Platelet Retention on Polymer Surfaces - Advances in Chemistry (ACS

4 Platelet Retention on Polymer Surfaces

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Some In Vitro Experiments E. W. MERRILL, E. W. SALZMAN, V. SA DA COSTA, D. BRIER-RUSSELL, A. DINCER, P. PAPE, and J. N. LINDON Massachusetts Institute of Technology, Department of Chemical Engineering, Cambridge, MA 02139 and Harvard Medical School and Beth Israel Hospital, Department of Surgery, Boston, MA 00215

Several polymers were evaluated in the form of a surface coat ing on glass beads packed in columns to determine their ability to retain platelets when whole human blood passes over the surface. This ability was measured as the platelet retention indexρ,the fraction of platelets retained on the column. Low est values of ρwere found for poly(ethylene oxide), poly(pro pylene oxide), poly(tetramethylene oxide) (in the form of polyurethanes), and polydimethylsiloxane. Highest values (around 0.8) were found for cross-linked poly(vinyl alcohol) and the copolymers of ethylenediamine with diisocyanates. Intermedi ate values were found for polystyrene and its copolymers with methyl acrylate, for polyacrylate, and for poly(methyl meth acrylate). The results are interpreted in terms of possible hy drophobic and hydrogen bonding interactions with plasma proteins.

T T n l i k e m e t a l l i c a n d c e r a m i c surfaces, p o l y m e r surfaces are m o b i l e (unless ^ v e r y h i g h l y cross-linked), a n d t h e i r m o l e c u l a r segments can rearrange side groups o r m a i n c h a i n groups i n response to the e n v i r o n m e n t (e.g., air, water) to m i n i m i z e free e n e r g y (I). P o l y m e r surfaces, exposed to b l o o d a n d b l o o d plasma, can f u n c t i o n like the stationary phase i n p a r t i t i o n l i q u i d chromatography, that is, the m o l e c u lar b l o o d elements (lipids, proteins) cannot o n l y adsorb o n , b u t may "dissolve i n t o " the surface layer o f the p o l y m e r . T h e arrangement o f m o l e c u l a r elements o f a p o l y m e r i c material at a b l o o d - p o l y m e r interface g e n e r a l l y is not k n o w n i n detail; x-ray photoelectron spectroscopy ( X P S , also c a l l e d E S C A ) indicates that for block c o p o l y m e r s , p o l y m e r s h a v i n g large side groups o f d i f f e r i n g polarity a n d polyelectrolytes, the surface c o m p o s i t i o n may b e q u i t e different f r o m the b u l k , stoichiometric c o m p o s i t i o n (2). 0065-2393/82/0199-0035$06.00/0 ® American Chemical Society Cooper et al.; 1982 Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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BIOMATERIALS: INTERFACIAL P H E N O M E N A A N D APPLICATIONS

T h i s c h a p t e r deals w i t h a specific test o f b l o o d - s u r f a c e interaction: i n vitro platelet r e t e n t i o n i n a c o l u m n o f beads (due to platelet adhesion a n d aggregation). P r o t e i n a d s o r p t i o n p r e c e d e s platelet adsorption, a n d thus the i n v i t r o platelet r e t e n t i o n test involves c o m p e t i t i v e a n d sequential adsorption of proteins, t h e o u t c o m e o f w h i c h p r o d u c e s surfaces having w i d e l y v a r y i n g degrees o f platelet r e t e n t i o n . E x c e p t i n t h e case o f t h r o m b i n (3), plasma p r o t e i n a b s o r p t i o n o n these surfaces has not b e e n s t u d i e d . W e h y p o t h e s i z e that the p l a s m a p r o t e i n molecules contain specific clusters of h y d r o p h o b i c g r o u p s , cationic groups, anionic groups, h y d r o g e n b o n d i n g groups, etc., over t h e i r surfaces that enable t h e m to recognize a n d adhere to the " f o r e i g n " surfaces, t h e r e b y r e c r u i t i n g platelets to adhere a n d aggregate u p o n contact. A c c o r d i n g to this hypothesis, h i g h surface concentration of certain p r o t e i n s , for e x a m p l e , f i b r i n o g e n o r f i b r o n e c t i n , or conformational change of the a d s o r b e d p r o t e i n c o u l d invoke platelet activity. T h e details o f these reactions a n d t h e i r relation to the i n vivo behavior o f p o l y m e r s r e m a i n to b e established.

