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 pH 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
2108
G . E. STONER,
s. SRINIVASAN, AND E. GILEADI
found to correspond to n surface charge density of -20 pC/cm'. The purpose of the present study w a y to examine the effect of hepnrin on the adsorption of fibrinogen from human plasma or from purified prepamtioris of fibrinogen in buffer solution on freshly cleaved micn surfaces. The experimental variables were the concentrntions of heparin nnd fibrinogen and the order of exposure of the surface to their solutions. It was nlso intended to find the effect of protnmine, n drug commonly employed clinically to neutrnlize the effect of hepnrin, on the nbility of heparin to inhibit adsorption of fibrinogen.
Experimental Section Mica surfaces (Tnngnnyiknn muscovite'), freshly cleaved immediately prior to immersion in solution, were used as substrates in all adsorption experiments. Adsorption of fibrinogen was followed by electron microscopy using a Type JEM 6A electron microscope. A shadowing technique, based on n modified Hall and Slayter procedure, was e m p l ~ y e d . ~ . ' ~ The supporting electrolyte for all solutions was 0.1 M ammonium acetate, adjusted to pH 7.0. Fibrinogen was prepnred from freshly drawn human plasma. Heparin wns "Liqunlmin Sodium"" lo00 USP units per cc, made up to desired concentrations. Solutions of protnmine sulfate were Grade 1 (from Snlmon).'2
Reaulta and Diacuaaion 1 . Adsorption o j Fibrinogen and Its Inhibition by Heparin. Fibrinogen is adsorbed very extensively on mien surfaces. Figure la shows the electron micrograph obtained when mica was dipped in n very dilute of phys concn) buffered solution of fibrinogen. Figure l b shows the results obtained for a solution containing fibrinogen at the physiological concentration. A high fractional degree of coverage is obtained in the solution of lower concentration and the individual fibrinogen molecules cnn he seen clearly on the electron micrograph. A t physiological concentration the surface is completely covered with fibrinogen which tends to form clusters and possibly multilnyers in certain areas. For this reason n dilute solution of phys concn (equal to ca. lo-' mol/l.) of fibrinogen wns used in all further experiments. The J o u d of Phwaiml Chmidru. Vol. 76. No. 14. 1871
Figure 2. Electrim mirmgmphs of: ( 8 ) partial inhibition cil fibrinogen by prior exposure to O.ooo2 rinits of heparin per ml; (b) and ( c ) total inhibition by prior exposure to U.OM and 2 units per ml, respectively; (d) mica surface not exposed to heparin; and (e) mira exposed to 2 unitti of heparin per ml.
In the experiments shown in Figure 2n, b, nnd c the mica surface was first exposed to solutions of various concentrations of heparin (in the range of 2 X lo-' to 2 units per cc = 6 X lo-" to 6 X lo-' mol/l.), washed with buffer solution, nnd then dipped into the fibrinogen solution for 1 min. At the lowest hepnrin concentrntion (Figure 2a) only pnrtinl inhibition of fibrinogen adsorption occurred, while nt n concentration of 6 X 10-'0 mol/l. or nbove, no fibrinogen was adsorbed on the surface (Figure 2b nnd e). The pattern seen in Figures 2b and c is caused by t,he adsorption of hepnrin (nlthough the individual heparin molecules nre too smnll t.o he seen by this technique). This is verified by comparing the electron micrographs for n hare micn surfnce (Figure 2d) with that for n mica surface exposed to heparin (Figure 2e). The structure of the basic tetrasaccharide unit in heparin is shown in Figure 8. This unit has a molecular weight of 986. Thus there nre nbout 20 snch units (8) Aeheville-Schoonmaker Mica Co.. Newport News. Vn. (8) C. E. Hall and H. 8. Slayter. J . f?&vhu&. Riochrm. Cufnl.. 5, 11 (1958).
(IO) R. R. Garman. G . E.Stoner. iind A. Cntlin. J . Phwo. C h m . . 75, 2103 (1871).
(11) nlganon IW.. west O ~ W .N. J. (12) Sigma Chemical
Co.. St. Louis. Mo.
COMPETITIVE ADSOWPIONOF FIBRINC-QEN AND HEPARIN ON MICA
2 109
*I:.om 0..
