Proteins at Interfaces - American Chemical Society

Chapter 10

Why Plasma Proteins Interact at Interfaces

Proteins at Interfaces Downloaded from pubs.acs.org by UNIV OF CALIFORNIA SANTA BARBARA on 04/08/19. For personal use only.

L. Vroman and A. L . Adams Interface Laboratory, Veterans Administration Medical Center, 800 Poly Place, Brooklyn, NY 11209

We found certain proteins able to deposit multimole cular layers between 2 surfaces separated at a dis ance inversely proportional to protein concentration. "Excess" deposited in wider gaps soon redissolves, perhaps by "self-removal". Blood plasma injected be tween a convex lens and a clot-promoting (glass or oxidized metal) surface leaves concentric rings of proteins. These apparently displace each other in the following sequence: albumin, immunoglobulin G, fibrin ogen and fibronectin, high molecular weight kininogen and factor XII - i.e. from most concentrated to least concentrated protein. Computer programs based on "self -removal" and physiological protein concentrations only, do yield realistic models; experiments with binary protein solutions indicate that more specific properties of each protein are involved in this interaction.

Each o f o u r p r o t e i n s e v o l v e d a l o n g w i t h t h e s t r u c t u r e s t h a t a r e r e p r o d u c i n g i t and a l o n g w i t h t h e m o b i l e c e l l membranes and subc e l l u l a r o b j e c t s t h a t i t must, from i t s b i r t h t o i t s d e a t h , e x i s t with. In sharp c o n t r a s t t o t h i n g s i n v i v o , most l a b o r a t o r y s u r f a c e s a r e c r e a t e d t o be a) u n i f o r m , so t h a t they w i l l a l l o w v e r y few c h o i c e s o f s c e n e r y f o r a p r o t e i n m o l e c u l e t o a t t a c h i t s e l f anywhere on them, and b) unchanging, r e m a i n i n g so r i g i d and so cons t a n t w i t h time t h a t they w i l l n o t adapt t o t h e s u b m o l e c u l a r des i r e s o f the p r o t e i n they a d s o r b : t h e p r o t e i n m o l e c u l e w i l l have to adapt t o t h e a r t i f i c i a l s u b s t r a t e i n s t e a d . Such l a r g e , u n i f o r m and uncompromising m a t e r i a l s u r f a c e s f o r c e t h e p r o t e i n s o l u t i o n t o d e p o s i t a measurably l a r g e number o f u n i f o r m l y a d a p t i n g m o l e c u l e s . T h e i r s y n c h r o n i z e d b e h a v i o r w i l l thus be a m p l i f i e d enough t o be measurable b u t may n o t r e p r e s e n t p h y s i o l o g i c a l e v e n t s . C e l l membranes have e v o l v e d t o be most u n l i k e any manmade s u r f a c e n o t o n l y by becoming v a r i a b l e and a d a p t a b l e on a m o l e c u l a r

This chapter not subject to US. copyright Published 1987 American Chemical Society

10. VROMAN AND ADAMS

Why Plasma Proteins Interact

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arid submolecular scale, but also by their becoming dotted with r e ceptors for s p e c i f i c proteins. Thus, l i v i n g c e l l s may adsorb and r e t a i n only s p e c i f i c proteins out of their physiological l i q u i d environment and only under very s p e c i f i c conditions. Caught within the r e l a t i v e l y dense structure of a c e l l , or i n the varying and often narrow spaces among c e l l s , a protein molecule must be expected to encounter a number of situations, each of which deserves to be imitated with simplifying models i n the laboratory. Limiting ourselves to 2 proteins (PI and P2), and 2 kinds of surfaces (Sa and Sb), we may l i s t the following choice of possible interactions (commas separating non-preadsorbed proteins). 1) PI, Sa and PI, PISa 2) PI, P2Sa (where P2 i s adsorbed onto Sa) 3) PI, P2, Sa (competition for 1 surface) 4) Sa, PI, Sa (1 protein i n a narrow space between i d e n t i c a l surfaces) 5) Sa, PI, P2, Sa (2 proteins competing i n a narrow space between i d e n t i c a l surfaces) 6) Sa, PI, Sb (2 d i f f e r e n t surfaces competing for 1 protein) 7) Sa, PI, P2, Sb 8) SaPl, SaPl (interaction among 2 mobile i d e n t i c a l p a r t i c l e s coated with the same protein) 9) SaPl, SaP2 10) SaPl, SbPl (interaction among 2 surfaces carrying the same protein) 11) SaPl, SbP2 12) SaPl, P2, SbPl Somewhat more physiological conditions can be created by r e placing at least 1 of the 2 proteins by blood plasma. The a b i l i t y of a surface to adsorb a certain protein, e.g. a c l o t t i n g factor, out of normal plasma, appears to be demonstrated by the following deceptively simple manipulations. Incubate normal citrated " i n t a c t " plasma (plasma that had never been i n contact with a negatively charged, high free energy surface such as glass) i n a glass test tube. Rinse the tube out and place citrated plasma i n i t from a patient who lacks c l o t t i n g factor XII. After adding calcium chloride, we w i l l find the c l o t t i n g time of this d e f i c i e n t plasma to be corrected by the adsorabte that the normal plasma had l e f t on the glass (1). We cannot help but conclude that normal plasma deposits factor XII on glass. Finding one protein deposited by plasma should not lead us to believe - as I unfortunately did for many years - that no other protein w i l l be present at other locations on the surface at the same time, or at the same location at other times. Having detected factor XII at coagulation-activating surfaces, i t took us 5 years plus Dr. S. Witte's suggestion before we started looking f o r f i b r i n ogen being deposited by plasma. We did find i t , and another 12 years l a t e r we found evidence of an entire s h i f t i n g population of proteins replacing one another at the solid/plasma interface. In the following description of early studies as well as of our recent findings, I w i l l use the c l a s s i f i c a t i o n of protein/surface interactions given above. Among the proteins to be discussed fibrinogen w i l l be prominent, not only because of our own work, but also because this work f i t s rather well within the context of others' studies showing protein, plasma and c e l l u l a r (platelet) interactions.

