THEADBORPT~ON OF FIBRINOGEN
21 03
Appendix Derj a g ~ i considered n~~ the repulsive energy between two spheres in a first approximation to be formed by the contributions of infinitesimal small rings parallel to each other at a distance d. The repulsive free energy between t x o parallel rings covered by adsorbed macroAFM. molecules is approximately given by A F V R The free energy of repulsion between two spheres of radius b at a minimal distance do (the distance between do) is then found the centers of the spheres being 2b by integrati0n.5~ The value of the integral does not
+
+
The Adsorption of Fibrinogen.
L:
VR = 2 ~ b
(AFVR
+ A F M ) dd
depend on b, so that TiR is proportional with b. The van der Waals attraction energy between two spheres is also proportional with b, so that the total free energy of interaction is proportional with b. The stability of the colloidal system is determined by the minimum in the free-energy curve, and of course also this minimal value of the interaction energy is proportional with b. (51) Reference 3b, Chapter IX, eq 54.
An Electron Microscope Study1
by R. R. Gorman, G. E. Stoner,* and A. Catlin Department of Materials Science, University of Virginia, Charlottesville, Virginia 22901
(Received September 26, 1970)
Publication costs assisted by The National Institute of Dental Research
The process of adsorption of fibrinogen onto mica and evaporated carbon has been characterized as completely diffusion controlled by the direct counting of individual molecules through the use of the electron microscope. As applied to adsorption on mica the method produced Hall and Slayter triad molecules, and the deposits were reproducibly and uniformly distributed over the specimens. Under the experimental conditions, the adsorption was strongly dependent on the compositsionof the substrate, and in the case of mica, it was insensitive t o the composition of the suspending buffer.
data by direct counting of the individual adsorbed Introduction molecules. The adsorption of proteins and macromolecules from aqueous suspensions has been investigated by Materials and Equipment means of several experimental techniques, including The bovine fibrinogen was the diagnostic product of streaming potentials,*&isotope and fluorescence labelthe General Diagnostics Division of Warner-Chilcott, ing,zbdepletion of s ~ s p e n s i o nellip~ometry,~ ,~ and visual packaged in vials as 6 mg of clottable protein, 17 mg of observation in the electron micro~cope.~Fibrinogen sodium chloride. adsorption is of special importance because of its unique configuration in the adsorbed stage, its strong tendency The human fibrinogen was prepared in this laborat o adsorb, and its possible involvement in the initial stages in the interaction of human blood in vivo with (1) This work supported by the Xational Institutes of Health under devices such as artificial heart valves and a r t e r i e ~ . * ~ ~ Grant ~ ~ ’ D E 02111-07. (2) (a) R. D. Falb, G. A . Grode, M .M.Epstein, B. G. Brand, and The objective of this investigation was to characterize R. I. Leininger, June 29, 1966. P. B. 168, 861, U. S. Dept. of Comthe adsorption of fibrinogen onto muscovite mica and merce: (b) R. D. Falb, G. A. Grode, M.T. Takshashi, and R. I. Leininger, March 30, 1967. P. B. 175, 668, U. S. Dept. of Comevaporated carbon at short adsorption times from a merce. dilute, unstirred suspension of the protein molecules, (3) A. D. McLaren, J. P h y s . Chem., 58, 129 (1954). and t o investigate the effect of the properties of the sus(4) L. Vroman, “Blood Clotting Enzymology,” W. H. Seegers, pending buffer on the adsorption. An additional Ed., Academic Press, New York, N. Y., 1967, p 279. objective was t o establish a methodology which could (5) D. Lang and P. Coates, J . Mol. Bid., 36, 137 (1968). (6) L. Vroman, J . Biomed. Mater. Res., 3, 669 (1969). be applied t o other proteins and solids. The method (7) D. J. Lyman, J. L. Brash, and K . G . Klein, Proceedings of the was the visualization of the adsorbed protein molecules Artificial Heart Conference, R. J. Hegyeli, Ed., Washington, D. C., in the electron microscope and the obtaining of kinetic 1969, pp 113-122. The Journal of Physical Chemistrv, Vol. 76, A’o. 14, 1971
2104
R. R. GORMAN, G. E. STONER, AND A. CATLIN
to$ from fresh citrnted human plasma. It was s e p mated from the plasma by a procedure involving adsorption of other components on magnesium sulfate, barium sulfate, and triethylaminoethyl cellulose, and glycine precipitation. The protein component of this preparation was assayed electrophoretically as being 95% fibrinogen. Total fibrinogenassays by absorbance at 280 mp and clottable protein assays indicated 100% clottability. Distilled water mas used throughout, and the mica \vas a clear, gray-green Tauganyikan muscovite. A Micros nutomatic valving vacuum evaporator, Model VE-IO, with an air-cooled diffusion pump was the principal unit used in the evaporation of carbon and platinum. The electron microscope was the JEM 6A with the 6C power supply. The usual accelerating voltage was 50 kV, the usual objective aperture, 50 pin diameter.
