Interaction Mechanism of Thrombin with Functional Polystyrene Surfaces

Chapter 13

Interaction Mechanism of Thrombin with Functional Polystyrene Surfaces: A Study Using High-Performance Affinity Chromatography 1

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X.-J. Yu , D. Muller , A. M . Fischer , and J . Jozefonvicz 1

Laboratoire de Recherches sur les Macromolécules, Greco 130048, Centre Scientifique Polytechnique, Université Paris—Nord, Avenue J . B. Clement, 93430 Villetaneuse, France Département d'Hématologie, Greco 130048, C.H.U. Necker-Enfants Malades, 156, Rue de Vaugirard, 75015 Paris, France

Downloaded by MONASH UNIV on November 1, 2014 | http://pubs.acs.org Publication Date: July 13, 1987 | doi: 10.1021/bk-1987-0343.ch013

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Insoluble sulfonated polystyrenes (PSSO ) and their L-arginyl methyl ester derivatives (PAOM) have been investigated by high-performance affinity chromatogra-phy. Highly active thrombin(Th) was found to be strongly adsorbed on both surfaces and was eluted by increasing ionic strength of the eluent. Compared to the PSSO surface, desorption from the PAOM resin occurred at a lower salt concentration and with a more specific enzyme-resin interaction. Masking of the enzyme's active site by its natural inhibitor antithrombin III (AT III) caused a total disappearence of its affinity for both types of surface. It was demonstrated that the active seryl residue of thrombin was involved in enzyme-PAOM interactions. In contrast, this same site of the enzyme was always available after binding to PSSO resin. This allowed a biospecific desorption procedure using AT III, heparin (Hep) and Hep-AT III complex. The present work provides further confirmation of the "AT III-like" and "heparin -like" properties, respectively, of PAOM and PSSO materials. 3

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In recent years, a g r e a t d e a l o f e f f o r t has been devoted t o t h e s t u d y o f a n t i t h r o m b o g e n i c p o l y m e r s ( 1 - 3 ) . I t h a s b e e n shown i n t h e present a u t h o r s ' l a b o r a t o r y (4-7) t h a t m o d i f i e d i n s o l u b l e p o l y s t y renes s u b s t i t u t e d e i t h e r with s u l f o n a t e o r amino a c i d s u l f a m i d e groups, e x h i b i t a n t i c o a g u l a n t a c t i v i t y , when s u s p e n d e d i n p l a s m a . This property c a n be a t t r i b u t e d t o t h e a d s o r p t i o n o f t h r o m b i n a n d a n t i t h r o m b i n I I I , a t the plasma-polymer i n t e r f a c e s (7-9). These a n t i c o a g u l a n t polymers may b e d i v i d e d i n t o t w o m a j o r c a t e g o r i e s , namely "AT I l l - l i k e " or "heparin-like" materials, a c c o r d i n g t o t h e i r d i f f e r e n t i n t e r a c t i o n mechanisms w i t h t h r o m b i n 0097-6156/87/0343-0197$06.00/0 © 1987 American Chemical Society

