Adsorption of plasma proteins and adhesion of platelets onto novel polyetherurethaneureas--relationship between denaturation of adsorbed proteins and platelet adhesion.

Adsorption of plasma proteins and adhesion of platelets onto novel polyetherurethaneureasrelationship between denaturation of adsorbed proteins and platelet adhesion Y. Ito, M. Skido,+ and Y. Imanishi Department of Polymer Chemistry, Faculty of Engineering and *The Research Center for Medical Polymers and Biomaterials, Kyoto University, Kyoto, Japan 606 Novel polyethemrethaneureas which have been synthesized by the present authors were chosen for the substrate polymers, on which adhesion of platelets was investigated. The number of adhered platelets and the amount of serotonin released from platelets adhered on the polymers and the protein-coated polymers were determined by rat ioisotope method. Both of them were ~qhancedwith increasing content of urea 1i.ikages in the polyetherurethaneureas. The platelet adhesion was discussed

in terms of the denaturation of plasma proteins upon adsorption, which was determined by Fourier-transform infrared spectroscopy. With increasing degree of protein denaturation, the platelet adhesion and the serotonin release were enhanced. This relationship was particularly evident in the case of albumin adsorption. It was shown that the surface properties of substrate polymers affect the protein adsorption, which in turn influences the adhesion of platelets.

INTRODUCTION

In considering the blood compatibility of a material, adhesion of platelets on the material is of primary importance. Prior to the platelet adhesion, plasma proteins are adsorbed on the material surface quickly and the proteins adsorbed affect profoundly subsequent blood-material interaction~.'-~ It has been investigated that the n a t ~ r e , ' , ~ distribution,8 -~ and orientation' of adsorbed proteins regulate the recognition of the protein-coated material surface by platelet. We have found by Fourier-transform infrared (FT-LR) spectroscopy, that the degree of denaturation of proteins adsorbed on the surface of polyetherurethaneureas depends on the surface properties.'" In the present investigation, we were interested in the effect of the degree of denaturation of adsorbed proteins on platelet adhesion. The adhesion of platelets on a material surface was investigated on the frequency of adhesion and on the release of serotonin from dense granule of adhered platelets.

Journal of Biomedical Materials Research, Vol. 24, 227-242 (1990) CCC 0021-9304/90/010227- 16$04.00 0 1990 John Wiley & Sons, Inc.

ITO, SISIDO, AND IMANISHI

228

EXPERIMENTAL

Preparation of polymers Preparative method of polyetherurethaneureas and their derivatives has been reported previously.*’ Poly(tetramethy1ene glycol) (PTMG) and polyaminoether (PAE) having the molecular weight 1257 and 2350, respectively, were mixed with 4,4’-diphenylmethane diisocyanate (MDI) in various molar ratios. Step-growth polymerization and the subsequent reaction with 1,2-diaminopropane (PDA) for chain extension yielded polyetherurethaneureas carrying tertiary amino groups in the backbone (PAEUU). Quaternization with benzyl chloride gave cationic polyetherurethaneureas (Q-PAEUU), and electrostaticbinding of heparin to Q-PAEUU yielded heparinized polyetherurethaneureas (H-PAEUU).” The characterization of the synthesized polyetherurethaneureas was performed as reported previously,1@12 and can be briefly summarized as follows. The content of amino groups in the polyaminoetherurethaneureas was determined by titration in phenol/ethanol (5:1 v/v) mixture with 0.1N dioxane solution of perchloric acid using bromophenol blue as an indicater. The water content of quaternized polymers and heparinized polymers was determined by immersion of the polymer membrane in distilled water or in an aqueous sodium heparinate solution, respectively, at 70°C for two days. The value of water content was calculated by dividing the weight of absorbed water by the weight of wet polymer. Heparin content in heparinized polymers was determined by the elemental analysis of sulfur, The polymers synthesized in this investigation are listed in Table I. Platelet adhesion Platelet adhesion experiments were carried out according to the procedures reported by us.12Canine blood was treated with a mixed aqueous solution of citric acid, sodium citrate monohydrate, and D-glucose dihydrate, and centrifuged at 180 g for 10 min to obtain platelet-rich plasma. Washedplatelet suspension was prepared by suspending platelets in a phosphatebuffered saline (PBS). After platelets were labeled with Na;*CrO, or 3Hserotonin, the labeled platelets were brought into contact for 10 min with test tubes which were coated with the polyetherurethaneureas or with plasma proteins adsorbed on the polymer coating. After rinsing with phosphate-buffered saline, the number of adhered platelets was determined by y-counting of 51Cr.The amount of serotonin remaining in adhered platelets was determined by the determination of 3H-labeled serotonin. For each polymer or protein coating, five samples were prepared and subjected to the platelet adhesion experiment. The experiment with each sample was repeated three times and a mean value was obtained. The sta-

