Langmuir 1995,11, 4001-4007
4001
Competitive Adsorption of High Molecular Weight Kininogen and Fibrinogen from Binary Mixtures to Glass Surface P. Dejardin,+SsP. ten Hove, X. J. Y u , ~and J. L. Brash* Department of Chemical Engineering, McMaster University, Hamilton, Ontario, Canada L8S 4L8, and Institut Charles Sadron, 6 rue Boussingault, 67083 Strasbourg Cedex, France Received January 17, 1995. In Final Form: June 12, 1995@ An investigation of adsorption from binary mixtures of fibrinogen and high molecular weight kininogen (HK)to glass is reported. Experimentswere performedusing radioiodinatedproteins in which the adsorption of each protein was measured from serial dilutions of mixtures having a ratio of the proteins approximately the same as in plasma (fibrinogenin excess). Fibrinogen adsorption passes through a maximum as solution concentrationincreases, analogous to its behavior in plasma as reported previously (Brash, J. L.; ten Hove, P. Thromb. Haemostasis, 1984,51,326). HK adsorption increases monotonically with concentrationsuch that fibrinogen is virtually excluded from the surface at the highest concentrations even though present in large excess. A kinetic model is developed in which initially adsorbed fibrinogen can either desorb spontaneously,relax to an irreversibly bound state, or exchange with HK. The model solution is obtained in terms of the ratio of the rate constants for exchange and relaxation, kex/k‘l,and is independent of the nature ofthe available surface function. Data fits to the model are excellent and give essentially invariant values of Kex/k’lfor different ratios of the two proteins in solution. The results of this study emphasize the importance of surface relaxation in competitive protein adsorption.
Introduction The competitive adsorption of proteins is of interest in a number of fields including chromatography, biocompatibility of biomaterials, and biosensors including solid phase immunoassays. In blood compatibility considerations, 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.’ Although surfaces used in the applications cited, e.g. affinity chromatography, often have specific protein binding ligands purposely incorporated so as to “capture” a particular protein, there is usually a significant proportion of nonspecific sites as well. The question of interest is how the proteins partition between the surface and the solution, i.e. whether there is a definite relationship between the compositions in the two phases. Also of interest is whether there is any correlation between layer composition and global surface properties such as wettability or charge.2 Competitive protein adsorption has been studied extensively in blood and p l a ~ m a , ~and - ’ ~phenomena referred to as the Vroman effect have been reported. The Vroman
* Author to whom correspondence should be sent.
’ Institut Charles Sadron.
0 Current address: Lqboratoire des Materiaux et Procedes Membranaires,8 rue de l’Ecole Normale, 34053 Montpellier Cedex 1, France. 8 Current address: Dept of Bioengineering, University of Washington, Seattle. Abstract published in Advance A C S Abstracts, September 15, @
1995. (1)Collins, W. E.; Mosher, D. F.; Tomasini, B. R.; Cooper, S. L. Ann. N.Y. Acad. Sci. 1987,516,291. (2)Elgersma, A. V.; Zsom, R. L. J.;Lyklema, J.;Norde, W. J. Colloid Interface Sei. 1992,152,410. (3)Vroman, L.; Adams, A. L; Fischer, G.; Munoz, P. Blood 1980,55, 156. (4)Adams, A. L; Fischer, G. C.; Munoz, P. C.; Vroman, L. J. Biomed Mater. Res. 1986,18,643. (5)Vroman, L.;Adams, A. L. J . Colloid InterfaceSci. 1986,111,391. (6)Brash, J. L.;ten Hove, P. Thromb. Haemostasis, 1984,51,326. (7) Wojciechowski, P.; ten Hove, P.: Brash, J.L. J. Colloid Interface Sci. 1986,111, 455.