In Proteins at Interfaces; Brash, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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(J_,2^_7)» Sulfonateand L-arginyl methyl ester-substituted p o l y s t y r e n e s have a specific affinity f o r thrombin, s i m i l a r to t h a t o f i t s n a t u r a l i n h i b i t o r AT I I I ( K ) ) . I n c o n t r a s t , s u l f o n a t e d p o l y s t y r e n e s a c t as a c a t a l y s t i n Th-AT I I I c o m p l e x f o r m a t i o n (1,2^), which i s g e n e r a l l y c o n s i d e r e d t o be a n i m p o r t a n t p r o p e r t y of h e p a r i n (1_1 ) . Thus, they have been d e s i g n a t e d h e p a r i n - l i k e m a t e r i a l s . I t has t h e r e f o r e been p o s t u l a t e d t h a t t h e thrombin s i t e i n v o l v e d i n t h e thrombin-PAOM i n t e r a c t i o n may be d i f f e r e n t f r o m that i n v o l v e d i n the thrombin-PSSO^ i n t e r a c t i o n . Based on t h e i r specific and r e v e r s i b l e interactions with t h r o m b i n , PAOM resins have been used as s t a t i o n a r y phases f o r a f f i n i t y chromatography o f t h e p r o t e a s e (12). Thus, i n a s i m p l e one-step chromatographic procedure, human t h r o m b i n was i s o l a t e d from a c t i v a t e d prothrombin complex c o n c e n t r a t e i n h i g h p u r i t y and yield (J_3). Because o f t h e i r e x c e l l e n t m e c h a n i c a l p r o p e r t i e s t h e r e s i n s can a l s o be u s e d a s s u p p o r t s f o r h i g h - p e r f o r m a n c e liquid a f f i n i t y chromatography (HPLAC). Thrombin a d s o r p t i o n and d e s o r p tion processes have been i n v e s t i g a t e d u s i n g t h i s method. S i n c e a s m a l l e r amount o f s a m p l e c a n be c h r o m a t o g r a p h e d i n a shorter time (14), t h i s HPLAC m e t h o d w o u l d be u s e f u l f o r f u r t h e r s t u d i e s o f t h e thrombin-polymer i n t e r a c t i o n mechanisms. In t h e p r e s e n t paper, we report high-performance a f f i n i t y c h r o m a t o g r a p h y o f t h r o m b i n i n p r e s e n c e o f AT I I I a n d H e p , u s i n g t w o t y p e s o f r e s i n s a s s t a t i o n a r y p h a s e s : e i t h e r h e p a r i n - l i k e PSSO^ o r AT I l l - l i k e PAOM. I n o r d e r t o d i f f e r e n t i a t e t h e i r mechanisms o f i n t e r a c t i o n w i t h t h r o m b i n , we e x a m i n e d t h e c h r o m a t o g r a p h i c b e h a v i o r of t h r o m b i n i n t h e p r e s e n c e , o r i n t h e a b s e n c e o f AT I I I a n d / o r h e p a r i n . F i n a l l y , t h r o m b i n was i n j e c t e d o n t h e c o l u m n s a t l o w i o n i c s t r e n g t h . The d e s o r p t i o n o f b o u n d thrombin f r o m t h e two s o l i d s u r f a c e s was t h e n c a r r i e d o u t u s i n g AT I I I , h e p a r i n a n d t h e AT I l l Hep c o m p l e x , to elucidate the s p e c i f i c i t y of the interactions involved. Experimental Reagents Human - t h r o m b i n ( 3 0 0 0 N I H U/mg) a n d a n t i t h r o m b i n I I I (3 IU/mg) w e r e p u r c h a s e d f r o m t h e C e n t r e N a t i o n a l de T r a n s f u s i o n Sanguine ( P a r i s , F r a n c e ) a n d f r o m t h e C e n t r e Régional de T r a n s f u s i o n Sanguine ( L i l l e , F r a n c e ) . Hog i n t e s t i n a l h e p a r i n ( H 1 0 8 ) , ( 1 7 3 IU/mg, MW 10700 d a l t o n s ) was s u p p l i e d b y I n s t i t u t CH0AY P a r i s , France). The c o l u m n loadings generally used w e r e 90 u n i t s o f t h r o m b i n i n 45 μΐ o f e l u t i o n b u f f e r ( 0 . 0 5 M p h o s p h a t e , 0.1 M N a C l , pH= 7.4) o r 0.25 u n i t o f a n t i t h r o m b i n I I I i n 10 u l o f b u f f e r . α

P r e p a r a t i o n of Complexes. 25 u l o f t h r o m b i n s o l u t i o n ( 5 0 N I H U) were mixed with AT I I I (1 I U ) o r h e p a r i n (25 u g ) i n a 4:1 m o l a r r a t i o of i n h i b i t o r o r p o l y s a c c h a r i d e t o e n z y m e Q_5). The m i x t u r e was i n c u b a t e d a t 37°C f o r 15 m i n ( 1 1 , 1 6 ) . H e p a r i n - A T I I I c o m p l e x was p r e p a r e d i n a 1:1 m o l a r r a t i o u n d e r s i m i l a r c o n d i t i o n s . P r e p a r a t i o n of Chromatographic Supports. Styrene-divinylbenzene c o p o l y m e r s ( B i o - B e a d s SX2, 200-400 mesh) f r o m B i o R a d , F r a n c e , were f i r s t c h l o r o s u l f onated (J_7 ). Further reaction of the c h l o r o -