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ONE P R O T E I N ON ONE SURFACE a) The a i r i n t e r f a c e Most p r o t e i n s o l u t i o n s tend t o form a monomolecular l a y e r a t t h e i r a i r / l i q u i d i n t e r f a c e a t a r a t e depending on t h e i r c o n c e n t r a t i o n . A p o l a r a m i n o a c i d r e s i d u e s w i l l become e x p o s e d o n t h e s u r f a c e of t h e f i l m t h a t f a c e s a i r . T h e r e f o r e , one c a n i m a g i n e t h a t a drop of p r o t e i n s o l u t i o n suspended i n a i r w i l l r a p i d l y form a s k i n i n w h i c h i t w i l l be t r a p p e d . I f p l a c e d onto a hydrophobic s o l i d , such a drop w i l l be h e l d by i t s hydrophobic s k i n s u r f a c e , and where i t i s f o r c e d t o r o l l a l o n g , t h e drop w i l l "break o u t o f i t s bag" o f s k i n , l e a v i n g p i e c e s o f s k i n s t u c k i n i t s t r a i l w h i l e f o r m i n g a new b a g . We d e t e c t e d t h e p h e n o m e n o n e . g . b y o b s e r v i n g t h e i n t e r f e r e n c e c o l o r p a t t e r n l e f t by p r o t e i n d r o p s l a n d i n g on and r o l l i n g o v e r f e r r i c stéarate p o l i s h e d a n o d i z e d t a n t a l u m s l i d e s . This Blodgett-Langmuir t r a n s f e r i s almost instantaneous (30). To o b s e r v e a d s o r p t i o n f r o m b u l k s o l u t i o n , we m u s t i n s e r t t h e h y d r o p h o b i c s u b s t r a t e i n t o t h e a q u e o u s medium b e f o r e a d d i n g p r o t e i n . Under s u c h c o n d i t i o n s , t h e r e c o r d i n g e l l i p s o m e t e r shows a d s o r p t i o n r a t e s o u t o f b u l k s o l u t i o n s onto hydrophobic s u b s t r a t e s that a r e n o t u n l i k e t h o s e o n h y d r o p h i l i c o n e s ( 2 ) . Where t h e h y d r o p h o b i c s o l i d s l i d e i s then d i p p e d deeper i n t o t h e c o n t a i n e r o f p r o t e i n , d a t a and r e c o r d i n g s show t h a t t h e s l i d e h a s g a i n e d t h i c k n e s s i n s t a n t a n e o u s l y a n d m u s t t h e r e f o r e h a v e d r a g g e d a s u r f a c e f i l m down i n t o t h e l i g h t path. T h i s f i l m may a p p e a r n o t much t h i n n e r t h a n t h e o n e t h a t h a d b e e n more s l o w l y a d s o r b e d o u t o f t h e b u l k s o l u t i o n ( 2 ) . P r o t e i n s adsorbed onto a hydrophobic s o l i d tend t o render i t m o r e h y d r o p h i l i c , w h i l e t h e same p r o t e i n s a d s o r b e d o n t a h y d r o p h i l i c s o l i d t e n d t o r e n d e r i t more h y d r o p h o b i c . On t h e d r i e d f i l m o f a d sorbate, the contact angle of a water d r o p l e t , o r the s c a t t e r i n g of l i g h t by condensing water vapor, appears t o demonstrate t h i s prop e r t y ( 3 ) , b u t t h e shape o f these s l o w l y s p r e a d i n g d r o p l e t s i s a f f e c t e d by p r o t e i n b e i n g "scooped up" a t t h e i r advancing a i r / w a t e r / solid interfaces (4). Among p l a s m a p r o t e i n s we s t u d i e d a t s o l u t i o n / a i r i n t e r f a c e s , f i b r i n o g e n behaved most r e m a r k a b l y . Compressed i n a s u r f a c e f i l m b a l a n c e ( 5 ) , f i b r i n o g e n tended t o form a c o h e s i v e f i l m t h a t c o u l d be l i f t e d o f f t h e i n t e r f a c e w i t h a h y d r o p h o b i c s l i d e ( 6 ) . The f i n d i n g s u g g e s t s t h a t f i b r i n o g e n may f o r m p o l y m e r - l i k e c o m p l e x e s a t c e r t a i n i n t e r f a c e s e v e n i n a b s e n c e o f t h r o m b i n ( 7 ) , t h e enzyme n o r m a l l y needed t o p o l y m e r i z e f i b r i n o g e n i n t o f i b r i n . b ) One p r o t e i n a t t h e l i q u i d / s o l i d i n t e r f a c e ( P I , S a a n d P I , P I S a ) T h o u g h we r e a l i z e d e a r l y ( 2 ) t h a t a d s o r p t i o n o f p r o t e i n s may b e l e s s r e v e r s i b l e t h a n we h a d e x p e c t e d , o t h e r t e c h n i q u e s t h a n o u r s w e r e r e q u i r e d t o show t h a t a t l e a s t f i b r i n o g e n a d s o r b e d o n t o c e r t a i n s u r f a c e s (8) w i l l be exchanged w i t h f i b r i n o g e n m o l e c u l e s i n s o l u t i o n Since e l u t a b i l i t y appears t o decrease w i t h increased time o f r e s i d e n c e a t t h e i n t e r f a c e ( 9 ) , some g r a d u a l c h a n g e i n c o n f o r m a t i o n may occur as t h e adsorbed molecule perhaps i n c r e a s e s i t s a r e a o f c o n t a c t w i t h t h e immobile s u b s t r a t e . We h a v e some e v i d e n c e t h a t f i b r i n o g e n , a d s o r b e d o n t o c e r t a i n s u b s t r a t e s , w i l l b e a b l e t o a d s o r b a mono-

10.

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c l o n a l a n t i b o d y t o p a r t o f i t s Ε d o m a i n , w h i l e t h e same a n t i b o d y does not b i n d f i b r i n o g e n i n s o l u t i o n . This i n d i c a t e s that adsorp t i o n can expose a group o f amino a c i d s t h a t i s n o r m a l l y not exposed i n s o l u t i o n (10). ONE

P R O T E I N ON TWO I D E N T I C A L SURFACES ( S a P l , F l , S a e t c . )