0743-7463/95/2411-4001$09.00/0
effect refers to kinetic phenomena by which abundant proteins of relatively low binding afinity, which are preferentially adsorbed a t short times, are later replaced by less abundant proteins of high binding afinity. This most likely results from the interplay of transport and binding, which favors abundant proteins initially and highaffinity proteins a t longer times. The Vroman effect has been observed by many investigators, particularly for fibrinogen adsorption, but there is evidence to suggest that it is a general effect potentially involving all the proteins in a given mixture such that the abundant, lowaffinity proteins are sequentially replaced by the scarce, high-affinity proteins. We have observed Vroman effects for IgG in plasma,14 and Vroman has provided evidence that in this medium there is a continuous change of composition a t the surface with more abundant proteins being replaced sequentially by less abundant ones.5 It has been observed both by Vroman et al.3 and Brash et al.*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. Although the Vroman effect is fundamentally kinetic in character, it is manifest in plasma also as a variation of adsorption with plasma concentration when the plasma is serially diluted.6-10 This has been explained by postulating that a t very high dilutions there is insufficient lower concentration components to cause displacement of initially adsorbed, abundant proteins. As the concentra(8)Brash, J. L.; Scott, C. F.; ten Hove, P.; Wojciechowski, P. W.; Colman, R. W. Blood 1988,71, 932. (9)Horbett, T. A. Thromb. Huemostasis 1984,51,174. (10)Slack, S.M.; Horbett, T. A. J. Colloid Interface Sei. 1988,124, 535. ~ . . (11)Breemhaar, W.; Brinkman, E.; Ellens, D. J.; Beugeling, T.; Bantjes, A. Biomaterials 1984,5,269. (12)Slack S. M.; Horbett, T. A. J. Colloid Interface Sci. 1989,133, 148. (13)Slack, S.M.; Horbett, T. A. In Proteins at interfaces; Horbett, T. A,, Brash, J. L., Eds.; ACS Symposium Series 602;American Chemical Society: Washington, DC, 1995;p 112. (14)Cornelius, R.M.; Wojciechowski, P. W.; Brash, J. L. J.Colloid Interface Sci. 1992,150, 121.
0 1995 American Chemical Society
Dtjardin et al.
4002 Langmuir, Vol. 11, No. 10, 1995 tion increases, the displacing components attain effective levels and may eventually be able to displace completely the more abundant components. The net effect is that adsorption of a given component passes through a maximum as a function of concentration at a fEed time. Vroman effects have been observed on many surfaces, and in general displacement decreases as hydrophobicity increases, presumably reflecting stronger binding on the more hydrophobic material^.^ We have also shown that some surfaces do not exhibit the Vroman effect.15 Sulfonated polyurethanes, for example, adsorb large amounts of fibrinogen from plasma without subsequent displacement. If the mechanism outlined above is correct, then displacement effects should be a property of adsorption not just from plasma but from any protein mixture in which there are abundant components of low binding affinity and scarce components of high binding affinity. Early preferential adsorption and later displacement of the more abundant components should be seen; correspondingly, high adsorption at high dilution and lower adsorption at lower dilution should be observed. In the work reported here, we sought an answer to the question whether the Vroman effect seen in plasma has its counterpart in simpler systems. We report on adsorption from fibrinogen/HK mixtures to glass. HKand fibrinogen were chosen because of the known behavior of these two proteins in plasma.
Materials and Methods Fibrinogenwas purchased from Kabi AB (Stockholm,Sweden) and treated as described previously.6 HK was purchased from Enzyme Research Laboratories (South Bend, IN) and used as received. It was in the intact single chain form with a molecular weight of 110 000 kDa as determined by SDS-PAGE. In this form HK is inactive as a cofactor in the contact phase of plasma c ~ a g u l a t i o n . ~Albumin ~J~ and IgG were respectively from Behringwerke (Marburg,Germany)and Sigma (St. Louis, MO). Proteins were labeled with lZ5Iusing the lactoperoxidasemethod as described elsewhere.ls Pyrexglass tubes oflength 1.5cm, i.d. 0.3 cm, and 0.d. 0.5 cm were cleaned in chromic acid cleaning mixture and rinsed thoroughly in distilled water. The water contact angle of the pyrex glass cleaned in this manner was on the order of 5-10'. Adsorption experimentswere carried out in isotonictris buffer, pH 7.4 under static conditions at ambient temperature. The tubes were first immersed in the buffer-filled wells of multiwell plates overnight to achieve complete hydration. They were then removed and drained but not dried. With the tubes in the empty wells, the protein solution (isotonictris, pH 7.4) was added and adsorption allowed to proceed usually for a period of 1h. This was found to be a long enoughtime to attain constant adsorption levels in these experiments. The tubes were retrieved from the wells so as to avoid passage through the air-solution interface by sequential dilution and decantation of the fluid in the well. They were thoroughly rinsed and the radioactivity on the tubes counted. Adsorption was calculated as described in previous reports.'g Experimental Results Mixtures of different compositions were studied in which either fibrinogen or HK was labeled. By analogy with plasma experiments,various initial mixtures were serially diluted with buffer and adsorption was measured after a (15) Santerre, J. P.; ten Hove, P.; Vanderkamp, N. H.; Brash, J. L. J. Biomed. Mater. Res. 1992,26, 39. (16) Mosher, D. F. Cardiovasc. Pathol. 1993,2,1495. (17)Asakura, S.; Hurley, R. W.; Skorstengard, K.; Ohkubo, I.; Mosher, D. F.J. Cell. Biol. 1992,116, 465. (18)Yu, X. J.;Brash, J. L. In TestProceduresforBlood Compatibility of Biomaterials; Dawids, S., Ed.; Kluwer Academic Publishers: Amsterdam, 1993; p 287. (19) Chan, B. M. C.; Brash, J. L. J. Colloid Interface Sci. 1981,82, 217.