Experimental Platelet Retention Test. The in vitro platelet retention assay device is shown in Figure 1 (4). Whole human blood, freshly drawn, citrated, and thermostatted to 37°C, was passed from the holding syringe by aliquots of 1 mL through columns packed with 0.2-mm diameter beads. The beads had been coated previously with the polymer to be tested. Each column offered a surface area of about 400 cm , with a void volume of about 0.5 mL. The platelet count was determined for the blood samples emerging, aliquot by aliquot, and was averaged for five aliquots and six different donors. This average, divided by the platelet count in the blood in the holding syringe, is the recovery index f, and from this the platelet retention index p = 1 — f, that is, the fraction of entering platelets retained on the column, was determined. Synthesis of Polymers. All polymers used in the study were synthesized in our laboratories, except for polyvinyl alcohol)(du Pont Elvanol 99.5% hydrolyzed). The acrylates and methacrylates were synthesized by batch polymerization in methyl ethyl ketone initiated by azobisisobutyronitrile. Molecular weights ranged between 60,000 and 150,000 (weight average). The copolymers of methyl acrylate and styrene and homopolymer polystyrene were synthesized at 60°C in solvent-free systems using benzoyl peroxide as initiator. Conversions were limited to 10% to maintain nearly constant composition of the polymer, and molecular weight averaged around 100,000 (weight average). Polymer composition (mole fraction of styrene) was determined by infrared spectroscopy and by material balance on residual monomer. Segmented polyurethanes containing polyethers were prepared by procedures given elsewhere (5). Aromatic polymers were prepared as alternating copolymers of 2,4-tolylene diisocyanate with ethylenediamine, and alternating copolymers of 4,4-diphenyl methyl diisocyanate with ethylenediamine (5, 6). Polymers synthesized in our laboratory were precipitated from the reaction medium and redissolved in appropriate solvent to remove initiator residues and oligomers. Polydimethylsiloxane (PDMS) was synthesized from the cyclic trimer hexamethyltrisiloxane "D3" in hexane using anionic ring-opening polymerization and dilithium stilbene as initiator (7). This material was terminated by chlorovinyldimethylsilane at a molecular weight M of 60,000. It was then precipitated from 2

w

Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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MERRILL ET AL.

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Platelet Retention on Polymer Surfaces

Figure 1. In vitro test for platelet retention, using plastic columns, packed with fine glass heads whose surfaces are thereafter coated with test polymer (ambient temperature = 37°C). hexane solution by alcohol, washed with alcohol-water to remove lithium chloride, and dried. Deposition of Polymer as a Test Surface. The test columns, packed with glass beads of 0.2-mm diameter to maximum density, were filled from below with a solution of the polymer, the concentration of which was adjusted (1 to 5 wt %) to insure that gravitational drainage in the subsequent step would be conveniently rapid. Drainage is the descent of the liquid-air meniscus through the packed column; it is "complete" when the meniscus leaves the lower end of the column. Obviously, the fluid film continued to flow down over the beads at a rate dependent on the solution viscosity, which in turn was a function of polymer concentration and molecular weight. For all polymers except polyvinyl alcohol), the column was then attached to a source of argon gas that provided a dust-free, nonoxidizing carrier into which the solvent could evaporate. As soon as the solvent started to evaporate, the viscosity of the film of polymer solution began to rise without limit, as polymer concentration increased, and thus drainage ceased. Upon completion of solvent evaporation, (hours to several days depending on volatility), the polymer was left as a continuous coating on the bead surfaces, joining them at their junctions. For each polymer the solvent was chosen to insure that the polymer film remained homogeneous as the solvent evaporated. In general, solvents of chromatographic grade purity were used to avoid adventitious contaminants in the polymer surface. For most polymers chloroform was used, but for the segmented polyurethanes, dimethylformamide was used. The final thickness of polymer coating on the bead was between about 0.1 and a few micrometers. Hydraulic resistance of the bed of beads after coating was substantially unchanged. (At most, a 10% increase in pressure drop for a fixedflowrate occurred.) Continuous coating of the glass surfaces, theoretically expected from the high critical surface tensions of acid-washed glass and the much lower surface tensions of the organic solution used, was confirmed by scanning electron microscopy combined with specific dye tests that would have revealed glass had it been exposed. Poly (vinyl alcohol) as a 5 wt % solution in water with 1% MgCl and 5% glutaraldehyde was coated onto the bead surfaces. After draining, the columns were heated to 60°C for 1 h, during which time the polyvinyl alcohol) was partially 2