Procedures Preparation of Solutions. The WnrneFChilcott bovine fibrinogen was obtained in the form of 6-mg freezedried batches, each packed in a small vial with 17 mg of NnCI. The fibrinogen was reconstituted by adding 2 ml of distilled water (pH 6-8) to the vial, and then diluted to of its original concentration with the appropriate buffer. A sample of this suspension was assayed for ultraviolet ndsorption a t 280 nm, and another portion was diluted to '/,w of its initial concentration, producing a final fibrinogen concentration of about 0.6 X lO-'mg/ml. The human fibrinogen was diluted in the same way, but in some cases was assayed before diluting. Adsorption and Rinsing. The adsorption was nccomplished by immeming 1-in. square pieces of freshly split mica in the unstirred suspensions. For the adsorption on carbon, the mica was coated with an evnporated carbon film after splitting. After the adsorption had proceeded for the desired length of time, the mica specimen was pulled quickly out of the suspension while protein-free huffer was poured over the specimen surface. This pouring was continued for 20 to 300 sec, at a rate of I50 to 300 ml per min. The buffer used for this rinsing was the same as that used to make up the suspension. When the suspending and rinsing buffer was citrate or saline phosphate, a final rinse was made with 50-150 ml of the ammonium acetate buffer a t 0.26 m, pH 7, because of its special usefulness in electron microscope preparations. The final step was the blowing off of the remnant film of buffer with room temperature air from a laboratory blower. An important variation of the standard procedure was the rinsing experiment, in which several specimens were immemed in the fibrinogen suspension for the same length of time and then rinsed for different times, ranging from 40 see to 5 min. The other variation was the splitting of the mica The J o u r 4 01Phu&d Chnnialru. Vol. 76. No. 14. 1871
Figure 1. Bovine fibrinown on mica.
while immersed in the fibrinogen suspension, in order t o produce a surface which had never been exposed to the atmosphere, and which had not been drawn through the airsuspension interface. Platinum Shadowing. The platinum shadowing tecbnique was in essence that applied to the visualization of fibrinogen by Hall and Slayter.' The specimens, with their deposits of adsorbed fibrinogen, were placed in a vacuum evaporator and were shadowed with platinum and then coated, if necessary, with a strengthening film of carbon. The specimens were then removed from the evaporator and the double film or pseudoreplica was stripped off on a water bath and mounted on electron microscope grids.
Results and D i m d o n Adsorption on Mica. The technique produced welldefined fibrinogen triads on mica, similar to those found by Hall and Slayter. Typical photomicrographs are shown in Figures 1and 2. The adsorbed fibrinogen molecules were found to be distributed uniformly over the mica surface. This was determined by photographing and counting the number of adsorbed molecules in several (five to seven) areas on each of nine different specimens. The average value of the number of fibrinogen molecules per electron image plate and the standard deviation of these count values were calculated for each specimen. I t is seen in Table I, below, that with one exception, the range of counts was within two standard deviations from the mean, and the values of the standard deviations were reasonably (8) W.L. Walker, submitted for publiention. (9) C. E. Hall and H. 8. Slayter, J . OiOnAu&. D i o c h m . Cuiol.. 5. 11 (1959).
2105
THEADSORPTION OF FIBRINOGEN
I
0
I
I 5
IO
IS
f (rnl"1
I
Figure 3. Adsorption kinetic data.
Figure 2. Hornan fibrinogen on mica; well-defined triads.
close to the square roots of the average counts, a relationship which would be expected of a Poisson distribution of countsper plate.
Table I: Variability of Raw Data Averwe L?o"r,t Of
moleeula VI
E. M.
plats
Squm .OOt of ."e"** 00""t
ObMrVsd slandard deviation 01 0O""td
n*ngo of obacrvsd
deviation.