3.45 3.03 2.71 2.23 0.0 1.94 3.45 4.65

Amine (wt%)

0.38 0.46 0.40 0.39 1.02 0.29 0.38 0.13

hIC

-

-

-

-

21.72 20.58 7.79 0.43

-

112.3 110.9 65.8 33.3

Heparin (wt%)

H-PAEUU”

H20 (wt%)

233.6 161.5 81.3 49.2

H,O (wt%)

Q-PAEUU”

“Abbreviations:PAEUU, polyaminoetherurethaneurea; Q, quaternized; H, heparinized. PTMG, poly(tetramethy1ene glycol); PAE, polyaminoether; MDI, 4,4’-diphenylmethane diisocyanate; PDA, 1,2-diaminopropane. bThe numerical ratios (1:0, 3: 1, etc.) represent the molar ratio of PTMG against PAE in the feed of polymerization. A, B, C, and D represent the molar ratio of MDI against PTMG + PAE in the feed of polymerization being 2, 3, 4, and 6, respectively. ‘Measured in dimethylformamide solution at 30°C and given in the unit of 100 cm3 g-’.

3:l-A 1: 1-A 1:3-A

1:l: 8: 6 1:1: 12: 10 1:o: 2: 1 3 : l : 8: 4 1:l: 4: 2 1:3: 8: 4

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1 1-A 1: 1-B

1: 1-c 1 :1-D 1:0-A

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TABLE I Compositions and Properties of PAEUU, Q-PAEUU, and H-PAEUU”

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230

tistical significance of experimental data on platelet adhesion was determined by using F-test.

Protein adsorption The protein adsorption experiment was carried out in the same procedures as reported previously." By employing the same procedures, we can discuss the platelet adhesion in relation with the properties of adsorbed proteins that have been reported previously." A test tube was coated with a polyetherurethaneurea and immersed in a protein solution (0.01M Tris-HC1 containing 0.9% NaC1, pH 7.4), which contains one of the plasma proteins in nearly physiological concentrations (4.5 gdL-l of bovine serum albumin, Sigma Co. No. A-6003; 1.6 gdL-' of bovine y-globulin, Sigma Co., No. G-3500; 0.3 gdL-' of bovine plasma fibrinogen, Sigma Co., No. F-4753) at 37°C for 1 h. Then the test tube was washed for 10 s with the same buffer solution, dried in vacuum, and subjected to the platelet adhesion test.

RESULTS

Interaction of platelets with plasma proteins adsorbed onto polyaminoetherurethaneureas The surface of polyaminoetherurethaneurea membrane was coated with one of the three plasma proteins and the protein-coated membrane was brought into contact with washed-platelet suspension. The number of platelets adhered and the amount of serotonin remaining in the platelets adhered to albumin-, globulin-, or fibrinogen-coated membrane of polyaminoetherurethaneureas with varying contents of urea linkages are shown in Figure l(a) and (b). The abscissa represents the molar ratio of the diisocyanate compound against the dihydroxyl compound in the feed of polymerization. On going from 2 to 6 on the abscissa, the content of urea linkages in the polymer increases. When the polymer surface was coated with albumin or fibrinogen, the number of adhered platelets increased and the amount of remaining serotonin decreased with increasing content of urea linkages, However, when the polymer surface was coated with globulin, the number of adhered platelets and the amount of remaining serotonin were not seriously influenced by the polymer composition. Figure l(c) and (d) show the effect of amine content in the polyaminoetherurethaneureas on the platelet adhesion. The content of amino groups in the polyaminoetherurethaneureas was decreased with decreasing ratio of polyaminoether, which is of high molecular weight and bulky, in the feed of step-growth polymerization. A decreasing content of amino groups necessarily increases the content of urea linkages in the polyaminoetherurethaneureas and vice versa. When the polymer surface was coated with