I
I
04.;
pi-*-
#$ "--,
HEPARIN
UNIT
I
I
Figure 3. The structure of the tetrmaccharide unit of heparin.
per molecule of heparin (molecular weight 17-20 X 10:). Assuming that each tetrasaccharide unit adsorbed on the surface occupies an area equal to that of four to five benzene molecules, the maximum surface concentration of heparin is (3.2-4) X mol/cm' (the maximum surface concentration for benzene is 3.2 X 10-'Omol/cm*).lx On the basis of this calculation it is easy to see why only partial inhibition could be achieved in the experiment shown in Figure 2s. The total area of the mica used in each experiment was m. 4 cmr and the volume of the solution \vas 50 ce. Thus, when the concentration of heparin was as low as G X lo-" mol/l., there \vae not enough heparin in solution to form a monolayer on the surface and total inhibition of fibrinogen adsorption could not be expected. It is rather remarkable that when the concentration of heparin was raised by one order of magnitude, complete inhibition of fibrinogen adsorption did occur. Although the total amount of heparin in this solution was ca. 2.5 times the amount required to form a monolayer, it is unlikely that a complete monolayer could indeed be formed in such dilute solutions in the short exposure time of 1 min. This may point to the eonclusion that even partial surface coverage by heparin may inhibit entirely the adsorption of fibrinogen, due to the negative charge imparted to the surface, as will be discussed below. 2. Displacement of Adsorbed Fibrimyen by Heparin. When a freshly cleaved mica surface was immersed in a solution containing both heparin and fibrinogen, the results were similar to these observed when the surface was first exposed to heparin and then to fibrinogen. Thus at concentrations of heparin of G X 1O-Io mol/l. or higher (Figures 4a and b) complete inhibition occurred, while at a concentration of G X lo-" mol/l. of heparin the adsorption of fibrinogen was only partially inhibited, as seen in Figure 4c. Comparing the molar concentrations of heparin and fibrinogen it is seen that complete inhibition of adsorption already occurs when the ratio of fibrinogen to heparin molecules is about 16. Thus the possibility that inhibition is due to some reaction between heparin and fibrinogen in the bulk of the solution is excluded. A similar ratio between the number of fibrinogen and heparin molecules
F i g r e 4. Electron micrographs of competitive adsorption from mlutions containing both fibrinogen and heparin: (a) 2 units of heparin/ml; (b) 0.002units/ml; (e) O.ooo2 units/ml. All fibrinogen concentrations are 0.001 physiological.
Figure ;1. k:Iertron micropapha of mira miner* first exposed to 0.001 physiological fibrinogen and then: (n)0.W. units of heparin per ml; mid (h) 0.oM units/ml.
also exists in the blood after a usual dmnge of heparin has been administered. Thus, inasmuch as the antithrombogenic properties of this drug in the adsorbed or bound state may depend on its effect on fibrinogen, there may be an interaction taking place on the surfaces of the blood vessels rather than in the bulk phase. Although the adsorption of fibrinogen is an important step in the formation of thrombi, one cannot conclude, on the basis of the above evidence only, that the physiological effect of heparin is due to the inhibition of this adsorption process. The drug may act in addition (or mainly) by interfering with a crucial enzymatic process leading to thrombos formation, e.&, with the conversion of protrombin into thrombin. Figures 5a and b show the results of an experiment in which the freshly cleaved mica surface was first exposed to a solution of fibrinogen (lo-* of phys concn) and then to solutions of heparin at different concentrations (6 X 10-10 and 6 X 10-9 mol/l., respectively). (13) This is a lower estimats. based on the assumption that the whole molecule of heparin lies Rat on the surfan.
The J o u d o / P h y d C h m i d r y . Vd.76. No. 14. 1871
G. E. h m a , 9. SRINIVASAN, AND E. GILEADI
2110
Figiire 7. Electron micrographs showing the eKect of protamine: (a) adiorption of fibrinogeii after exposure to 2 units of protamine/ml; and (h) after exposure to 2 iinits of heparin per ml neutralized with protamine. All fibrinogen concentrations are 0.001 physiological.
Figure 6. I?lectron micrographs of mica siirfacex exposed to plllsmn: (a) o.nn1 physiological (diluted with hoRer); (h) physiological concentration; (e) same ns (a) but with prior exposure to 2 units of heparin per ml; (d) same a- (b) hut with prior exposure to 2 units of heparin per ml.