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s u l f o n a t e groups with L - a r g i n y l methyl ester v i a formation of s u l f a m i d e bonds or total hydrolysis of this chlorosulfonated p o l y m e r i n 2 M NaOH s o l u t i o n l e a d s t o PAOM o r PSSO^ r e s i n s , r e s p e c t i v e l y ( 4 , 1 2 ) . The p e r c e n t a g e o f t h e u n s u b s t i t u t e d , s u l f o n a t e d a n d L-arginyl methyl e s t e r - s u b s t i t u t e d monomer u n i t s a r e r e p r e s e n t e d r e s p e c t i v e l y b y Χ,Υ,Ζ i n F i g u r e . 1 . Chromatographic Procedure B e f o r e p a c k i n g , t h e r e s i n s were washed i n 2 M aqueous sodium chloride s o l u t i o n and then i n M i c h a e l i s buffer 0.026 M sodium barbital, 0.026 M s o d i u m a c e t a t e , 0.1 M s o d i u m c h l o r i d e , ( p H = 7 . 3 ) . The f i n e p a r t i c u l e s w e r e e l i m i n a t e d b y f l o t a t i o n . The c h r o m a t o g r a p h i c c o l u m m s ( 25 χ 0.4 cm I D ) w e r e p a c k e d u s i n g t h e s l u r r y method, w i t h a s u s p e n s i o n o f about 3g o f r e s i n i n a h i g h i o n i c s t r e n g t h s o l u t i o n p h o s p h a t e b u f f e r 0.05 M, N a C l 2 M, ( p H = 7 . 4 ) . The c o l u m m was e q u i l i b r a t e d f o r s e v e r a l h o u r s b y w a s h i n g with the chromatographic eluent buffer at a flow-rate of lml/min. The HPLC a p p a r a t u s c o n s i s t e d o f a t h r e e - h e a d (120°) c h r o m a t o g r a p h i c pump ( M e r c k LC 2 1 B ) , c o n n e c t e d t o a Rheodyne 7126 i n j e c tion valve ( S a m p l e l o o p 100 u l ) . A v a r i a b l e - w a v e l e n g t h UV-Visible d e t e c t o r (Merck-LC 313) and t h e g r a d i e n t system were c o n n e c t e d t o a n E p s o n QX-10 c o m p u t e r . The c h r o m a t o g r a p h i c s i g n a l was m o n i t o r e d , i n t e g r a t e d and s t o r e d by t h e computer. These v a r i o u s components were o b t a i n e d from M e r c k - C l e v e n o t (Nogent-sur-Marne, France). In a f i r s t s e r i e s o f chromatographic experiments, thrombin, ThAT I I I o r Th-Hep samples i n 0.1 M N a C l p h o s p h a t e b u f f e r w e r e i n j e c t e d on t h e columns and then e l u t e d by i n c r e a s i n g t h e s a l t concentration i n a linear gradient 0.1 M t o 2 M N a C l i n 0.05 phosphate b u f f e r (pH=7.4). I n a second s e r i e s o f chromatographic experiments, thrombin was f i r s t i n j e c t e d a t low i o n i c strength. D e s o r p t i o n o f t h e bound thrombin from t h e s o l i d s u r f a c e s was performed by i n j e c t i n g an excess o f AT I I I , Hep o r AT I l l - H e p c o m p l e x . T h e f l o w - r a t e was d e c r e a s e d t o 0.2 m l / m i n . The e l u t i o n was stopped f o r 5 minutes just after injection of the desorbing s u b s t a n c e . T h i s p r o c e d u r e was d e s i g n e d t o m i n i m i z e p o s s i b l e k i n e t i c effects i n this process. Results