0.30
1
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0
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40
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60
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Concentration ( 0 )
Figure 1. Adsorptionoffibrinogen to glass from HWfibrinogen mixtures of varying composition. Adsorption time, 1h. Inset gives solution concentrations fibrinogen/HK (mg/mL) corresponding to 100%. The solid lines do not represent fits to a model and are shown only to aid visual presentation of the data.
0.25
i
t
0
20
40
60
80
100
Concentration (%)
Figure 2. Adsorption of HK to glass from HWfibrinogen mixtures of varying composition.Adsorption time, 1h. Inset gives solution concentrations fibrinogen/HK(mg/mL)corresponding to 100%. The solid lines do not represent fits to a model and are shown only to aid visual presentation of the data.
given time. The data are thus in what may be referred to as the "concentration domain", as opposed to the "time domain", where adsorption is given as a function of time at fixed solution concentrations. Figure 1shows data for four experiments in which fibrinogen was labeled. The mixture compositions were chosen such that the ratio of the proteins was similar to that in plasma (3 mg/mL fibrinogen, 0.07 mg/mL, HK), but the total concentration was considerably smaller, namely that of plasma. As seen in Figure 1,Vroman-like effects are evident in the mixtures, and the fibrinogen adsorption passes through a maximum. The effect is amplified as the ratio of HK to fibrinogen increases, and at higher concentrations adsorption deviates more and more from that observed in pure fibrinogen at the same concentration. The corresponding data showing the behavior of HK in these experiments are given in Figure 2. These data show more conventional adsorption behavior in the sense that adsorption increases monotonically with concentration. Adsorption of HK increases slightly as the ratio of HK to fibrinogen increases. Data in the absence of fibrinogen
Langmuir, Vol. 11,No. 10, 1995 4003
Competitive Adsorption to a Glass Surface HK
0.35
FIB
0.30 h
0.25
3. 2 0.20 v
C
.-
5* 0.15
2
Figure 5. Model for competitive adsorption of fibrinogen and HK.
0.10 0.05 i
0.00 20
40
60
80
100
Concentration (%)
Figure 3. Adsorption of HK and fibrinogen to glass from a binary mixture of composition (100%):CFib = 0.3 mg/mL, CHK = 0.03 mg/mL. Adsorption time, 1 h. The solid lines do not represent fits to a model and are shown only to aid visual presentation of the data.
E
0.15
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Fibrinogen
0
HK
3
Q
0.10
0.05 t
0
-
A
1 v
albumin and IgG is almost completely suppressed even though these are the most abundant components in the solution. Fibrinogen shows a maximum in adsorption at about 5%, while HK shows similar domination of the surface as in the binary system.
A Kinetic Model for Competitive Adsorption We consider a kinetic model consisting of the interactions shown in Figure 5 , i.e. adsorption and desorption of fibrinogen, relaxation of bound fibrinogen to an irreversibly adsorbed state, adsorption of HK, and exchange of fibrinogen and HK. In the following discussion, 1refers to fibrinogen and 2 to HK, r to interfacial concentration, and C to bulk concentration. Q, is the available surface function,which is assumed to be the same for both molecules. The model provides for three populations of adsorbed molecules varying with time: r2 for HK, rl for exchangeable fibrinogen, and for irreversibly adsorbed, nonexchangeable fibrinogen. Molecules in population rl can be desorbed (constant kdl), can exchange with HK (constant kex),or can be transformed to an irreversibly adsorbed state (constant k'l). kal and ka2 are the adsorption rate constants of fibrinogen and HK, respectively. The governing kinetic equations for these populations are
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Concentration (S)
Figure 4. Adsorption from a solution of albumin (4 mg/mL), IgG (1.0mg/mL), fibrinogen (0.3 mg/mL), and HK (0.01 mg/ mL)diluted to differentextents. Adsorptiontime, 1 h. The solid lines do not represent fits to a model and are shown only to aid visual presentation of the data.