231

ADSORPTION OF PLASMA PROTEINS

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(4 Figure 1. Platelet adhesion and amount of serotonin remaining in platelets adhered from plasma-free suspension on to protein-coated polyaminoethemrethaneureas: (a) the effect of the content of urea linkages in PAEUU on platelet adhesion; (b) the effect of the content of urea linkages in PAEUU on the amount of remaining serotonin; (c) the effect of the content of amino groups in PAEUU on platelet adhesion; (d) the effect of the content of amino groups in PAEUU on the amount of remaining serotonin; (0)albumin coating,).( globulin coating, (A) fibrinogen coating; bars indicate the standard deviation; (*) P < 0.05, (**) P < 0.01, (***) P < 0.005.

232


albumin, the number of adhered platelets increased and the amount of remaining serotonin decreased with decreasing content of amino groups or with increasing content of urea linkages in the polyaminoetherurethaneureas. On the othel hand, when the polymer surface was coated with globulin or fibrinogen, the platelet adhesion was unaffected by the amine content or by the content of urea linkages in the polyaminoetherurethaneureas. Washed-platelet suspension was brought into contact with polyaminoetherurethaneureas which are not coated with plasma proteins in advance, and the experimental results are shown in Figure 2(a)-(d). The experimental results are similar to those obtained in the interaction of the washed-platelet suspension with albumin-coated polyaminoetherurethaneureas, i.e., the number of adhered platelets increased and the amount of remaining serotonin decreased with increasing content of urea linkages or with decreasing content of amino groups in the polyaminoetherurethaneureas. Uncoated polymers were also brought into contact with platelet-rich plasma, and the experimental results are shown in Figure 3(a)-(d). The results are similar to those obtained for the interaction of washed-platelet suspension with uncoated polymers, i.e., the number of adhered platelets increased and the amount of serotonin remaining in adhered platelets decreased with increasing content of urea linkages or with decreasing content of amino groups in polyaminoetherurethaneureas.

Interaction of platelets with plasma proteins adhered on quaternized polyaminoetherurethaneureas The interaction of platelets with quaternized polyaminoe therurethaneurea coated with one of the three plasma proteins was investigated, and the experimental results are shown in Figure 4. In these cases, with increasing content of urea linkages in the polymer, the water content decreased, as shown in Table 1. For any kind of proteins coated, the number of adhered platelets increased and the amount of serotonin remaining in adhered platelets decreased with increasing content of urea linkages in the quaternized polymers. When uncoated membranes of quaternized polyaminoetherurethaneurea were brought into contact with washed-platelet suspension or platelet-rich plasma, the number of adhered platelets increased and the amount of serotonin remaining in adhered platelets decreased with increasing content of urea linkages, or with increasing water content of the quaternized polymers (data not shown).

Interaction of platelets with plasma proteins adsorbed on heparinized polyaminoetherurethaneureas Heparinized polyaminoetherurethaneurea membrane was coated with one of the three plasma proteins and protein-coated membranes were

233


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(4 Figure 2. Platelet adhesion and amount of serotonin remaining in platelets adhered from plasma-free suspension on to uncoated polyaminoetherurethaneureas: (a) the effect of the content of urea linkages in PAEUU on platelet adhesion; (b) the effect of the content of urea linkages in PAEUU on the amount of remaining serotonin; (c) the effect of the content of amino groups in PAEUU on platelet adhesion; (d) the effect of the content of amino groups in PAEUU on the amount of remaining serotonin; bars indicate the standard deviation; (*) P < 0.05, (**) P < 0.01, (***) P < 0.005.

brought into contact with washed-platelet suspension. The experimental results are shown in Figure 5. With reference to the heparinized polyaminoetherurethaneureas, an increasing content of urea linkages in the polymers


234

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0

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MDI / (PTMG PAE )