At the higher concentration of heparin fibrinogen was completely replaced from the surface while at the lower concentration it was only partially replaced." This is a somewhat unexpected result in view of the observation of Gorman, et al.,lo that adsorption of fibrinogen on mica was very irreversible and the adsorbed layer could not be removed by prolonged washing in buffer solution. Two possible mechmisms oould be proposed for the replacement process: (i) heparin molecules may interact with adsorbed fibrinogen to form a compound which can be easily desorbed from the surface; (ii) initial adsorption of a few heparin molecules may cause desorption of neighboring fibrinogen molecules due to the negative charge on heparin. This makes place for further adsorption of heparin which then causes further desorp tion of fibrinogen and RO on, until the whole surface layer is replaced. S. Adsorption of Blood Proleins from P l n m . Freshly cleaved mica surfaces exposed to plasma a t physiological concentration (Figure 6a) and at IO-' of phys eoncn (Figure 6b) show extensive adsorption of blood proteins, and primarily of fibrinogen. Preexpw sure of the surface to heparin solution inhibits the adsorption of all plasma proteins, as seen in Figures 6c and d. 4. The Effect oj Prolamine on Ihe Adsorptia of Fibrinogen. Protamine is n drug used clinically to counteract or neutralize the antithrombogenic effect of heparin in the blood. Exposure of a freshly cleaved mica surface to protamine had no appreciable effect on the subsequent adsorption of fibrinogen from a buffer solution on the same surface, as seen in Figure 7s. Moreover, exposure of the surface to a solution containing equivalent amounts of heparin and protamine (both T h e J a c d of Phu&
Chnnidw. Vd.76. No. 14. lsll
at a concentration which would result in the blood after a regular dosage has been administered) had no inhibiting effect on the adsorption of fibrinogen (Figure 7b). Here again a correlation exists between the thrombogenic and antithmmbogenic properties of the drugs tested and their ability to inhibit or enhance the adsorption of fibrinogen on mica. 6. Adsorption Mechanism for the Activity oj Antithrmnbog.enic and Thrombogenic Drugs. From electrokinetic studies on the effects of drugs on the surface charge of the blood vessel wall and of blood cells, it was shown" that antithrombogenic drugs increase the magnitude of the negative charge densities of these surfaces while thrombogenic drugs decrease it nnd often cause a reversal of sign of the surface charge. Of a large number of drugs investigated to date, heparin and pro& amine have the largest effect. It was proposed" that the mechanism of drug action in preventing or accelerating thrombosis is by the adsorption of these compounds on the blood vessel wall nnd the blood cells. The present work lends further support for this view. On the other hand, many more surfaces and drugs will have to be tested before it can be stated with certainty that the physiological activity of thrombogenic and antithrombogenic drugs is due in part to their effect on the adsorption of fibrinogen on the blood vessel wall. Considering the structure of heparin (Figure 3) it is seen that six negative charges are associated with each tetrasaccharide unit, or M. 120 unit charges per molecule. Thus, a surface covered with a monolayer of heparin will have an average charge density of 43 &/em' (based on an average value of 3.6 X lo-'* mol/cm' of heparin). In a recent study' of the adsorption of fibrinogen on a mercury electrode, complete desorption occurred when the surface charge density was more negative than -20 pC/cm'. By correlation it may be estimated about half a monolayer of heparin on the surface should be enough t,o inhibit completely the (14) The same Eoncsntration of heparin caused complete inhibition of adsorption of f i b h 0 5 " when the surface WIYI a x i d first to heparin or to II mixture of heparin and fibrinogen. (15) 8. SrinivsMn. It. Aaron. P. S. Chopra. T. Lucas. nnd 1'. N. S a v e r . Surocry, 64,827 (1968).
COMPETITIVE ADSORPTION OF FIBRINOGEN AND HEPARIN ON MICA adsorption of fibrinogen. This explains the effectiveness of heparin at very low concentrations, as shown above. The high charge density on the heparin molecule also supports the second mechanism proposed above for the replacement of fibrinogen by heparin from a mica surface. Thus, since the charge on one heparin molecule is sufficient to replace fibrinogen from an area twice as large as that taken up by the molecule itself, it may cause the desorption of a neighboring fibrinogen molecule, which makes space for the adsorption of several more heparin molecules. The process of replacement of fibrinogen by heparin probably spreads laterally from a relatively small number of points resembling nucleation centers.
Conclusions Adsorption of fibrinogen is probably an important factor in the adhesion of thrombos deposits on the surface of prosthetic materials in contact with blood. A mica surface was used to study the effect of certain drugs on the adsorption of fibrinogen. Heparin which is a potent antithrombogenic drug was found to inhibit completely the adsorption of fibrinogen on mica. Protamine which is a drug used to neutralize the effect of
2111
heparin in the blood acts similarly with respect to adsorption of fibrinogen. Thus, no inhibition of adsorption was observed when the surface was exposed to a solution containing both heparin and protamine, or to one containing protamine only. The correlation found here between the antithrombogenic activity of a drug and its effect on adsorption of fibrinogen on mica will be tested for other drugs and on other surfaces. The present finding points to the possible importance of adsorption onto either prosthetic materials or the blood vessel wall during the action of antithrombogenic drugs.
Acknowledgments. Financial support from the National Institute of Dental Research, Grant DE-2 111-02 (for Glenn Stoner) and from the Artificial Heart Program, National Heart Institute National Institutes of Health, Contract No. PH43-68-75, (for S. Srinivasan) is gratefully acknowledged. S. Srinivasan is the recipient of a Career Scientist Award from the Health Research Council, City of Kew York, Contract No. I 542. E. Gileadi wishes to thank the Center of Advanced Studies a t the University, sponsored by the National Science Foundation, for the award of a University Lecturership.
T h e Journal of Physical Chemistry, Vol. 76, N o . 14, 1971