and D i s c u s s i o n

Elution of α-thrombin. H i g h l y a c t i v e enzyme was c h r o m a t o g r a p h e d on t h e t w o r e s i n s u n d e r g r a d i e n t e l u t i o n c o n d i t i o n s ( F i g u r e s 2A a n d 2 B ) . The c h r o m a t o g r a m s show t h a t t h r o m b i n i n 0.1 M s o d i u m c h l o r i d e i n phosphate buffer was s t r o n g l y adsorbed on b o t h supports. D e s o r p t i o n o f t h e b o u n d enzyme f r o m PAOM r e s i n o c c u r r e d a t a r o u n d 1.7 M s a l t c o n c e n t r a t i o n ( F i g u r e 2 A ) . A s p r e v i o u s l y r e p o r t e d ( 1 4 ) , the i o n i c s t r e n g t h r e q u i r e d f o r d e s o r p t i o n v a r i e d w i t h t h e c h e m i c a l and p h y s i c a l p r o p e r t i e s o f t h e r e s i n s and w i t h t h e e l u t i o n c o n d i t i o n s , b u t n e v e r exceeded 2 M N a C l . However, t h r o m b i n a d s o r b e d on PSSO^ s u r f a c e c o u l d n o t be e l u t e d a t i o n i c s t r e n g t h l o w e r t h a n 2 M NaCl ( F i g u r e 2B). This r e s u l t suggests t h a t thrombin-PSSO^ i n t e r a c t i o n s a r e s t r o n g e r t h a n t h o s e o f t h r o m b i n -PAOM a n d i s c o n s i s t e n t w i t h the r e l a t i v e magnitudes o f t h e a f f i n i t y constants o f thrombin f o r t h e s e p o l y m e r s . These were p r e v i o u s l y d e t e r m i n e d from Langmuir i s o t h e r m s , t o b e 10 1 M a n d 10 1 M f o r P S S 0 a n d PAOM 3

resins,

respectively

(1,2,7,18).



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F i g u r e 1 : S t r u c t u r e and p e r c e n t a g e o f d i f f e r e n t monomer u n i t s of sulfonated polystyrene (PSSO ) and i t s L-arginyl methyl e s t e r d e r i v a t i v e (PAOM).



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Figure 2 : E l u t i o n o f human α - t h r o m b i n a t 25°C o n PAOM (A) a n d PSSO^ (B) r e s i n s with a l i n e a r g r a d i e n t f r o m 0.1 t o 2 M s o d i u m c h l o r i d e i n 0.05 M phosphate buffer, pH = 7.4 ; flow-rate, 1 ml/min.


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Stepwise E l u t i o n of Antithrombin I I I o r Heparin Both types o f m a t e r i a l have a f f i n i t i e s f o r s e v e r a l o t h e r plasma p r o t e i n s (6,10). A n t i t h r o m b i n I I I , f o r example, has an a f f i n i t y constant o f about 10 1 M~ f o r e i t h e r PAOM o r P S S 0 s o l i d s u r f a c e (8,1S>). C o n s e q u e n t l y , r e t e n t i o n on these r e s i n s was o b s e r v e d i n l o w - p r e s s u r e a f f i n i t y c h r o m a t o g r a p h y , a n d o n l y a t a l o w i o n i c s t r e n g t h (10). I t s h o u l d be n o t e d that t h e a d s o r p t i o n o f t h r o m b i n was f a r g r e a t e r (100-fold) than that o f i t s i n h i b i t o r u n d e r t h e same e x p e r i m e n t a l conditions.


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U n d e r HPLAC c o n d i t i o n s , s i m i l a r e l u t i o n c u r v e s f o r AT I I I a n d H e p a r i n were observed o n PAOM a n d P S S 0 ~ s t a t i o n a r y p h a s e s . A n t i t h r o m b i n I I I was immediately eluted b y 0.1 M s o d i u m c h l o r i d e s o l u t i o n a n d no d e t e c t a b l e p r o t e i n d e s o r p t i o n was o b s e r v e d a t 2 M i o n i c s t r e n g t h ( F i g u r e 3 A ) . T h u s t h e i n h i b i t o r was n o t r e t a i n e d b y e i t h e r PAOM o r PSSO^. C o m p a r e d w i t h t h e f i n d i n g s m e n t i o n e d a b o v e , t h i s c a n be e x p l a i n e d i n t e r m s o f k i n e t i c e f f e c t s . By d e c r e a s i n g the e l u t i o n f l o w - r a t e and s i m u l t a n e o u s l y o v e r l o a d i n g t h e columms, r e t e n t i o n o f a s m a l l amount o f AT I I I ( 17 ) was o b s e r v e d a t t h e same i o n i c s t r e n g t h , i . e . 0.1 M N a C l . 0