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cross-linked via acetal bond formation. The columns were then washed exhaustively with water to remove the MgCl (the catalyst) and unreacted aldehyde. As a consequence, polyvinyl alcohol) was rendered insoluble and its crystallinity was reduced significantly. Upon conditioning in isotonic saline, prior to contact with blood, the polyvinyl alcohol) coatings swelled but remained integral and adherent to the bead surfaces. 2

Results Ten p o l y m e r s w e r e s t u d i e d i n respect to platelet retention, as s u m m a r i z e d i n F i g u r e 2. T h e first t h r e e , p o l y e t h e r s i n the f o r m o f the "soft s e g m e n t " o f segm e n t e d p o l y u r e t h a n e s , are d e s c r i b e d i n detail i n another chapter i n this v o l u m e (6), as is the n i n t h o f the list, i d e n t i f i e d as aromatic p o l y u r e a , an

PLATELET 0

(SPU)

POLYETHYLENE

OXIDE

POLYPROPYLENE

OXIDE

(SPU) P O L Y T E T R A M E T H Y L E N E

OXIDE

(SPU)

P O L YDIM E T H Y L

SIL0XANE

POLYMETHYL

ACRYLATE

POLYHEXYL

ACRYLATE

0.2

RETENTION

0.4

0.6

INDEX

0.8

1.0

j]

JJ]

PPP

in mzzm PPP

POLYMETHYL

METHACRYLATE PPP POLYSTYRENE

AROMATIC

POLYUREA

POLYVINYL

(SPU)

tzzzzzzzzzzzz rrr/77J77A

T

ALCOHOL

,

0 • NO

PLATELETS

RETAINED

ALL

PLATELETS

RETAINED

Figure 2. Composite display of average platelet retention index p for selected polymer types. The acrylates, hut not polystyrenes, are significantly passivated by pretreatment with plasma (n = ^6). Key: *, lowest value observed in each case; and PPP, preincubated in platelet-poor plasma.


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MERRILL ET AL.

analogue

39


of the " h a r d s e g m e n t " of the polyurethanes

that contain

the

polyethers. T h e acrylates (fifth a n d sixth i n the list of F i g u r e 2) are part of a series i n w h i c h the alkane side chain l e n g t h was v a r i e d over a range of 1 (methyl) to 16 (hexadecyl). T h e e x p e r i m e n t s are d e s c r i b e d c o m p l e t e l y elsewhere

(8),

and i n c l u d e a s i m i l a r series on the methacrylates one of w h i c h p o l y ( m e t h y l methacrylate) ( P M M A ) , is shown as seventh i n the list of F i g u r e 2. N o t r e p o r t e d e l s e w h e r e are results on p u r e P D M S (fourth i n F i g u r e 2), pure p o l y s t y r e n e (eighth i n F i g u r e 2), cross-linked polyvinyl alcohol) (tenth in F i g u r e 2), a n d c o p o l y m e r s of styrene w i t h m e t h y l aery late ( F i g u r e 3). W e tested the hypothesis that the platelet retention index s h o u l d i n crease as p o l y m e r c o m p o s i t i o n v a r i e d f r o m 100 m o l % m e t h y l acrylate to 100 m o l % styrene, the respective platelet retention indices p b e i n g about

0.25

and 0.55 for the h o m o p o l y m e r s . T h e results are shown i n F i g u r e 3, w h e r e i n p increases to a m a x i m u m near 4 0 % styrene. W h e n the surfaces were i n c u bated i n platelet-poor p l a s m a before contact w i t h w h o l e b l o o d , the values of p were m u c h r e d u c e d (from 0.25 to 0.05 for m e t h y l acrylate) for copolymers containing u p to 60 m o l % styrene. C o p o l y m e r s of h i g h e r styrene content were not r e n d e r e d significantly less retentive b y plasma

pretreatment.