The f a c t t h a t a f i l m o f p r o t e i n a d s o r b e d o n t o 1 s u r f a c e c a n still adhere t o another s u r f a c e i s proved simply by e x p o s i n g the f i l m t o a s u s p e n s i o n o f s o l i d p a r t i c l e s , e.g. o f a m e t a l o x i d e s u s p e n s i o n . Many p r o t e i n f i l m s a r e i n t e n s e l y " s t a i n e d " ( c o a t e d ) b y c h r o m i u m o x i d e o r i r o n o x i d e s u s p e n s i o n s (2, 29). We s t u d i e d t h e e f f e c t o f a much w i d e r s p a c e b e t w e e n t w o i d e n t i c a l s u r f a c e s upon a d s o r p t i o n o f a p r o t e i n a s f o l l o w s . We p l a c e d a p l a n a r - c o n v e x l e n s ( r a d i u s o f c u r v a t u r e between about 150 and 2 0 0 0 mm) o n a g l a s s s l i d e , a n d i n j e c t e d a s o l u t i o n o f t h e p r o t e i n b e t w e e n them. A f t e r i n c u b a t i o n a t room t e m p e r a t u r e f o r a b o u t 10 m i n , we t i l t e d t h e s l i d e a n d r i n s e d i t w i t h b u f f e r w h i l e t h e l e n s was a l l o w e d t o s l i p o f f . The d e n s i t y d i s t r i b u t i o n p a t t e r n o f a d s o r b e d p r o t e i n c o u l d b e ( b u t o f t e n need not be) e n h a n c e d b y e x p o s u r e t o a m a t c h i n g a n t i s e r u m ; a f t e r r i n s i n g a g a i n the s l i d e was u s u a l l y s t a i n e d w i t h c o n c e n t r a t e d Coomassie Blue. Under t h e s e c o n d i t i o n s albumin, c e r t a i n preparations o f p u r i f i e d f i b r i n o g e n anda l l prepar a t i o n s o f p u r i f i e d i m m u n o g l o b u l i n G ( I g G ) we t e s t e d , d e p o s i t e d not o n l y a p r e s u m a b l y m o n o m o l e c u l a r l a y e r , b u t a l s o a r i n g o f much g r e a t e r t h i c k n e s s a t a d i s t a n c e from the l e n s c e n t e r c o r r e s p o n d i n g t o a n a b o u t 10 t o 20 m i c r o n s t h i c k n e s s o f l i q u i d f o r s o l u t i o n s c o n t a i n i n g a b o u t 8 mg/ml o f t h e p r o t e i n ( 1 1 ) . More d i l u t e d s o l u t i o n s deposited rings o f greater diameter. D i r e c t observation o f IgG i n j e c t e d as described, and s e r i a l experiments i n t e r r u p t e d from 1 t o about 4 sec a f t e r i n j e c t i o n , s h o w e d t h a t a t h i c k l a y e r i s d e p o s i t e d o u t o f a n a p p r o x i m a t e l y 8 mg/ m l b u f f e r s o l u t i o n (pH 7.4) w i t h i n a b o u t 2 t o 3 s e c e x c e p t n e a r t h e c e n t e r , w h e r e t h e l i q u i d l a y e r i s s o t h i n t h a t t h e amount o f I g G per surface area i s b a r e l y s u f f i c i e n t t o deposit a monolayer and i s then depleted. T h i s c r e a t e s the " h o l e i n the doughnut". Ati t s immediate p e r i p h e r y , the l i q u i d l a y e r i s t h i c k enough t o s u p p l y t h e amount o f p r o t e i n n e e d e d f o r " e x c e s s " d e p o s i t i o n o f t h e t h i c k r i n g , a t w h i c h p o i n t the l i q u i d above t h i s a r e a i s a l s o d e p l e t e d and the thick ring i s stabilized. More p e r i p h e r a l l y , a d d i t i o n a l p r o t e i n m o l e c u l e s are a v a i l a b l e t o c o l l i d e w i t h the t h i c k d e p o s i t and desorb the excess. I t s h o u l d be noted that the c o n d i t i o n s r e q u i r e d t o y i e l d a rather stable multimolecular l a y e r are s t r i c t l y l i m i t e d t o r e l a t i v e l y high concentrations o f p r o t e i n i n spaces over a narrow r a n g e ( e . g . more t h a t 10 a n d l e s s t h a n 20 m i c r o n s f o r s o l u t i o n s o f a b o u t 5 mg/ml f o r a t l e a s t s e v e r a l p r o t e i n s ) . These f i n d i n g s would support those o f o t h e r s t h a t p r o t e i n s such as f i b r i n o g e n (12) a n d p h o s p h o r y l a s e b (13) can i n t e r c h a n g e a d s o r b e d molecules w i t h those i n s o l u t i o n even though, f a c i n g a p r o t e i n - f r e e s o l u t i o n , the a d s o r b e d m o l e c u l e s do not d e s o r b s p o n t a n e o u s l y . TWO P R O T E I N S ON ONE SURFACE a)

One p r o t e i n p r e a d s o r b e d

Turnit

demonstrated

that

(SaPl, P2)

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i s s u i t a b l e f o r showing

