4. Discussion

In vivo screening tests for determining the thrombogenicity of materials have been proposed [4, 6]. Previous in vitro systems, even though simpler and less expensive, have not been readily adapted because of some experimental drawbacks such as blood-air interface, exposure to materials other than the one under study, and the need for anticoagulants [5, 7]. The kinetic closed cell test described herein minimizes these drawbacks [11]. Furthermore, the kinetics of the coagulation process can be followed; thereby determining the beginning, slope, and the end point of the reaction. This is not possible with in vivo tests. Moreover, in vivo testing involves variables which are extremely difficult to control, such as the blood flow patterns on the surface of the material analyzed.

The test uses material in sheet form; the need for special configurations is eliminated. Also, the ratio of material surface to the amount of blood is increased over previous methods [7]. The same cells can be utilized for analyzing other parameters of material blood compatibility. Preliminary data (fig. 7) illustrates the decrease in platelet counts induced by the listed materials, showing a significant dif ference between pericardium and the other three.

The variability of the blood properties requires that all tests be run with a simultaneous control. Silastic was selected as a control material, since it

and clinical results obtained by using the same materials in blood contacting applications remains to be demonstrated.

These materials can be rated quantitatively as to the rate of clot formation on their surface. Natural rubber proved to be the least desirable material with a direct modulus of 0.66 min. The blood compatibility of natural rubber is considered to be inferior to the other materials tested [2, 3. 8, 9, 10]. The direct clot modulus of polyurethane. Hydron, and glutaraldehyde treated pericardium was similar. The indirect modulus for the glutaraldehyde treated pericardium, however, was twice that of the polyurethane, suggesting a higher percentage of white clot formation.

The authors wish to thank T. Agishi, I. Koshino. and C. Carse for their technical assistance. Dr. J. Urzua provided advice in the preparation of the manuscript.

5. References

[1] Boretos, J. W., and Pierce, W. S., Segmented polyurethane: A polyether polymer, J. Biomed. Mater. Res. 2, 121-130 (1968).

[2] Boretos, J. W., Detmer, D. E., and Donachy, J. H., Seg mented polyurethane, II. Two years experience, J. Biomed. Mater. Res. 5, 373–387 (1971).

[3] Bruck, S. D., Rabin, S., Fergusin, R. J., Evaluation of biocompatible materials, Biomat. Medical Devices and Artificial Organs 1, 191-222 (1973).

[4] Gott, V. L., Keopke, D. E., Daggett, R. L., Zarnstorff, W.. and Young, W. P., The coating of intravascular prosthesis with colloidal graphite-The relative importance of electrical charge in the prevention of clot formation, Surgery 50, 382 (196).

[5] Imai, Y., and Nosé, Y., A new method for evaluation of antithrombogenicity of materials, J. Biomed. Mater. Res. 6, 165 (1972).

[6] Kusserow, B., Larrow, R., Nichols, J., Analysis and measurement of the effects of materials of blood, Proceedings of the Artificial Heart Program Conference (R. J. Hegyeh. Ed.), (National Institutes of Health, Bethesda, Md.. June 9-13, 1969), U.S. Government Printing Office. Washington, D.C., p. 233.

[7] Lee, R., and White, P., A clinical study of the coagulation time of blood, Am. J. Med. Sci. 145,495 (1913). [8] Leininger, R. I., Polymers as surgical implants, CRC Critical Reviews in Bioengineering (October 1972). [9] Levowitz, B. S., La Guerre, J. N., Calem, W. S., Gould. F. E., Scherrer, J., and Schoenfield, H., Biologic compatibility and applications of Hydron, Trans. Amer. Soc Artif. Int. Organs 14, 82-87 (1968).

[10] Nosé, Y., Tajima, K., Ogawa, H., Klain, M., von Bally, K.. and Effler, D. B., Cardiac prosthesis utilizing biological material, J. Thor. Cardiov. Surg. 62, 714-724 (1971) [11] Nosé, Y., Kambie, H., Kiraly, R., Komai, T., and Urzua. J., In vitro test of compatibility of blood with various natural and synthetic surfaces in: P. Didisheim, T. Shimamoto, and H. Yamazaki, Editors, Platelets, Throm bosis, and Inhibitors, Seminar Proceedings, Honolulu, Hawaii, Dec. 19-21, 1973 (Stuttgart-New York: F. K Schattauer Verlag, 1974), pp. 87-95.