- 1 to + 3 -5 to +3 - 4 to +9 -14 to +17
12 15
3 4
2
39 4R
6
7
5 I1
59
8
11
- 14 to +20
90
9
113
11
18.5 266
14
15 14 19 41
-22 -lR -3R -93
16
3
to +20 to +24 to +22 to +63
The data for the time and concentration dependence of the adsorption of bovine fibrinogen on mica are presented graphically in Figure 3. The observed average numbers of molecules, N, divided by CO,the bulk fibrinogen concentrations in molecules per milliliter, are plotted against adsorption time in seconds. The parabolic form of this graph suggests that the adsorption may be a completely diffusion-controlled process, in which there is no significant energy barrier to adsorption nor any significant desorption at the interface. Such a process is described in ref 10
tl'2,d'2,
Figure 4. Fit of adsorption data to diffusion ciirve.
where D is the diffusion coefficient for the molecule. To test this hypothesis, the observed values of N/Co were plotted against t'/', and this graph is presented in Figure 4. The value of N for a given experiment was obtained by dividing the average number of molecules per standard electron image plate by the standard plate area, which waa 2.02 X lo-* cm'. The values for the bulk concentrations of fibrinogen, COin terms of molecules per milliliter, were obtained from the uv adsorption at 280 nm, using for the extinction coefficient 1.6 em-', multiplied by the factor 1.1 as an estimate of the effect of the impurity level reported by the supplier, and taking 350,000 for the molecular weight. A straight line fits the data within the uncertainty (IO) F. C. G d r i c h . J . C h m . Phys.. 22,
M(8
(1054).
The J o u d of Phyriml Chnniscru. Vol. 76. No. 14. 1871
R. R. GORMAN, G.E. STONER, AND A. CATLIN
2106 R b k 11: Reaulta of Fibrinogen Adsorption on Mica
Slumonding buRsr
0.26 M CH&QONH, (ammonium acetate) 0.26 M CHsCOONH. 0.26 M 0.26 M CHEOONH. 0.26 M CHsCGQNH, 0.40 M NaCl 0.014 M NaHrPO, 0.022 M NarHPO, 0.055 M sodium citrate HCI 0.15 M CHCQONH, 0.47 M CHSCOONH, 0.47M CHAXONH, 0.47 M CHGOONH,
CHICOON&
+ +
+
moboda/om*
mdml
sourn
pH 6 . 7
2.8 X IO'
0.50 x 10-1
Bovine
30
pH 7.1 pH 7 . 6 pH 7 . 0 pH 7.1 pH 6.4
2.1 x 2.3 X 2.6 X 2.6 X 2.7 X
0.55 x 0.66 x 0.47 x O.9R x 1.16 x
Bovine Bovine Bovine Bovine Human
40 40 40 40 60
pH R.3
2 . 7 X IO'
0.73
Human
35
PH 7 PH 7 PH 7 PH 7
2.6 X IO' 3 . 7 x IO' 3.6 X IO* 3 . 5 x lo*
0.51 X IO-' 0.9 x 10-1
Bovine Human Human Human
30 40
IO' 10' 10' 10' 10'
0.9
lo-* lo-'
IO-' lo-' lo-*
x IO-' x
10-2
0.9 x 10-
e c c
120
300
established by the spread of the measurements. The diffusioncoefficient D is found to be
D = 0.91 X lo-' cmz/sec This is about half that obtained by Shulman" from his sedimentation experiments. He suspended fibrinogen in 0.40 m NaCI, 0.05 m sodium phosphates at pH 6.2, and obtained D = 2.2 X lO-'cm'/sec. A reason for this discrepancy may lie in the use of 350,000 for the molecular weight of fibrinogen in eq 1. This value has been reported by several investigators,".lz but others have found 369,000," 392,000," and even 580,000." Given a pure, monodisperse suspension of fibrinogen and an accurate value for D , this experiment would be an excellent means for measuring the molecular weight. Taking D as 2.2 X lo-' cm'/ sec and taking for N , the average experimental value at 1 min of 26 X 1IY molecules/cm' one obtains mol mt =
2c, N
(11
'I' I'/'X
6.02 X 10" = 570,000
The effects of variations in buffer composition and pH and in rinsing time are shown in Table 11. The standard 1-min counts of fibrinogen molecules per square centimeter are a convenient way of comparing the results of experiments. They were obtained by converting the counts observed a t various adsorption times and concentrations into the values which would be expected at 1 min and a fibrinogen concentration of 0.6 X lo-' mg/ml, by means of eq 1. The adsorption counts are seen to have been insensitive to the variations which were made in the buffer composition and in rinsing time. The results from the experiment in which the mica specimen was split while immersed in the fibrinogen suspension shonred no difference when compared with the The J o u d olPhyriml Chmi*ru,
Vd. 76. No. 14, 1871
resulta obtained by splitting the mica in air and then immersing it. In no instance was an equilibrium reached in the adsorption onto mica. The maximum coverage observed and counted was roughly 1 X lolo molecules/cm*, or 6 X g/m*. A t higher coverages it became difficult, because of overlapping, to distinguish and count individual molecules. (11) 8. Shulrnan. J . Amw. C h m . Soc.. 75. 5840 (1953). (12) E. A. Kaspary and H. A. Keswiek. Oiochem. J . . 67, 4 1 (1957). (13) B. Blombaek and T.C. Laurent. Ark. K m i . 12. 137 (1957). (14) E. F. Cas-. J . Phya. Chrm.. 60, 926 (1956). (15) J. L. Oneley. G. Sentehmd. and A. Brown. ibid.. 51. 1x4 (1947)
COMPETITIVE AD~ORPTION O F FIBRINOGHN AND HEPARIN ON
Adsorption on Carbon. The results from the few experiments which were performed with a vacuum evaporated carbon film as the adsorber were qualitatively and quantitatively different from the mica resuIts. The adsorbed deposit did not (Figure 5 ) , for the most part, have the triad form, and the adsorption apparently reached equilibrium, the counts for 5- and
MICA
2 107
lO-min exposure to the standard fibrinogen concentration (0.6 X lom9mg/ml) both being about 2 x 1 0 9 molecules/cm2. This is in marked contrast to the adsorption of fibrinogen on mica, where equilibrium was not reached and where the number of molecules adsorbed increased with time in an apparently diffusion-controlled process.
