Schaaf, P.; Talbot, J. J. Chem. Phys. 1989, 91, 4401. (45) Heinrich, L.; Mann, E. K.; Voegel, J. C.; Koper, G. J. M.; Schaaf, P. Langmuir 1996, 12, 4857.

Langmuir, Vol. 14, No. 12, 1998 3363

process is often considered in the literature; it was analyzed some years ago for the adsorption from simple solutions of fibrinogen with the scanning angle reflectometry technique.46 The rearrangement is more likely to occur when the surface is quite crowded as changes of orientation of one molecule with respect to the surface could create available space for a neighboring molecule. As stated in ref 47 where greatest effects of HK were seen at long times such as 2 h, it may be that at shorter times competition effects would be less evident as the surface is not completely filled. These changes of orientation and interaction between both proteins could be a consequence of the direct contact of one of them with the surface, and vice versa. Measurements by atomic force microscopy would be useful to check the different orientations of the proteins. The same work mentioned the possibility of a possible switch from a one-chain to two-chain HK at the interface, induced by trace amounts of impurities such as kallikrein. It has been however suggested that, on the supports examined in ref 47, glass among them, any formation of two-chain HK that may occur had no influence on the results. Moreover, when rinsing with buffer, our technique gives access to the continuous change in the interfacial concentration. We observed that the lack of a continuous arrival of proteinaceous materials in the system did not change the tendency for the fibrinogen interfacial concentration to decrease, as already observed in plasma, and for the kininogen interfacial concentration to be constant, emphasizing an interfacial rearrangement. We have however strictly no indication about the state, single chain or two chain if any, of HK at the interface. If the histidine-lysine rich domain of HK is likely to provide a high affinity between this protein and the surface,33 we lack information to decide whether kininogen keeps almost its native conformation or is largely denatured by the contact with the surface when fibrinogen leaves the interface. Acknowledgment. We are indebted to the Fondation de la Recherche Me´ dicale for a grant and to the Hospal R&D International company (Lyon, France) for financial support. We are grateful to M. Thomas and P. Valette (Hospal) for providing advice in the manufacture of bundles of silica fibers. We are indebted to one reviewer for signaling ref 47. Appendix We start from the expression of the rate of adsorption given in ref 43. k is the kinetic constant of the global process at distance x from the entrance of a slit or capillary of large thickness or radius, respectively, and ka is the kinetic constant of the interfacial reaction.

k ) kag(X)

X ) x/Lco

(A1)

]

(A2)

with

Lco ) 3

[

Γ′(2/3) Γ′(1/3)

3

D3γ ka3

D is the diffusion coefficient of the solute, γ the wall shear rate, and Γ′ the classical Gamma function. The prime is (46) Schaaf, P.; De´jardin, P.; Johner, A.; Schmitt, A. Langmuir 1992, 8, 514. (47) Cornelius, R. M.; Brash, J. L. J. Biomed. Mater. Res. 1997, 37, 314.

3364 Langmuir, Vol. 14, No. 12, 1998

Leˆ and De´ jardin

added to distinguish this from the interfacial concentration. The numerical prefactor is 0.387.

g(X) ) e-X + G(2/3,X) - G(1/3,X)

X)

(

ka 1 kLev Γ′(2/3)

(A3)

)

3

.1

k-1 ≈ kLev-1 + 0.684ka-1

(A5)

k-1 ≈ ka-1 + 0.827kLev-1

(A6)

with

∫0Xzn-1ez dz

Γ′(n) G(n,X) ) e-X

X,1

(A4)

It is preferable to approximate the inverse of k. We found43:

LA9713513