Issued May 1975).

1. Introduction

Proteins and enzymes localized at interfaces play an important role in many biological phenomena. Matrix insolubilized enzymes are utilized clinically and commercially [1], glyco-proteins on the cell surface can promote and control both cellular aggregation [2] and cellular growth [3], the cytochrome enzyme system for oxidative phosphorylation is localized on the mitochondrion membrane [4], while the deleterious effect of materials on blood resulting in surface-induced coagulation has long been recognized [5-7]. The possible effects of a given surface on a protein mixture include permanent or reversible adsorption with or without denaturation, preferential adsorption of specific proteins, and changes in the protein microenvironment. The details of these interactions can best be understood by determining the conformation of the adsorbed molecule and the conformational changes concomitant with adsorption.

Various techniques have been utilized for the determination of the conformation of adsorbed proteins. Adsorbed molecular area and solution dimensions have been compared by Bull [8] for egg albumin using film balance techniques, and by

Figures in brackets indicate the literature references at the end of the paper.

Brash and Lyman [9] for numerous serum proteins using infrared internal reflection spectroscopy. Perturbations in the transmission fluorescence spectrum arising upon adsorption of trypsin and chymotrypsin were analyzed by Katchalski [10] to demonstrate conformational changes, while Loeb and Harrick [11] induced fluorescence of adsorbed serum albumin using internal reflection techniques and showed that the protein is apparently native when adsorbed. Kochwa and coworkers [12] applied potentiometric titrations to show that yGglobulin apparently unfolds upon adsorption to a polystyrene latex under conditions of low surface coverage and acquires an antigenicity similar to that of heat denatured material. Ellipsometry has been used qualitatively by Vroman [13] to study sequential, competitive adsorption of blood proteins. As indicated by these studies, protein adsorption is a complex process and each technique provides only partial answers. As a result, a consistent model of protein adsorption at the solid-solution interface does not exist at present.

In general, an adsorbed polymer is attached to the surface at a number of locations along the chain. Adsorbed segments can occur singly or in runs with attached portions separated by loops of unattached segments which extend away from the surface into the solution as shown in figure 1. The number and arrangements of the attached portions and the size and distribution of the unattached

loops and tails define the conformation of the adsorbed polymer molecule [14]. In the case of proteins, numerous internal constraints limit the accessible conformations, both in solution and on the surface, and make the prediction of the adsorbed conformation more difficult.

In this paper we review some of our studies [15] of the direct measurement of both the number of protein carbonyl group-surface attachments made using infrared difference spectroscopy, and the average dimensions of the adsorbed protein layer determined ellipsometrically for a number of blood proteins and materials. These results are viewed as providing essential information on the conformation and conformational changes of the adsorbing molecule. These studies have been carried out in situ on individual proteins as a function of the amount adsorbed, time of adsorption, and surface energy of the adsorbent. Additional information on blood-protein-material interactions is essential to an understanding of surface-induced coagulation and the development of a rational approach to the selection of thromboresistant materials.

2. Techniques

The interaction of the chromophores of an adsorbed molecule with a surface frequently results in a shift of their characteristic spectral absorption bands. For the protein studies presented here, a shift of 20 cm-1 of the amide I band for free and bound carbonyl groups was typically observed. Since there is a significant overlap of these two bands, difference techniques must be utilized. From the data obtained, one determines the bound fraction, i.e., the fraction of carbonyl groups of an adsorbed protein directly interacting with the surface. The number of carbonyl attachments may then be calculated using the known amino acid composition of the protein.