Adsorption Inhibition as a Mechanism for the Antithrombogenic Activity
of Some Drugs. I.
Competitive Adsorption of Fibrinogen and Heparin on Mica
by G. E. Stoner,* S. Srinivasan, and E. Gileadi Department of Materials Science, School of Engineering and Applied Science, The University of Virginia, CharlottesvQle, Virginia, and Electrochemical and Biophysical Laboralory, Department of Surgery a d Surgical Research, State University of New York, Downstate Medical Center, Brooklyn, New York: 11,908 (Received September 28, lD70) Publication costs assisted by The Center of Advanced Studies, University of Virgina
Heparin has been shown to inhibit the adsorption of fibrinogen on mica surfaces. This effect was observed over several orders of magnitude of heparin concentration and for fibrinogen concentrations ranging from physiological concentration up to physiological concentration. The inhibition effect of heparin is destroyed when the drug is neutralized with protamine. The results are explained by an electrostatic desorption of fibrinogen by the highly negatively charged drug. This is consistent with adsorption studies of fibrinogen on metals which show desorption on highly negative surfaces. Some insight is given into the mechanism of heparin action in the adsorbed or bound state.
Introduction Fibrinogen is known to adsorb entensively on most surfaces exposed to blood, plasma, or a bufferedsolution containing it.’ The possible importance of this adsorption in the overall processes of formation of thrombi has been discussed out by numerous investigator^.^-^ Recent studies“ of adsorption of fibrinogen (from purified buffer solutions) onto freshly cleaved mica surfaces a t different pH and concentrations showed nearly complete coverage on the surface even when the concentration in solution was reduced to 0.001 physiological concentration (phys concn). The rate of adsorption was found to be diffusion controlled in the concentration range of to phys concn (lO-*-lO-lo mol/l.). It was also noted in the same study that fibrinogen was held very tJenaciouslyto the surface, and it could not be removed by repeated rinsing, when performed within the normal temperature and p H range. The mechanism of antithrombogenic activity of drugs such as heparin, when adsorbed on the surface (or bonded in the surface phase by a suitable chemical process) is of major interest. Heparin is a polysaccharide containing a relatively large number of negative
groups (six negative charges per tetrasaccharide unit). A surface covered with a monolayer of this substance will hence appear as a negatively charged surface having a relatively high charge density. Recent work on the adsorption of fibrinogen on mercury as a function of concentration and potential7 showed heavy adsorption at positive and at low negative potentials. However, at potential more negative than -1.6 V v5. (sce) adsorption of fibrinogen did not occur, even at the highest solution concentration studied. This potential was
(1) L. Vromon, J . Biorned. Mater. Res., 3 , 669 (1969). (2) L. Vromon and A. L. Adams, Thrornb. Diath. Haemorrh., 18, 510 (1967). (3) D. J. Lyman, J. 2.Brash, and K. G. Klein, “Proceedings of the Artificial Heart Conference,” R. J. Hegyeli, Ed., U. S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Washington, D. C., 1969, pp 113-122. ( 4 ) E. Gugler and E. F . Luscher, Thromb. Diath. Haemorrh., 1 4 , 361 (1965). (5) R . E. Baier and It. C. Dutton, J. Bwmed. Mater. Res., 3 , 191 (1969). (6) It. Gorman, Ph.D. Thesis, University of Virginia, Charlottesville, Va., 1969. (7) G. E. Stoner, J . Bbmed. Mater. Rea., 3 , 645 (1969).
The Journal of Physical Chemistry, Vol. 76, No. 14, 1971