(not shown)are little different from those for the mixtures at the same HK concentration,suggesting that adsorption of HK is virtually independent of the presence of fibrinogen. The adsorption levels for HK are high, with values at 100% concentration between 0.2 and 0.3 pg/cm2. Although the shape and size of HK are not known, such values are probably in the monolayer range for a protein of about 100 000 kDa. Considering the low concentrations in solution, the afinity of HK for the glass surface must be relatively high. A composite diagram showing the adsorption of both proteins for the 0.3/0.03, fibrinogen/HK mixture is shown in Figure 3. The sum of the two curves is effectively an isotherm for total adsorptionand shows the characteristics of classical adsorption behavior. It is noteworthy that the peak in fibrinogen adsorption occurs near the concentration where the surface appears to be full. Clearly there would be no reason for a significant displacement to occur on a surface which is not well covered. It is also interesting to note that the fibrinogen peaks (Figure 1) occur a t the same total adsorption of about 0.21 ,ug/cm2 for all three HWfibrinogen mixtures. Figure 4 shows adsorption data for the quaternary system albumin/IgG/fibrinogen/HK. The adsorption of
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+
drddt = ka2C2Q, ke,T1C2
(2) (3)
We assume that after a relatively long time the final population of reversibly adsorbed, exchangeablefibrinogen molecules goes to zero (rlf= 0), and only the two populations rlf and r2fare present. These conform to the relation @(I"lf,r2f) = 0. The exact available surface function could also depend on the process history. As the model includes irreversible adsorption of both molecules, the final state should correspond to a well-covered surface. Data for the highest dilution ("concentration" = 5%) were therefore removed from the present analysis since they probably represent partial coverage due to low, diffusioncontrolled rates a t low concentration. Integrating eqs 1, 2,and 3 between times zero and infinity gives, respectively, eqs 4, 5 , and 6:
I"lf = k'lhmrl dt
(5)
r2f= ka2C2hmQ,dt + kexC2~mI'l dt
(6)
The two integrals are connected through eq 4, thus giving r'lfand r2fin terms of a common unknown finite integral. This integral is not easy to evaluate unless one assumes a linear function of interfacial concentrations
Dkjardin et al.
4004 Langmuir, Vol. 11, No. 10, 1995
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7
Table 1. Estimation of K.#l and Average Molecular Areas from Regression of Data According to Equation 10 and Figure 2 (First Line) or Equation 19 and Figure 4 (Second Line)”
I
Fibrinogen keX/k’l HK [HKI (mg/mL) ([Fib]= 0.3 mg/mL) (mL/mg) (nm2/molecule) (n”Vmolecu1e) 0.01
210
0.02b
160b
0.03
200
30 24 62b
261 274 23gb 26!jb 323 272
5Bb
a
0.0
0.2
0.4
0.6
0.8
1.0
51 52
MW(fibrinogen) = 340 000 g/mol. MW(HK) = 110 000 g/mol. = 0.8 and 1.0 were not taken into account.
Data at d
d Figure 6. Ratio of interfacial concentrationsof HKfibrinogen versus dilution, d , for three HK solution concentrations.From bottom to top: C Z , , ,= , ~0.01;0.02; 0.03mg/mL. Fibrinogen bulk concentration Cl,max = 0.30 mg/mL.
for Q as in the Langmuir model. In this case the system of eqs 1-3 is linear with respect to interfacial concentrations. It is more useful to calculate the ratio of these two populations, which for a fibrinogen molecule should reflect, a t least partly, the competition between the two processes of change of conformation and exchange with HK. Dividing eq 6 by eq 5, we arrive a t the following expression, valid whatever the exact nature of the available surface function, Q:
0 ’ 0.0
I 0.2
0.4
0.6
08
1.0
d
Figure 7. Inverse ofinterfacialfibrinogen concentrationversus dilution, d, for three HK solution concentrations.From bottom to top: C P , , , ,=~ ~0.01; 0.02; 0.03 mg/mL. Fibrinogen bulk
concentration C I , =~0.30 ~ ~mg/mL. When no exchange occurs, the ratio of the interfacial concentrations is proportional to the ratio of the bulk concentrations. The second term in eq 7 gives the contribution of exchange to the ratio of the interfacial concentrations. For various dilutions d (0 5 d I1)of a n initial solution of the two proteins, assuming a small surface-to-volume ratio (and therefore negligible solution depletion), the ratio of the solution concentrations CdC1 will remain constant while the individual concentrations vary: CZ= C Z , , and ~ C1 = Cl,maxd.Based on eq 7 ,Tzf/T’lf data are plotted against d in Figure 6 for three values of C2,max/Cl,max. The ratio r2pTlfclearlyvanes with dilution in a linear fashion in all three cases. Equation 7 further implies that the slopes of the lines should be proportional to C2,max, and since this is confirmed by the data (Figure 61, we infer that in our experiments kazCdkalC1