the

158


i n t e r a c t i o n s o f a p r e a d s o r b e d enzyme w i t h a s o l u t i o n o f i t s p r o t e i n s u b s t r a t e ( 1 3 ) . We f o u n d t h a t p r e a d s o r b e d t h r o m b i n w i l l a d s o r b w h a t appears t o be a monomolecular l a y e r o f f i b r i n o g e n o u t o f f i b r i n o g e n s o l u t i o n , b u t w i l l c o n t i n u e t o c r e a t e f i b r i n monomer s o t h a t t h e s o l u t i o n w i l l c l o t ( 2 ) , m a i n l y o r most r a p i d l y a t t h e s i t e o f a d s o r b ed t h r o m b i n . A n t i b o d i e s a r e d e p o s i t e d out of s o l u t i o n onto preadsorbed a n t i gens a s p a r t o f c o u n t l e s s r o u t i n e s , i n c l u d i n g e l l i p s o m e t r y , i n meth ods i n t e n d e d t o i d e n t i f y t h e p r e a d s o r b e d a n t i g e n o r t o q u a n t i t a t e antibody potency o r antigen concentration. Where a n t i g e n i s m o r e densely packed than t h e d e t e c t i n g immunoglobulin can be, t h e p r o p o r t i o n a l i t y between s u r f a c e c o n c e n t r a t i o n o f a n t i g e n and i t s a b i l i t y to adsorb antibody i s disturbed. I n general, preadsorbed antigens r e t a i n t h e i r a b i l i t y t o a d s o r b p o l y c l o n a l a n t i b o d i e s , a n d we r e l y o n t h i s a b i l i t y r o u t i n e l y (4, 11, 14). In our experience, unless the preadsorbed p r o t e i n has a s p e c i f i c (enzyme/substrate, antigen/antibody) r e l a t i o n s h i p with the d i s s o l v e d o n e , t h e p r e a d s o r b e d p r o t e i n w i l l a d s o r b l i t t l e o r no other protein. Where t h e e l l i p s o m e t e r shows i n c r e a s e d o p t i c a l f i l m t h i c k n e s s upon exposure o f , e . g . , a p r e a d s o r b e d l a y e r o f a l b u min t o f i b r i n o g e n , such i n t e r a c t i o n s o c c u r a t t h e expense o f t h e a b i l i t y o f the preadsorbed albumin t o adsorb antibody t o albumin (14). T h i s i n d i c a t e s t h a t exchange - i n t h i s case by t h e l a r g e r species - took place. A n o t a b l e e x c e p t i o n may b e t h e a b i l i t y o f plasma t o d e p o s i t a thus f a r u n i d e n t i f i e d p r o t e i n onto preadsorbed i m m u n o g l o b u l i n s ( I g G ) ( 1 5 ) , b u t h e r e t o o , some s p e c i f i c i n t e r a c t i o n s may t a k e p l a c e . O u t o f t h e h u n d r e d s o f p r o t e i n s t h a t o u r p l a s m a c o n t a i n s , we must e x p e c t t h a t f o r a random c h o i c e o f most p a i r s , t h e d i s s o l v e d species of protein facing a surface completely occupied with the p r e a d s o r b e d s p e c i e s o f p r o t e i n , c a n o n l y be d e p o s i t e d by d i s p l a c i n g the preadsorbed species. Many e x p e r i m e n t s h a v e b e e n p u b l i s h e d t o s h o w how o n e p r o t e i n c a n " i n h i b i t " s u r f a c e a c t i v a t i o n o f a c l o t t i n g f a c t o r such as h i g h molecular weight k i n i n o g e n o r f a c t o r X I I , w h i l e i n f a c t t h e s u r f a c e i s merely b l o c k e d n o n s p e c i f i c a l l y by preadsorption of a protein that i snot easily displaced. b) B o t h p r o t e i n s d i s s o l v e d i n i t i a l l y ( S a , P I , P 2 ) . In s e v e r a l s e r i e s of binary mixtures, r e l a t i v e c o n c e n t r a t i o n o f e a c h p r o t e i n i n s o l u t i o n d e t e r m i n e d w h i c h was a d s o r b e d most ( 1 5 ) , but r e l a t i o n s h i p s were n o t s i m p l y p r e d i c t a b l e . F o r example, judged by o p t i c a l t h i c k n e s s o f d e p o s i t e d a n t i b o d y , a d s o r p t i o n o f f i b r i n o g e n i n p r e s e n c e o f I g G r a p i d l y r o s e t o i t s maximum a s t h e r e l a t i v e c o n c e n t r a t i o n o f f i b r i n o g e n was i n c r e a s e d , w h i l e i n p r e s e n c e o f a l b u m i n f i b r i n o g e n a d s o r p t i o n increased almost l i n e a r l y as t h e p r o p o r t i o n o f f i b r i n o g e n t o a l b u m i n i n s o l u t i o n was i n c r e a s e d ( 1 5 ) . Those o f u s v e n t u r i n g o v e r t h e edge o f p u r e p r o t e i n s t u d i e s i n t o t h e f i e l d o f l i f e by s t u d y i n g more t h a n one p r o t e i n a t a t i m e , w i l l soon face c r i t i c i s m from workers i n both f i e l d s : that o u r s y s t e m s a r e n o t s i m p l e enough t o a p p l y known l a w s o f a d s o r p t i o n , and t h a t t h e y a r e n o t complex enough t o m i m i c t h e b e h a v i o r o f t h e same p r o t e i n s i n p r e s e n c e o f b l o o d o r b l o o d p l a s m a . Most work p u b l i s h e d r e c e n t l y b y o t h e r s , some o f w h i c h i s i n c l u d e d i n t h i s

10.

VROMAN AND ADAMS

159


i s s u e , u s e s more s o p h i s t i c a t e d t o o l s a n d a n a l y s e s t h a n o u r s , a l l o w i n g a view o f i n c r e a s i n g l y submolecular d e t a i l and r e v e a l i n g more and more i n d i v i d u a l d i f f e r e n c e s i n r a t e a n d e x t e n t o f c o n f o r m a t i o n changes upon a d s o r p t i o n and i n t e r a c t i o n . ONE

OR TWO PROTEINS ON TWO SURFACES

(SaPl, SbPl

and SaPl,

SbP2)

We f o u n d t h a t o n e s u r f a c e ( S a ) s u c h a s F e 2 0 3 p o w d e r w i l l a d h e r e t o f i b r i n o g e n t h a t i s adsorbed out o f a s o l u t i o n onto a second s u r f a c e (Sb) s u c h a s a g l a s s s l i d e . Fibrinogen adsorbed onto a f l a t surface e v e n c o a t s i t s e l f t o some d e g r e e w h e n t h e c o a t i n g p o w d e r a l s o h a d been coated w i t h e i t h e r f i b r i n o g e n o r another p r o t e i n . The powders we t e s t e d ( i r o n o x i d e s , c h r o m i u m o x i d e a n d u l t r a m a r i n e ) a d h e r e d m o s t to surfaces c a r r y i n g f i b r i n o g e n , l e s s t o those c a r r y i n g IgG and l e a s t t o those c a r r y i n g albumin, and t h i s a b i l i t y t o coat surfaces appeared t o be reduced by c o a t i n g the p a r t i c l e s w i t h a p r o t e i n . Though t h e r e i s a p o s s i b i l i t y t h a t the p r o t e i n a d s o r b e d o n one s u r f a c e "sees through" the p r o t e i n c o a t i n g o f the other s u r f a c e , i t seems more l i k e l y t h a t i t s e e s t h r o u g h h o l e s i n t h e i m p e r f e c t coat ing. H o l e s may a f f e c t s t u d i e s o f S a P l , S a P l i n t e r a c t i o n s ( 1 6 ) , a n d cause aggregation o f p a r t i c l e s w h i l e being coated (17). V e r y s i m p l e e x p e r i m e n t s a r e a b l e t o show t h e r a t h e r c o m p l e x e v e n t s o c c u r r i n g when a p r o t e i n i s e x p o s e d t o 2 s u r f a c e s a t o n c e . F o r e x a m p l e , when f i b r i n o g e n s o l u t i o n i s i n t r o d u c e d t o a g l a s s t e s t tube c o n t i n i n g an i r o n oxide suspension, the s u s p e n s i o n w i l l tend to aggregate andboth s i n g l e andaggregated p a r t i c l e s w i l l adhere t o the w a l l . U s i n g o u r n o t a t i o n s ( S a r e p r e s e n t i n g t h e p a r t i c l e s , Sb t h e t e s t t u b e w a l l , a n d P I t h e f i b r i n o g e n ) , we may a s s u m e t h e f o l lowing i n t e r a c t i o n s take place. S a , S b , P I -> S a P l , S b P l , P I -> SaPISb (Sa a d h e r e s t o P I S b a n d S a P l a d h e r e s t o S b ) , S a P l S a ( p a r t i c l e s c l u m p ) -> SaPlSaPlSb

(clumps adhere t o p r o t e i n - c o a t e d

t e s t tube w a l l ,

PISb).