Amine content in polymer(%)

(4 Figure 3. Platelet adhesion and amount of serotonin remaining in platelets adhered from platelet-rich plasma on to uncoated polyaminoetherurethaneureas: (a) the effect of the content of urea linkages in PAEUU on platelet adhesion; (b) the effect of the content of urea linkages in PAEUU on the amount of remaining serotonin; (c) the effect of the content of amino groups in PAEUU on platelet adhesion; (d) the effect of the content of amino groups in PAEUU on the amount of remaining serotonin; bars indicate the standard deviation; (*) P < 0.05, (**) P < 0.01, (***) P < 0.005.

leads to a decrease of heparin and water content, as shown in Table I. When a membrane of heparinized polymer was coated with albumin or globulin, the number of adhered platelets increased, but the amount of serotonin remaining in adhered platelets was little affected with increasing content of urea linkages in the polymer. When a membrane of heparinized polymer was coated with fibrinogen, the polymer composition did not affect either the platelet adhesion or the serotonin release.

235


MDI I ( PTMG PAE)

(a)

MDI / ( PTMG+PAE )

(b) Figure 4. The effect of the content of urea linkages in polyaminoetherurethaneurea on (a) the number of platelets and @) the amount of serotonin remaining in platelets adhered from plasma-free suspension on to quateralbumin, (e)globulin, nized polyaminoetherurethaneurea coated with (0) or (A) fibrinogen; bars indicate the standard deviation; (*) P < 0.05, (**) P < 0.01, (***) P < 0.005.

The interaction of uncoated membrane of heparinized polymer with washed-platelet suspension or platelet-rich plasma was investigated, too. In either case, with increasing urea linkages in the heparinized polymers the number of adhered platelets increased and the amount of serotonin remaining in adhered platelets decreased (data not shown).

Relationship between denaturation of adsorbed proteins and interaction with platelets The relationship between the degree of denaturation of adsorbed proteins and the platelet adhesion and the serotonin release is shown in Figure 6(a)-(f) for all kinds of polymers investigated in the present investi-


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gation. It is evidently shown that on the albumin-coated material surface, more platelets were adhered and more serotonin was released from the adhered platelets, as adsorbed protein was more extensively denatured. Definite conclusion cannot be drawn for globulin- and fibrinogen-coated materials, because only a narrow range of protein denaturation was investigated. However, a trend similar to albumin-coated material was observed. Keeping the degree of denaturation constant, comparison shows that the platelet adhesion and the serotonin release were suppressed according to the nature of adsorbed proteins in the order albumin L globulin > fibrinogen (Fig. 6). However, when the degree of denaturation was variable, this relationship did not hoId.

237


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Figure 6 . Dependence of the number of adhered platelets (a)-(c) and the amount of serotonin remaining in adhered platelets (d)-(f) on denaturation of adsorbed proteins: (a) and (d) albumin, (b) and (e) globulin, (c) and (f) fibrinogen.