Fontana and Thomas [16] originally described a method whereby a polymer is adsorbed from

solution on a high surface-area powder, the suspension centrifuged, and an infrared difference spectrum recorded for the resulting gel. To prevent the introduction of uncertainties in polymer concentration, this method has been modified [17] to permit a direct analysis of the suspension. This modification has been used by a number of investigators [18-20] to study the conformation of synthetic polymers adsorbed from organic solvents.

For the determination of the bound fraction for an adsorbed protein, it is necessary to use D2O solutions of deuterated proteins to obtain a window in the 1650 cm-1 region. The difference spectrum resulting from a protein solution-silica suspension in the sample beam versus the same protein solution in the reference beam (fig. 2) was resolved using a Beer's Law analysis of the optical density of the unbound peak.

OUTPUT (VOLTMETER)

discussed [22]. Briefly, ellipsometry is an optical technique in which changes in the state of polarization of light upon reflection from a surface (fig. 3) are analyzed to characterize the surface and to determine the thickness and refractive index of a thin film on the surface. The thickness of a protein film on a surface is related to the extension and distribution of loops as shown in figure 1. This average extension of the adsorbed molecule normal to the surface is therefore a measure of the dimensions of an undisturbed protein layer on the surface. Calculations of the average_extension utilize an iterative solution [23] of the Drude equations [24]. Additionally, one calculates the amount of protein adsorbed using the experimentally measured film refractive index and the refractive index increment for the protein at the wavelength of light of the measurements. These calculations are, however, based upon the model of a homogeneous film of constant refractive index and discrete boundaries. Since an adsorbed protein film would probably be an inhomogeneous film with refractive index decreasing with distance from the surface, the calculated values reported represent an upper bound for the thickness. Appropriate inhomogeneous film models [25] may be utilized to relate the average extension to actual molecular dimensions.

3. Experimental Detail

The proteins selected for study are either major constituents of blood plasma or are implicated as being important in the clotting process.

3.1. Ellipsometry

Human serum albumin (4× crystallized) and bovine prothrombin (Cohn III-2) were obtained from Nutritional Biochemicals (NBC)2 and used without

"Certain commercial materials and instruments are identified here and elsewhere in this publication in order to adequately specify the experimental procedure. In no case does such identification imply recommendation or endorsement by the National Bureau of Standards, nor does it imply that the equipment or instruments identified are necessarily the best available for the purpose.

further treatment. Human fibrinogen (NBC) was purified by the procedure of Batt, et al. [26], a modification of a procedure developed by Laki [27].

Commercial ferrotype plate consisted of chromium electroplated on either brass or stainless steel. Platinum was 99.95 percent pure and rolled to a mirror finish. The quartz was fused silica. These materials were cleaned before use with hot 50:50 HNO3: H2SO4 followed by three rinses in boiling distilled water. They were then heated for 5 minutes at 500° in a muffle furnace and placed while still warm into the adsorption cell filled with buffer.

Low temperature laminar (LTL) type pyrolytic graphite was kindly supplied by Dr. Jack Bokros of General Atomic Corp. Commercial polyethylene sheet was cleaned in ethanol and cast under vacuum at 180° between plate glass sheets. Several samples were prepared from NBS Standard Reference Material 1475 linear polyethylene which has a density of 0.978 g/cm3, also by casting against glass under vacuum. All results on these two types of polyethylene were identical. The carbon and polyethylene samples were cleaned before use by refluxing with ethanol for two weeks in a Soxhlet extractor followed by a similar washing with distilled water.

All solutions were made up in pH 7.4 phosphate buffer and concentrations were determined by UV spectroscopy. All adsorption measurements were made in a cell thermostatted at 37±0.1 °C.

3.2. Infrared Difference Spectroscopy

Bovine serum albumin (4× crystallized), bovine fibrinogen (Cohn fraction I, 60% clottable), and bovine prothrombin (fraction III-2) were obtained from NBC. The serum albumin was deuterated [28], lyophilized, and stored in vacuo at 4 °C. The prothrombin was dissolved in 0.1M D2O phosphate buffer pD 7.4, dialyzed overnight against buffer, and filtered through a well washed 0.8 μm pore size filter just prior to use. Fibrinogen was purified by the method of Laki [27] with the following modifi