PLASMA AT I N T E R F A C E S As we e x p a n d o u r o b s e r v a t i o n s f r o m t h e b e h a v i o r o f a few p r o t e i n s t o t h a t o f p l a s m a , we may b e g i n t o f e e l j u s t i f i e d i n a s k i n g w h y t h e s y s t e m p e r f o r m s r a t h e r t h a n how i t d o e s . T h e q u e s t i o n "Why d o plasma p r o t e i n s i n t e r a c t a t i n t e r f a c e s ? " can t h e n be i n t e r p r e t e d t o mean: " w h a t a s p e c t s o f t h e b e h a v i o r a n d i n t e r a c t i o n s among p u r i f i e d plasma p r o t e i n s can be seen a s w e l l i n t h e i r n a t u r a l h a b i t a t - t h e plasma -, a n d can t h e s e a s p e c t s be ' e x p l a i n e d ' a s b e i n g b e n e f i c i a l t o o u r s u r v i v a l ? " What t h u s f a r h a d a p p e a r e d a s s e n s e l e s s l y com p l e x b e h a v i o r o f p u r i f i e d p r o t e i n s a t i n t e r f a c e s may b e c o m e m o r e r e a s o n a b l e i n t h e c o n t e x t o f many p l a s m a p r o t e i n s i n t e r a c t i n g a t t h e m o t t l e d s u r f a c e s o f c e l l s i n a way t h a t w i l l a l l o w the s u r v i v a l of t h e i r host. M e a n w h i l e , we w i l l d i s c o v e r t h a t p u r i f i e d p r o t e i n s behave u n l i k e t h e i r s i b b l i n g s i n v i v o and that i n the eyes o f o u r plasma a p u r i f i e d p r o t e i n adsorbed out o f an a r t i f i c i a l s o l u t i o n w i l l n o t l o o k l i k e a f i l m o f t h e same p r o t e i n d e p o s i t e d b y t h e plasma i t s e l f .


160 a)

P l a s m a a t one i n t e r f a c e .

Our own s t u d i e s o f human c i t r a t e d i n t a c t p l a s m a a t a c t i v a t i n g surfaces ( g l a s s , anodized tantalum, o x i d i z e d s i l i c o n ) r e s u l t e d i n the f o l l o w i n g f i n d i n g s . I n t h e e l l i p s o m e t e r , p l a s m a e v e n when d i l u t e d a b o u t 1 5 0 - f o l d , deposits immunologically i d e n t i f i a b l e fibrinogen but the f i l m loses i t s a b i l i t y t o adsorb antibody t o f i b r i n o g e n w i t h i n minutes under these c o n d i t i o n s . The l o s s i s p r o b a b l y n o t caused by t h e p l a s m a ' s p r o t e o l y t i c a c t i v i t y ( 1 8 ) a n d o c c u r s m o r e s l o w l y a t 10C t h a n a t 37C ( 1 9 ) . A t room t e m p e r a t u r e , u n d i l u t e d p l a s m a d e p o s i t s f i b r i n o g e n w i t h i n a b o u t 2 s e c a n d t h e l o s s o c c u r s w i t h i n 30 s e c . M o r e r e c e n t l y , we f o u n d t h a t t h e f i b r i n o g e n i s r e p l a c e d b y h i g h m o l e c u l a r w e i g h t k i n i n o g e n (HMK) ( 2 0 , 2 1 ) . S t u d i e s w i t h r a d i o l a b e l e d f i b r i n o g e n rather than w i t h a n t i b o d i e s t o f i b r i n o g e n r e c e n t l y c o n f i r m e d t h a t plasma d e p o s i t s f i b r i n o g e n and t h e n removes i t ( 2 2 , 2 3 ) . We f o u n d t h e e v e n t d o e s n o t o c c u r o n v a r i o u s h y d r o phobic s u r f a c e s : o t h e r s found i t d i d ( 2 4 ) . These experiments imply t h a t f i b r i n o g e n , a p r o t e i n present a t a c o n c e n t r a t i o n o f a b o u t 2 t o 3 mg p e r m l p l a s m a , i s d e p o s i t e d a n d t h e n r e p l a c e d b y a p r o t e i n (HMK) p r e s e n t a t a c o n c e n t r a t i o n about 2 orders o f magnitude l e s s . The p o s s i b l e s i g n i f i c a n c e o f t r a c e p r o t e i n s and t h e i r i n a b i l i t y t o cover o r d i s p l a c e c o a t i n g s o f r e l a t i v e l y abundant p r o t e i n s i n narrow spaces i s d i s c u s s e d below. b)

P l a s m a between two s u r f a c e s . In spaces between a c t i v a t i n g s u r f a c e s t h a t a r e l e s s than about 20 m i c r o n s b u t m o r e t h a n a b o u t 3 m i c r o n s a p a r t , n o r m a l i n t a c t p l a s m a w i l l s t i l l c o n t a i n enough f i b r i n o g e n t o c r e a t e a c o n f l u e n t c a r p e t , b u t w i l l l a c k t h e c o n c e n t r a t i o n o f HMK p e r u n i t o f s u r f a c e a r e a t o displace this fibrinogen film. A s a r e s u l t , t h e p l a s m a when i n j e c t e d between a f l a t a c t i v a t i n g s u r f a c e and a convex l e n s r e s t i n g b e l l y - d o w n upon t h e s u r f a c e w i l l l e a v e a d i s k o f f i b r i n o g e n where t h e p l a s m a o c c u p i e s a s p a c e o f a b o u t 3 t o 20 uM w i d e ( 2 5 ) . Dilu t i o n o f plasma causes i t t o leave a correspondingly l a r g e r d i s c of f i b r i n o g e n ( r e q u i r i n g a t h i c k e r l a y e r o f plasma t o c o n t a i n enough HMK p e r s u r f a c e a r e a ) w i t h a c e n t r a l h o l e t h a t c o n t a i n s o t h e r p r o teins (26). M o r e d e t a i l e d s t u d i e s ( u s i n g l e n s e s o f a b o u t 147mm r a d i u s o f curvature) indicated that a s e tof concentric rings of proteins i s l e f t by plasma under these c o n d i t i o n s . D i l u t e d t o a b o u t .02 t o .01% i t w i l l l e a v e a l b u m i n c e n t r a l l y , s u r r o u n d e d by i m m u n o g l o b u l i n s ( I g G ) : a t a b o u t 1%, i t w i l l l e a v e a c e n t r a l d i s k o f I g G s u r r o u n d e d by f i b r o n e c t i n a n d f i b r i n o g e n , w h i l e a t a b o u t 2 0 % , i t w i l l l e a v e a s o l i d d i s c o f f i b r i n g o e n w i t h a c e n t r a l h o l e c o n t a i n i n g IgG s u r rounded by a f i n e l i n e o f f i b r o n e c t i n ( 1 1 ) . c)