238 DISCUSSION

Interaction of platelets with polyaminoetherurethaneureas The present investigation showed that the interaction of platelets with the polymers was weakened according to the properties of the polymer in the order, untreated polymer > quaternized polymer > heparinized polymer, which is in the same order as that previously reported for other series of polyaminoetherurethaneurea derivatives.I2 The present investigation also showed that the platelet adhesion and serotonin release on the polyaminoetherurethaneureas increased with increasing content of urea linkages of polyaminoetherurethaneureas [Figs. l(a) and (b), 2(a) and (b), and 3(a) and (b)]. The same trend was observed in the platelet adhesion on quaternized polymers (Fig. 4) and heparinized polymers (Fig. 5). However, the water content of the quaternized polymers, and the water content and the heparin content of the heparinized polymers decreased with increasing content of urea linkages (Table I). Therefore, the enhanced adhesion and activation of platelets may be caused not only by the increasing content of urea linkages, but also by the reduced content of water and heparin. Reduced adhesion and activation of platelets on hydrophilic and/or negatively charged surfaces have been r e p ~ r t e d . ’ ~ ” ~ The relationship between the proportion of soft segment (or hard segment) on the surface of polyurethane membrane and platelet adhesion has been investigated over the last d e ~ a d eHowever, .~ disagreement has been reported on this problem. Some articles have reported that the platelet adhesion increased with increasing content of soft segment, and others have reported the opposite conclusion. It has also been reported that platelet adhesion is most suppressed at an optimum concentration of soft segment. A recent article” has reported that crosslinked polyether does not strongly stimulate platelets. A diversity of factors determining the platelet adhesion may be responsible for these variations of experimental results. One of the possible factors should exist in different experimental conditions employed such as the nature of platelets, the kind of source animal, the evaluation method (in vitro, in vivo, or ex vivo), etc. Another possible factor should be that the surface properties of matrix polymers are too complex to be determined by a single method of spectroscopy such as electronic spectroscopy for chemical analysis (ESCA). We have reported that in untreated polyaminoetherurethaneureas either the increasing feed of MDI or the decreasing content of amino groups leads to the increasing content of urea linkages and the enhanced hydrogenbonding properties.” In the present investigation it was found that in untreated polyaminoetherurethaneureas either the increasing feed of MDI or the decreasing content of amino groups led to adhesion and activation of platelets in the presence of albumin or total plasma proteins, but that they did not influence strongly adhesion and activation of platelets in the case of coating with globulin or fibrinogen.


239

With regard to the surface structure of polyetherurethanes, Yoon and Ratner16 have reported that the concentration of polyether segments increases on the membrane surface, as the phase separation in the bulk proceeds. On the other hand, Hearn et al.I7 have reported that the hard segment still exists on the surface layer in the depth of less than 1.0-1.5 nm, even after the phase separation in the bulk, These reports indicate that the bulk composition is not necessarily the same as the surface composition. However, taking into account the fact that urea acts as reagent of protein denaturation, it is feasible to say that plasma proteins are denatured on the membrane surface containing urea linkages to induce subsequent interactions of denatured proteins with platelets. Interaction of platelets with adsorbed proteins Following our observation,” Lenk et al.19 observed the formation of the @-sheetstructure in bovine serum albumin denatured upon adsorption using attenuated total reflection Fourier-transform infrared spectroscopy. They observed different behaviors on the surface of Ge plate and on the surface of polyurethane membrane, but the effect of the composition of polyurethane was not detected because of poor sensitivity. On the other hand, in the present investigation, we determined the adsorbed proteins with sensible transmission Fourier-transform infrared spectroscopy, and the relationship between the degree of denaturation of adsorbed proteins and the platelet adhesion is shown in Figure 6 . It was found that the platelet adhesion is determined not only by the nature of adsorbed proteins, but also by the conformation of adsorbed proteins. The same conclusion has been reached in our previous investigation.*‘In the same article, it has also been reported that the free energy of protein-coated surface was influenced neither by the nature nor by the degree of denaturation of adsorbed proteins.12 Therefore, it is feasible to say that the conformational properties of adsorbed proteins influence the interaction with platelets. The denatured site of a protein molecule may be agonistic to platelets. In fact, it has been shown that a cell-adhesive protein, fibronectin, does not interact with cells until a conformational change occurs upon adsorption on substrate. On the other hand, fibrinogen has been reported to interact with platelets more strongly in its native form than in the denatured form.” It is therefore considered that a conformational change of adsorbed proteins does not necessarily form the platelet recognition site. The interaction of uncoated polyaminoetherurethaneurea derivatives with platelet-rich plasma (Fig. 3) was similar to that of protein-coated polymers with washed-platelet suspension (Fig. 1). Here, we should pay attention to the possibility of differences that may result from using canine platelets and bovine protein species. The primary structure of the same kind of plasma protein may differ from one species to other species. In fact, Absolom et al.” have reported a higher hydrophilicity