X P S was u s e d to evaluate the surface c o m p o s i t i o n of these copolymers in relation to the b u l k c o m p o s i t i o n (fractions of m e t h y l acrylate and styrene). Surface c o m p o s i t i o n was essentially i d e n t i c a l to the b u l k c o m p o s i t i o n . T h u s , for example, w h e n the b u l k c o m p o s i t i o n is 40 m o l % styrene, the surface percentage of styrene is about 40. S u c h a surface appears to be as active as polystyrene h o m o p o l y m e r . O f the p o l y m e r s l i s t e d i n F i g u r e 2, cross-linked polyvinyl alcohol) was the least w e l l c h a r a c t e r i z e d . It swells to nearly twice its d r y v o l u m e w h e n i n e q u i l i b r i u m i n isotonic saline. A n u n d e t e r m i n e d fraction of its secondary h y d r o x y l groups is c o n v e r t e d to acetal linkages b y the d i a l d e h y d e .

Discussion T h e m o b i l i t y of groups attached to p o l y m e r segments i n response to interfacial conditions p r o b a b l y has m u c h to do w i t h the response of plasma proteins, and then platelets, to these surfaces. F o r example, H o f f m a n a n d Ratner d e s c r i b e d X P S spectra s h o w i n g that (9, 10) a c r y l a m i d e grafted to silicone r u b b e r by radiation w i l l p r e d o m i n a t e at the surface after e q u i libration i n water, but that d i m e t h y l s i l o x a n e w i l l p r e d o m i n a t e i n the surface if it has b e e n e q u i l i b r a t e d i n air. G r e g o n i s et al. (11) showed that even glassy P M M A , after l o n g exposure to water, w i l l change its contact angle (receding) from 80° to 20°, i n d i c a t i n g significant m o t i o n of side groups. T h u s , t i m e of water exposure p r i o r to c h a l l e n g i n g platelets is p r o b a b l y important, but the matter needs f u r t h e r study. O u r tests suggest that i n creasing the h y d r o p h o b i c side g r o u p length increases platelet retention; i n these experiments surfaces w e r e exposed to isotonic saline solution for less


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ALL 1.0 RETAINED

0.8

0.6

P 0.4

0.2 NONE RETAINED

0 0

0.2 MOL

0.4 FRACTION

0.6 0.8 STYRENE

1.0

Figure 3. Average platelet retention index p as a function of mole fraction of styrene in a series of copolymers of methyl acrylate and styrene. Key: —, control and —, pretreated in platelet-poor plasma. than 30 m i n p r i o r to testing. V e r y l o n g exposures to isotonic saline c o u l d result i n a significant rearrangement o f the ester linkage and alkane chain. P r e i n c u b a t i o n o f surfaces w i t h platelet-poor plasma substantially r e d u c e d the platelet retention index o f polyacrylates and polymethacrylates, but hardly altered the r e t e n t i o n index o f p o l y s t y r e n e o r c o p o l y m e r s w i t h m e t h y l acrylate that are r i c h i n styrene. E v e n w i t h o u t plasma pretreatment, the surface, w h e n exposed to w h o l e b l o o d , was p r o b a b l y first contacted b y m o l e c u l a r elements ( i n c l u d i n g the proteins) before the c e l l u l a r elements arrived. W h a t then is the m o d e o f action o f plasma pretreatment? Several hypotheses, none conclusive, can b e advanced, such as: (1) L i p i d adsorption d u r i n g i n c u b a t i o n alters the adsorptive capacity for certain proteins effective i n b i n d i n g platelets. (2) P l a t e l e t - b i n d i n g proteins desorb from the surface d u r i n g i n c u bation i n favor o f proteins that w h e n adsorbed d o not b i n d platelets. (3) T h e p o l y m e r surface itself, i n response to the l i p i d s and/or proteins to w h i c h it is exposed d u r i n g i n c u b a t i o n , undergoes t i m e - d e p e n d e n t conformational changes (hydrophobic side groups m o v e d to o r from the surface, for example) i n such a way that a layer o f proteins h a v i n g m o r e o r less affinity for platelets is achieved. T h e exchange o f proteins ( a l b u m i n , g l o b u l i n , and fibrinogen) and the t i m e course o f a d s o r p t i o n - d e s o r p t i o n f r o m p o l y m e r s have b e e n s t u d i e d b y


4.