Plasma a t a p r o t e i n - c o a t e d i n t e r f a c e An o v e r v i e w o f o u r d a t a o n t h e e f f e c t o f p l a s m a o n p r e - a d s o r b e d f i b r i n o g e n ( e . g . 1 4 , 1 5 , 28) shows i n t a c t r a t h e r t h a n a c t i v a t e d plasma i s a b l e t o d i s p l a c e preadsorbed f i b r i n o g e n , b u t under c e r t a i n c o n d i t i o n s (degree o f p a c k i n g ? ) p r e - a d s o r b e d f i b r i n o g e n i s removed more s l o w l y t h a n t h e f i b r i n o g e n t h a t h a d b e e n d e p o s i t e d by t h e plasma i t s e l f . M o s t r e m a r k a b l e w e r e t h e i n t e r a c t i o n s we f o u n d o c c u r r i n g w h e n p l a s m a came i n c o n t a c t w i t h I g G p r e a d s o r b e d o n t o a w e t t a b l e ( o x i -

10.

VROMAN AND ADAMS


161

d i z e d s i l i c o n c r y s t a l ) s u r f a c e i n t h e e l l i p s o m e t e r . The plasma d e p o s i t e d a l a y e r about a s o p t i c a l l y t h i c k a s was t h e o r i g i n a l I g G f i l m ; t h e n , i f t h e p l a s m a was i n t a c t , i t w o u l d remove more t h a n t h e amount i t h a d d e p o s i t e d ( 1 5 ) . The m a t t e r d e p o s i t e d was n o t removed i f t h e plasma had been a c t i v a t e d , and c o u l d n o t be d i s t i n g u i s h e d i m m u n o l o g i c a l l y f r o m t h e u n d e r l y i n g I g G . I n r e t r o s p e c t , we b e l i e v e t h e e v e n t may b e r e l a t e d t o t h e d e p o s i t i o n o f I g G o n t o p o f I g G t h a t we o b s e r v e d i n o u r l e n s - o n - s l i d e e x p e r i m e n t s d e s c r i b e d a b o v e : i n p r e s e n c e o f s u f f i c i e n t I g G , i t removed i t s e l f . This would n o t e x p l a i n why a c t i v a t e d p l a s m a w a s u n a b l e t o r e m o v e i t s own d e p o s i t f r o m the IgG s u b s t r a t e . d.

More complex systems i n v o l v i n g plasma When c o n t a c t o f p l a s m a i s i n t e r r u p t e d a f t e r i t h a s depos i t e d f i b r i n o g e n b u t b e f o r e i t r e p l a c e d t h e f i b r i n o g e n w i t h HMK, a n d the s u r f a c e i s then coated w i t h a metal o x i d e (one o f t h e i r o n o x i d e s , o r chromium o x i d e ) powder, subsequent re-exposure t o i n t a c t plasma w i l l cause t h e oxide t obe l i f t e d o f f wherever i t had been deposited on t h e f i b r i n o g e n f i l m . T h i s l i f t - o f f c a n s e r v e t o demon s t r a t e displacement o f one p r o t e i n by another as f o l l o w s . I n t a c t plasma i s i n j e c t e d between l e n s and s l i d e , incubated andr i n s e do f f as d e s c r i b e d e a r l i e r . A t t h i s p o i n t , t h e e n t i r e s l i d e w i l l have b e e n e x p o s e d t o p l a s m a u n d e r v a r i o u s c o n d i t i o n s . Where t h e p l a s m a had r e s i d e d i n t h e p e r i p h e r a l (wide) a r e a under t h e l e n s , t h e r e w i l l b e HMK. C e n t r a l l y ( i n t h e n a r r o w a r e a ) , a s w e l l a s w h e r e c o n t a c t was b r i e f ( d u r i n g t h e r i n s e , b e y o n d t h e a r e a o f r e s i d e n c e a n d i n t h e r e f i l l a b l e " s c r a t c h " l e f t by the lens w h i l e s l i d i n g o f f ) , f i b r i n o g e n w i l l be present. Such a s l i d e w i l l c o a t i t s e l f e n t i r e l y when e x posed t o a suspension o f t h e metal oxide. Subsequent exposure o f the e n t i r e coated s l i d e t o plasma a s d e s c r i b e d above w i l l cause r e moval o f o x i d e where i t h a d r e s i d e d on f i b r i n o g e n b u t n o t where i t h a d r e s i d e d o n HMK. A s a r e s u l t , o n l y a r i n g o f o x i d e r e m a i n s , c o r r e s p o n d i n g t o t h e l o c a t i o n o f HMK t h a t h a d b e e n d e p o s i t e d b y t h e o r i g i n a l ( i n j e c t e d ) plasma. P l a s m a l a c k i n g k i n i n o g e n s was u n a b l e t o remove t h e o x i d e ( 2 9 ) . e.

Plasma i n f l o w between s u r f a c e s When a 1 % s o l u t i o n o f n o r m a l i n t a c t p l a s m a i n V e r o n a l b u f f e r was i n j e c t e d b e t w e e n a n a n o d i z e d t a n t a l u m - s p u t t e r e d s l i d e a n d a c o n v e x l e n s o f a b o u t 2000mm r a d i u s o f c u r v a t u r e , t h e p l a s m a l e f t a teardrop-shaped r i n g o f albumin on t h e s l i d e , i t s " t a i l " p o i n t i n g away f r o m t h e s i t e o f i n j e c t i o n ( 2 6 ) . I na stagnation point flow chamber p l a s m a c r e a t e d t h e f o l l o w i n g p a t t e r n . I n t h e narrow areas around t h e metal t i p s t h a t supported t h e cover s l i p , and along l i n e s ( " t a i l s " ) p o i n t i n g t o t h e p e r i p h e r y o f t h e chamber t h u s i n d i c a t i n g the d i r e c t i o n o f f l o w , u n d i l u t e d i n t a c t plasma took longer than i t d i d e l s e w h e r e t o d e p o s i t f i b r i n o g e n a n d t h e n t o o k l o n g e r (more t h a n 5 m i n ) t o remove i t ( 2 7 ) . Under i d e n t i c a l c o n d i t i o n s , h e p a r i n i z e d b l o o d d e p o s i t e d p l a t e l e t s where plasma would l e a v e f i b r i n o g e n . WONDERING HOW AND WHY P R O T E I N S I N PLASMA AT SURFACES INTERACT I f o u r o b s e r v a t i o n s a r e c o r r e c t , plasma d e p o s i t s a sequence o f pro t e i n s , t h e more abundant b e i n g a d s o r b e d f i r s t a n d t h e n b e i n g r e -