240

of human albumin than bovine albumin. However, Packham et a1.6 have reported the absence of the effect of source species of plasma proteins upon adhesion of platelets, after investigating the interactions of human or bovine proteins with human or porcine proteins. Furthermore, there have been many investigations on the interaction between proteins and platelets from different species, e,g., canine platelets and human albumin," human platelets and human or bovine protein~,'~ and human platelets and bovine protein^.'^ In the application of protein-coated material to vascular prostheses, the biocompatibility and the applicability of protein-coated material would be more important than the difference of species. The similarity between Figures 1 and 3 indicates that the major plasma proteins such as albumin, globulin, and fibrinogen play an essential role in platelet adhesion. Other proteins such as fibronectin, factor VIII/vWF, and glycoproteins, which exist in blood in low concentrations, may participate in platelet adhesion. However, it has been reported that fibronectin does not promote cell adhesion efficiently in the presence of a large amount of alb ~ m i n . Therefore, '~ it is considered that the participation in platelet adhesion of the minor plasma proteins may be less important than that of the major plasma proteins. Washed-platelet suspension interacted similarly with protein-uncoated polymers (Fig. 2) and with protein-coated polymers (Fig. 1). The similarity suggests that upon adhesion the membrane proteins of platelet undergo conformational change similar to that occurring in adsorbed plasma proteins, leading to platelet activation. On protein-uncoated materials, more platelets were adhered, but less serotonin was released from the washedplatelet suspension (Fig. 2) than from the platelet-rich plasma (Fig. 3). This indicates that the stimulation received by platelets in the plasma-free suspension was different from that in the plasma-containing suspension. Finally, the relationship between the platelet adhesion and the serotonin release should be discussed. The present investigation and previous investigations showed that the more the adhered platelets, the more the released serotonin. However, platelets in plasma-free suspension and in plasmacontaining suspension behaved differently toward protein-uncoated polyaminoetherurethaneurea derivatives as stated above. Furthermore, no clear relationship was found on the platelet adhesion and the serotonin release on some block In order to understand the mechanism of platelet-material interactions in detail, it should be necessary to investigate "platelet reactivity" proposed by Whicher and Brash,27or to investigate the release of P-thromboglobulin as reported by us.13

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2. S. W. Kim, R. G. Lee, H. Oster, D. Coleman, J. D. Andrade, D. J. Lentz, and D. Olsen, "Platelet adhesion to polymer surfaces," Trans. Amer. SOC. Artif. Intern. Organs, 20, 449-455 (1974). 3. Y. Ito and Y. Imanishi, "Blood compatibility of polyurethanes," CRC Critical Rwiews in Biocompatibility, 5, 45-104 (1988). 4. M. D. Lehlah and S. L. Cooper, "Polyurethanes in Medicine," CRC Press, Boca Raton, FL, 1986. 5. R. G. Lee and S. W. Kim, "Adsorption isotherms and kinetics," J. Biomed. Muter. Res., 8, 251-259 (1974). 6. M.A. Packham, G. Evans, M. F. Glynn, and J. F. Mustard, "The effect of plasma proteins on the interaction of platelets with glass surfaces," J. Lab. Clin. Med., 73, 686-697 (1969). 7. C. S. P. Jenkins, M. A. Packham, M. A. Guccione, and J. F. Mustard, "Modification of platelet adherence to protein-coated surfaces," J. Lab. Clin. Med., 81, 280-290 (1973). 8. T. Okano, S. Nishiyama, I. Shinohara, T. Akaike, Y. Sakurai, K. Kataoka, and T. Tsuruta, "Effect of hydrophilic and hydrophobic microdomains on mode of interaction between block polymer and blood platelets," J. Biomed. Muter. Res., 15, 393-402 (1981). 9. K. Kataoka, M. Maeda, T. Nishimura, Y. Nitadori, T. Tsumta, T. Akaike, and Y. Sakurai, "Estimation of cell adhesion on polymer surface with the use of column-method," J. Biomed. Muter. Res., 14, 817-823 (1980). 10. T. Sanada, Y. Ito, M. Sisido, and Y. Imanishi, "Adsorption of plasma proteins to the derivatives of polyaminoetherurethaneurea: The effect of hydrogen-bonding property of the material surface," J. Biomed. Muter. Xes., 20, 1179-1196 (1986). 11. R. Shibuta, M. Tanaka, M. Sisido, and Y. Imanishi, "Synthesis of novel polyaminoetherurethaneureas and development of antithrombogenic materials by their chemical modification," J. Biomed. Mafer. Res., 20, 971-987 (1986). 12. Y. Ito, M. Sisido, and Y. Imanishi, "Platelet adhesion onto proteincoated and uncoated polyetherurethaneurea having tertiary amino groups in the substituents and its derivatives," J. Biomed. Muter. Res., 23, 191-206 (1989). 13. I.-K. Kang, Y. Ito, M. Sisido, and Y. Imanishi, "Serotonin and /3-thromboglobulin release reaction from platelet as triggered by interaction with polypeptide derivatives," J. Biomed. Muter. Res., 22, 595-611 (1988). 14. Y. Ito, M. Sisido, and Y. Imanishi, "Synthesis and antithrombogenicity of anionic polyurethanes and heparin-bound polyurethanes," J. Biomed. Muter. Res., 20, 1157-1177 (1986). 15. L. van der Does, J. G. F. Bots, and A. Bantjes, "Crosslinked polyether blends as biomaterials," Transactions of the 3rd World Biomaterials Congress, XI, 58 (1988). 16. S. C. Yoon and B. D. Ratner, "Surface structure of segmented poly(ether urethanes) and poly(ether urethane ureas) with various perfluoro chain extenders. An X-ray photoelectron spectroscopic investigation," Macromolecules, 19, 1068-1079 (1986). 17. M. J. Hearn, 8. D. Ratner, and D. Briggs, "SIMS and XPS studies of polyurethane surfaces. 1. Preliminary Studies," Macromolecules, 21, 2950-2959 (1988). 18. Y. Ito, M. Sisido, and Y. Imanishi, "Adsorption of plasma proteins to the derivatives of polyetherurethaneurea carrying tertiary amino groups in the side chains," J. Biomed. Muter. Res., 20, 1139-1155 (1986). 19. T. J. Lenk, B. D. Ratner, R. M. Gendreau, and K. K. Chittur, "IR spectral changes of bovine serum albumin upon surface adsorption," J. Biomed. Mater. Res., 23, 549-569 (1989).