MERRILL ET AL.

various researchers

41


(12-14). C o n t i n u a l transition of, or e v o l u t i o n of, the

surface u p o n p r o l o n g e d contact w i t h plasma, separate p r o t e i n solutions, or b l o o d appears to o c c u r often. T h e e v i d e n c e s u m m a r i z e d i n F i g u r e 2 shows lowest platelet r e t e n t i o n indices for poly(ethylene oxide) ( P E O ) , p o l y ( p r o p y l e n e oxide) ( P P O ) , and P D M S . T h i s result i m p l i e s l o w degrees of adsorption of specific critical plasma proteins and/or adsorption w i t h o u t conformation alteration of the p r o t e i n . T h e m o l e c u l a r attributes these three p o l y m e r s have i n c o m m o n appear to be: (1) A b s e n c e of i o n i c groups (2) A b s e n c e of h y d r o g e n atoms that can f o r m h y d r o g e n bonds (3) Relatively s m a l l n o n p o l a r groups (e.g., m e t h y l , - C H - ) 2

(4) Regular alternation of the n o n p o l a r groups w i t h a polar group (the siloxane a n d ether oxygens) (5) Segmental m o b i l i t y (at 37°C), that is, " l i q u i d - l i k e " m o t i o n of units and side groups (6) Surface activity: ( P D M S can be spread as a monolayer on water; P E O w i l l concentrate at an o i l - w a t e r interface). T h e s e observations indicate a n e e d to refine the m e a n i n g of h y d r o p h o b i c i t y i n c o n n e c t i o n w i t h p r o t e i n interaction a n d platelet r e t e n t i o n . Reports f r o m recent conferences sponsored b y the D e v i c e s a n d T e c h nologies B r a n c h of the N a t i o n a l H e a r t , L u n g , and B l o o d Institute (15, 16) indicate that silicone r u b b e r a n d s e g m e n t e d polyurethanes are a m o n g the most i n e r t materials s t u d i e d b y ex v i v o or i n vivo methods, whereas p o l y e t h y l e n e a n d p o l y t e t r a f l u r o e t h y l e n e are m o r e active. I n these latter two p o l y m e r s , no c o n f o r m a t i o n a l change of the surface can result that w i l l expose less h y d r o p h o b i c groups, whereas w i t h P E O , P P O , a n d P D M S , s u c h a result is i n e v i t a b l e , since h i g h c h a i n a n d segment m o b i l i t y occur i n the p o l y m e r s , p e r m i t t i n g response to the t h e r m o d y n a m i c r e q u i r e m e n t of m i n i m u m free energy easily a c h i e v e d b y c h a n g i n g oxygen for - C H or - C H - at the surface. 3

2

P o l y s t y r e n e beads are k n o w n for t h e i r capacity to b i n d i m m u n o g l o b i n G globulins nonspecifically, b y h y d r o p h o b i c interaction, so that the i m m u nospecificity of the F b r e g i o n is p r e s e r v e d . Because of the d i p o l a r nature of the p h e n y l r i n g , p o l y s t y r e n e p r o b a b l y represents a surface capable of extraordinary i n t e r a c t i o n w i t h h y d r o p h o b i c regions of proteins. a

T h e u n u s u a l p r o p e r t i e s of P E O [ i n c l u d i n g poly(ethylene glycol) ( P E G ) ] in m i n i m i z i n g a d s o r p t i o n w e r e d e s c r i b e d b y H i a t t et al. (17) w i t h respect to rabies v i r u s , a n d b y G e o r g e (18) w i t h respect to platelets. W h i c h e r and B r a s h (13, 14) n o t e d the l o w degree of a d s o r p t i o n b y s e g m e n t e d polyurethanes c o n t a i n i n g P E G of a l b u m i n or f i b r i n o g e n , a n d the l o w degree of platelet r e t e n t i o n o n these surfaces. W a s i e w s k i et al. (19) r e p o r t e d that adsorption of t h r o m b i n to glass can be p r e v e n t e d b y a d d i n g P E G o f 6 0 0 0 m o l e c u l a r w e i g h t .