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p l a c e d by l e s s a b u n d a n t o n e s - a t l e a s t o n s u r f a c e s t h a t c a n a c t i vate c l o t t i n g . A t t h e h i g h e s t d i l u t i o n s t e s t e d (.05 t o . 1 % ) , n o r m a l p l a s m a a p p e a r s t o d e p o s i t a l b u m i n f i r s t , and s l o w l y r e p l a c e s i t w i t h immunoglobulins (IgG). We s u g g e s t t h a t i n u n d i l u t e d p l a s m a t o o , a l b u m i n w o u l d be d e p o s i t e d f i r s t , t o be r e p l a c e d by I g G , w h i c h i s r e p l a c e d by f i b r i n o g e n and f i b r o n e c t i n , a l l w i t h i n a f e w s e c o n d s : and f i n a l l y , t h e l a t t e r a r e r e p l a c e d by HMK and f a c t o r X I I i n a m e a s u r a b l e amount o f t i m e . Thus, the behavior of a very complex m i x t u r e s u c h as p l a s m a d i f f e r s m a r k e d l y f r o m t h a t d e s c r i b e d f o r more simple mixtures (31). On t h i s r a t h e r n a r r o w b a s i s o f t h e 4 t o 6 p r o t e i n s s t u d i e d s o f a r , we c a n b u i l d t h e h y p o t h e s i s t h a t t h e r e i s a c o r r e l a t i o n b e t w e e n t h e c o n c e n t r a t i o n o f e a c h p r o t e i n i n t h e p l a s m a and i t s r a t e s o f b o t h a d s o r p t i o n and d e s o r p t i o n . Two p o s s i b l e c a u s e s o f t h i s c o r r e l a t i o n can t h e n be considered. a) T h e r e i s an i n v e r s e c o r r e l a t i o n b e t w e e n t h e n o r m a l l y o c c u r r i n g c o n c e n t r a t i o n s o f p l a s m a p r o t e i n s and t h e i r d e s o r p t i o n constants. T h i s w o u l d mean o u r p r o t e i n s e v o l v e d i n s u c h a way t h a t t h e r a r e ones would s t i c k l a t e s t but l o n g e s t : i t would imply t h a t t h e r e i s some p u r p o s e o f t h i s d i c t a t e d s e q u e n c e t o t h e s u r v i v a l o f t h e h o s t . b ) I n s t e a d , t h e r e may be a s i n g l e m e c h a n i s m t h a t c a n n o t h e l p b u t l i n k each p r o t e i n ' s c o n c e n t r a t i o n w i t h i t s r a t e s of a d s o r p t i o n and d e s o r p t i o n , e . g . b y means o f two r u l e s : 1) E a c h a d s o r b e d p r o t e i n m o l e c u l e c a n o n l y be r e m o v e d b y one o r m o r e m o l e c u l e s o f t h e same s p e c i e s p r e s e n t i n t h e b u l k s o l u t i o n , perhaps b r i e f l y forming a dimer or l a r g e r aggregate w i t h the ad s o r b e d mate a f t e r w h i c h the p a i r or complex i s d e s o r b e d . This m o d e l w o u l d be s u g g e s t e d by o u r e x p e r i m e n t s on p r e s u m a b l y p u r e p r o t e i n s f o r m i n g t h i c k r i n g s b e t w e e n l e n s and s l i d e , as d e s c r i b e d ( u n d e r "one p r o t e i n o n two i d e n t i c a l s u r f a c e s " ) . 2) The l o n g e r a p r o t e i n m o l e c u l e r e s i d e s o n a s u r f a c e , t h e s t r o n g e r i t i s bound and t h e more e n e r g y ( e . g . c o l l i s i o n s o r t i m e s p e n t w i t h a mate) i s r e q u i r e d f o r d e s o r p t i o n . We h a v e w r i t t e n a s i m p l e home c o m p u t e r p r o g r a m i n c o r p o r a t i n g t h e s e 2 r u l e s , so t h a t a t more o r l e s s n o r m a l c o n c e n t r a t i o n s o f a l b u m i n , I g G , f i b r i n o g e n , HMK a n d f a c t o r X I I t h e s e p r o t e i n s w i l l d i s p l a c e each other i n the sequence found e x p e r i m e n t a l l y . The pro gram d i s p l a y s the p r o t e i n s as i f g r a d u a l l y c o l l a p s i n g w i t h i n c r e a s e d t i m e o f r e s i d e n c e , a n d r e c o v e r i n g w h e n h i t and j o i n e d w i t h a m a t e u n t i l the p a i r i s desorbed. T h i s model r e p r e s e n t s the s i m p l e s t of p o s s i b l e s i t u a t i o n s : p l a s m a on a u n i f o r m , f l a t s u r f a c e w i t h m i n i m a l flow. I f p l a s m a d i l u t i o n , n a r r o w s p a c e s and l e s s " a c t i v a t i n g " (more a p o l a r ? ) s u r f a c e s a l l d e l a y t h e s e q u e n c e o f p r o t e i n i n t e r a c t i o n s a t t h e i n t e r f a c e , t h e n t h e s e e v e n t s w i l l be l e s s s y n c h r o n i z e d on p h y s i c a l l y r o u g h and c h e m i c a l l y n o n - u n i f o r m d e v i c e s e v e n when i n t r o d u c e d r a p i d l y t o b l o o d , and e v e n i f t h e 2 s i m p l e r u l e s g i v e n above were the o n l y ones g o v e r n i n g these i n t e r a c t i o n s . I t a p p e a r s m o s t l i k e l y t h a t t h e r e l a t i o n s h i p we p r e s u m e d t o e x i s t between the p h y s i o l o g i c a l c o n c e n t r a t i o n s of p r o t e i n s i n our p l a s m a and t h e i r s u r f a c e p r o p e r t i e s does n o t h o l d t r u e f o r most o f t h e p r o t e i n s we h a v e n o t y e t s t u d i e d . The f a c t t h a t p l a s m a i n j e c t e d b e t w e e n a l e n s and a s l i d e l e a v e s c o n c e n t r i c r i n g s o f a l l p r o t e i n s we h a v e l o o k e d f o r - e v e n i n c l u d i n g t r a c e s o f c o m p l e m e n t f a c t o r s , p l a s m i n o g e n and p r e a l b u m i n ( u n p u b l i s h e d f i n d i n g s ) - s u g g e s t s t h a t

10.