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20. J. N. Lindon, G. McManama, L. Kushner, E. W. Merrill, and E. W. Salzman, "Does the conformation of adsorbed fibrinogen dictate platelet interactions with artificial surfaces?" Blood, 68, 355-362 (1986). 21. D. R. Absolom, W. Zingg, Z. Policova, and A. W. Neumann, "Determination of the surface tension of protein coated materials by means of the advancing solidication front technique," Trans. Amer. SOC.Artif. lnt. Organs, 29, 146-151 (1983). 22. L. I. Friedman, H. Liem, E. F. Grabowski, E. F. Leonard, and C. W. McCord, "Inconsequentiality of surface properties for initial platelet adhesion," Trans. Amer. SOC. Artif. lnt. Organs, 16, 63-73 (1970). 23. A. W. Neumann, M. A. Moscarollo, W. Zingg, 0.S. Hum, and C . K. Chang, "Platelet adhesion from human blood to bare and proteincoated polymer surfaces," J. Polym. Sci., Polym. Symp. Ed., 66, 429-441 (1979). 24. S. W. Kim and E. S. Lee, "The role of adsorbed proteins in platelet adhesion onto polymer surfaces," J. Polym. Sci., Polym. Symp. E d . , 66, 429-441 (1979). 25. Y. Ito, M. Sisido, and Y. Imanishi, "Attachment and proliferation of fibroblast cells on polyetherurethaneurea derivatives," Biomaferials, 8, 464-472 (1987). 26. A. Mori, Y. Ito, M. Sisido, and Y. Imanishi, "Interaction of polystyrene/ poly(y-benzyl L-glutamate) and poly(methy1 methacrylate)/poly(y-benzyl L-glutamate) block copolymers with plasma proteins and platelets," Biomaterials, 7, 386-392 (1986). 27. S. J. Whicher and J. L. Brash, "Platelet-foreign surface interactions: Release of granule constituents from adherent platelets," J. Biomed. Muter. Res., 12, 181-201 (1978).

Received October 7, 1986 Accepted October 19, 1989

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PEO triblock copolymers.

Platelets and blood vessels: the adhesion event.

Proteomic analysis of bone proteins adsorbed onto the surface of titanium dioxide.