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B e f o r e l o n g - t e r m h e m o c o m p a t i b i l i t y can b e expected for any m a t e r i a l , the nature o f p o l y m e r s u r f a c e - p r o t e i n int e ra c tion must b e established i n m o r e d e t a i l : t h e w a y i n w h i c h t h e p o l y m e r surface alters itself (rotation o f segments,

side g r o u p s , c h a i n r e f o l d i n g , etc.) i n response to the p r o t e i n

species; t h e way t h e p r o t e i n is a l t e r e d i n c o n f o r m a t i o n (if at all) u p o n a d sorption b y t h e surface; a n d h o w this conformational change provokes platelet r e t e n t i o n . O f course, l o n g e r - t e r m ex v i v o o r i n vivo studies also w i l l b e necessary.

Acknowledgments T h e s u p p o r t o f U n i t e d States P u b l i c H e a l t h Service u n d e r G r a n t H L 20079 is gratefully a c k n o w l e d g e d . J . J . W u s y n t h e s i z e d the P M M A a n d the polyacrylates. T h e c r o s s - l i n k e d polyvinyl alcohol) was p r e p a r e d b y J o h n F r e n z as part o f his M . S . thesis ( M I T , D e p a r t m e n t o f C h e m i c a l E n g i n e e r i n g , January 1980).

Literature Cited 1. Andrade, J. D.; Coleman, D. L.; Didisheim, P.; Hanson, S. R.; Mason, R.; Merrill, E. W. Trans. Am. Soc. Artif. Intern. Organs, in press. 2. Thomas, H. R.; O'Malley, J. Macromolecules 1979, 12, 323. 3. Sa da Costa, V.; Brier-Russell, D.; Trudell, G., III; Waugh, D. F.; Salzman, E. W.; Merrill, E. W. J. Colloid Interface Sci. 1980, 76, 594. 4. Lindon, J. N.; Rodvien, R.; Brier-Russell, D.; Greenberg, R.; Merrill, E. W.; Salzman, E. W. J. Lab. Clin. Med. 1978, 92, 904. 5. Sa da Costa, V.; Brier-Russell, D.; Salzman, E. W.; Merrill, E. W.J.Colloid Interface Sci. 1981, 80, 445. 6. Chapter 8 in this book. 7. Meyers, K. O.; Bye, M. C.; Merrill, E. W. Macromolecules 1980, 13, 1045. 8. Brier-Russell, D.; Salzman, E. W.; Lindon, J. N.; Handin, R.; Merrill, E. W.; Dincer, A. K.; Wu, J. S. J. Colloid Interface Sci. 1981, 81, 311. 9. Hoffman, A. S.; Ratner, B. D. In "Synthetic Biomedical Polymers," Szycher, M.; Robinson, W. J., Eds.; Technomic: Westport, CT, 1980; pp. 133-150. 10. Ratner, B. D. J. Biomed. Mater. Res. 1980, 14, 665. 11. Gregonis, D. E.; Smith, L. M.; Andrade, J. D. ACS, Div. Org. Coat. Plast. Chem., Prepr. (New York, August, 1981). 12. Cooper, S. L.; Ihlenfeld, J. V.; Mathis, T. R.; Barber, T. A.; Mosher, D. F.; Riddle, L. M.; Hart, A. P.; Updike, S. J. Trans. Am. Soc. Artif. Intern. Organs 1978, 24, 727. 13. Whicher, S. J.; Brash, J. L. J. Biomed. Mater. Res. 1978, 12, 181. 14. Brash, J. L.; Uniyal, S. J. Polym. Sci., Polym. Symp. 1979, 66, 377. 15. Contractors Meeting Proceedings, Devices and Technologies Branch, National Heart, Lung, and Blood Institute, (December 1979). 16. Ibid. (December 1980). 17. Hiatt, C. W.; Shelvkov, A.; Rosenthal, E. J.; Galimore, J. N.J.Chromatogr. 1971, 56, 362. 18. George, J. N. Blood 1972, 40, 862. 19. Wasiewski, W.; Fasco, M. J.; Martin, B. M.; Detwiler, J. C.; Fenton, J. W. Thromb. Res. 1976, 8, 881. RECEIVED for review January 16, 1981. ACCEPTED August 10,

1981.


Platelet Retention on Polymer Surfaces - Advances in Chemistry (ACS

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