VROMAN AND ADAMS


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many p r o t e i n s we h a v e n o t y e t l o o k e d f o r a r e a l s o d e p o s i t e d , i n t h e i r own t i m e , c o n d i t i o n s o f f l o w a n d m i c r o m e t e r s o f s p a c e b e t w e e n interfaces. The p h y s i o l o g i c a l s i g n i f i c a n c e o f t h e i r presence a t t h e i n t e r f a c e w i l l l i e i n t h e i r t i m e o f a r r i v a l : i t may c o i n c i d e w i t h the a r r i v a l o f a c e l l c a r r y i n g r e c e p t o r s f o r t h a t p r o t e i n . The adsorbed p r o t e i n m o l e c u l e , l i k e a word o f t e x t , w i l l then be s t r e s s e d by the c e l l and the c o n t e x t o f the t e x t w i l l change. An i n c r e a s e d l y c o m p l e x s c e n e r y w i l l grow o u t o f more o r l e s s s y n c h r o n i z e d e v e n t s t h a t o c c u r upon i n t r o d u c t i o n o f a u n i f o r m but rough s u r f a c e i n t o the blood. T h i s g r o w t h c a n b e d e p i c t e d ( f i g . 1) a s i f p r o g r e s s i n g from near t o f a r d i s t a n c e , w i t h i t s d i s t a n t complex i t y o f c e l l behavior and f i b r i n formation m e r c i f u l l y b l u r r e d by p e r s p e c t i v e and environmental haze.

F i g u r e 1. A d r a w i n g s h o w i n g e v e n t s t h a t may o c c u r w h e n b l o o d comes i n c o n t a c t w i t h a r o u g h , a c t i v a t i n g s u r f a c e . T i m e i s shown a s i f p r o g r e s s i n g f r o m n e a r e s t t o d i s t a n t s c e n e r y . A l b u m i n (drawn egg-shaped) b e g i n s t o be d e p o s i t e d i n the most open a r e a s , then a l s o i n the narrow v a l l e y s . I t i s followed by i m m u n o g l o b u l i n s (IgG) (drawn Y - s h a p e d ) . On o p e n a r e a s w h e r e I g G has r e p l a c e d a l b u m i n , g r a n u l o c y t e s a d h e r e . F a r t h e r away, where IgG i s d i s p l a c e d b y f i b r o n e c t i n (drawn a s a n a r r o w band o f f a t h o o k s ) , m o n o c y t e s a d h e r e , w h i l e more d i s t a n t l y f i b r i n o g e n h a s d i s p l a c e d I g G , p l a t e l e t s become a t t a c h e d a n d b e g i n t o l i b e r a t e their products. On t h e f a r m o u n t a i n p e a k s , h i g h m o l e c u l a r w e i g h t k i n i n o g e n has d i s p l a c e d f i b r i n o g e n and i n t e r a c t s w i t h o t h e r f a c t o r s and w i t h p l a t e l e t p r o d u c t s t o f o r m s t r a n d s o f p l a s m a blown f i b r i n .

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U n f o r t u n a t e l y , i t i s t h i s b l i s s f u l haze t h a t w i l l keep us from s e e i n g t h e d e t a i l s we need f o r u n d e r s t a n d i n g the beauty o f t h i s scenery.

Literature Cited 1. Vroman, L. Ph.D. Thesis, Rijksuniversiteit Utrecht, Netherlands, 1958. 2. Vroman, L. Thrombos. Diathes. Haemorrh. 1964, 10, 455-493. 3. Vroman, L. Nature 1962, 196,476-477. 4. Adams, A.L.; Fischer, G.C.; Vroman, L. J. Colloid Interface Sci. 1978, 65, 468-478. 5. Vroman, L; Kanor, S.; Adams, A.L. Rev. Sci. Instrum. 1968, 39, 278-279. 6. Vroman, L.; Adams, A.L.; Klings, M.; Fischer, G.C. DCIEM Conf. Ρroc.#73-CP-960,Downsview, Ont., 1973, 49-70. 7. Copley, A.L. Ann. New York Acad. Sci. 1983, 416, 377-395. 8. Brash, J.; Davidson, V.J. Thromb. Res. 1981, 9, 249-259. 9. Bohnert, J.L.; Horbett, T. J. Colloid Interface Sci. 1986, 111, 363-377. 10. Kudryk, B.J.; Vroman, L. To be published. 11. Vroman, L; Adams, A.L. J. Colloid Interface Sci. 1986, 111, 391-402. 12. Brash, J.L; Uniyal, S. Plastics Med. Surg. 1979, 3, 29-1-29-7. 13. Jennissen, H.P. J. Colloid Interface Sci. 1986, 111, 570-577. 14. Vroman, L . ; Adams, A.L; Klings, M. Feder. Proc. 1971, 30, 1494-1502. 15. Vroman, L.; Adams, A.L.; Klings, M.; Fischer, G.C. Adv. Chem. Series 1975, 145, 255-289. 16. Klein, J. J. Colloid Interface Sci. 1986, 111, 305-313. 17. Silberberg, A. J. Colloid Interface Sci. 1986, 111, 486-495. 18. Vroman, L.; Adams, A.L. Thromb. Diathes. Haemorrh. 1967, 18, 510-524. 19. Vroman, L.; Adams, A.L. J. Biomed. Mater. Res. 1969, 3, 43-67. 20. Vroman, L.; Adams, A.L.; Fischer, G.C.; Munoz, P.C. Blood 1980, 55, 156-159. 21. Schmaier, A.H.; Silver, L . ; Adams, A.L.; Fischer, G.C.; Munoz, P.C.; Vroman, L. Thromb. Res. 1983, 33, 51-67. 22. Brash, J.L.; ten Hove, P. Thromb. Haemostas. 1984, 51, 326330. 23. Horbett, T.A. Thromb. Haemostas. 1984, 51, 174-181. 24. Horbett, T.A.; Cheng, C.M.; Ratner, B.D.; Hoffman, A.S.; Hanson, S.R. J. Biomed. Mater. Res. 1986, 20, 739-772. 25. Adams, A.L; Fischer, G.C.; Munoz, P.C.; Vroman, L. J. Biomed. Mater. Res. 1984, 18, 643-654. 26. Vroman, L.; Adams, A.L.; Fischer, G.C.; Munoz, P.C; Stanford, M. Adv. Chem. Series 1982, 199, 265-276. 27. Stanford, M.F.; Munoz, P.C.; Vroman, L. Ann. N.Y. Acad. Sci. 1983, 416, 504-512. 28. Vroman, L. Bull. N.Y. Acad. Med. 1976, 48, 302-312. 29. Vroman, L.; Adams, A.L; Brakman, M. Haemostasis 1985, 15, 300-303. 30. Blodgett, K.B.; Langmuir, I. Phys. Rev. 1937, 51, 964. 31. Horbett, T.A. Personal communication, 1986. RECEIVED January 3, 1987

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