F i g u r e 3B i l l u s t r a t e s t h a t h e p a r i n was n o t r e t a i n e d o n e i t h e r o f t h e two s u p p o r t s i n 0.1 M N a C l . T h i s e f f e c t may b e a t t r i b u t e d t o electrostatic repulsion a t t h e i n t e r f a c e between the anionic m u c o p o l y s a c c h a r i d e and t h e s t r o n g l y a n i o n i c s u l f o n a t e d r e s i n s . S t e p w i s e E l u t i o n o f Th-AT I I I C o m p l e x . I t has been w i d e l y r e p o r t e d that thrombin i n h i b i t i o n by a n t i t h r o m b i n I I I r e q u i r e s a number o f c r i t i c a l amino a c i d r e s i d u e s i n e a c h o f t h e two p r o t e i n s ( 1 1 , 2 0 ) . These b i n d i n g s i t e s a r e e s s e n t i a l l y an a c t i v e - c e n t e r s e r i n e o f t h e enzyme a n d a n a r g i n y l r e s i d u e o f t h e i n h i b i t o r . I n a d d i t i o n , t h e inherently slow formation of thrombin-antithrombin I I I complex i s a c c e l e r a t e d by h e p a r i n (1_1). Although t h e mechanism of this catalysis i s s t i l l under i n v e s t i g a t i o n ( J^,21_,22^), i t h a s b e e n shown t h a t t h e b i n d i n g s i t e s i n v o l v e d i n t h e t h r o m b i n - a n t i t h r o m b i n III interaction d i f f e r f r o m t h o s e o f h e p a r i n f o r t h e two p r o t e i n s (23,24,25). The PAOM r e s i n s were designed t o mimic antithrombin I I I a f f i n i t y f o r t h r o m b i n by p a r t i a l l y s u b s t i t u t i n g , on t h e backbone o f the s y n t h e t i c polymer, one o f t h e m a j o r binding sites ofthe i n h i b i t o r , namely L - a r g i n y l m e t h y l e s t e r . T h i s s t r a t e g y w o u l d be justified i f a real involvement of the active seryl residue of t h r o m b i n w e r e f o u n d i n t h e enzyme-PAOM i n t e r a c t i o n s a n d n o t i n t h e enzyme-PSSO^ i n t e r a c t i o n s . I n j e c t i o n o f t h e Th-AT I I I c o m p l e x o n PAOM o r P S S 0 supports was p e r f o r m e d a t 0.1 M s a l t c o n c e n t r a t i o n . The c h r o m a t o g r a m s o n both supports show only one peak which a p p e a r s a t 0.1 M N a C l s o l u t i o n . No p e a k i s detected at high ionic strength (2 M NaCl), indicating that t h e Th-AT I I I complex and t h e excess o f a n t i thrombin I I I are eluted t o g e t h e r a t 0.1 N a C l b u f f e r ( F i g u r e 4 A ) . Consequently, i t i s concluded t h a t t h r o m b i n w i t h a masked s e r y l s i t e loses i t s a f f i n i t y f o r both r e s i n s .


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F i g u r e 3 : S t e p w i s e e l u t i o n o f a n t i t h r o m b i n I I I (A) a n d h e p a r i n ( B ) o n PAOM o r PSS0„ r e s i n s a t 25°C. B o t h r e s i n s h a v e same c u r v e . E l u e n t : 0.05 M p h o s p h a t b u f f e r (pH = 7 , 4 ) ; N a C l 0.1 M a n d 2 M; flow r a t e , 1 ml/min.



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Figure 4 : S t e p w i s e e l u t i o n o f Th-AT ( A ) a n d Th-Hep ( B ) c o m p l e x e s on PAOM o r P S S 0 r e s i n s a t 25°C. C o n d i t i o n s a s i n F i g u r e 3. B o t h r e s i n s h a v e same c u r v e . 3


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Assuming t h a t t h e a c t i v e s e r y l r e s i d u e o f thrombin i s r e s p o n sible f o r thrombin-resin interactions, a lack of a f f i n i t y ofthe i n a c t i v a t e d enzyme f o r t h e s e r e s i n s w o u l d be e x p e c t e d . However, the thrombin b i n d i n g s i t e ( s ) i n v o l v e d i n t h e enzyme-resin i n t e r a c t i o n s may b e d i f f e r e n t f r o m t h e a c t i v e s e r y l c e n t e r , b u t may b e located nearby. A simple steric r e s t r i c t i o n could thus prevent adsorption. The t h r o m b i n - h e p a r i n c o m p l e x was a l s o i n j e c t e d o n t o b o t h t h e PAOM a n d PSSO^ s u p p o r t s , t o i n v e s t i g a t e T h / H e p / r e s i n i n t e r a c t i o n s . The c h r o m a t o g r a p h i c profiles on each o f t h e s o l i d phases were a l m o s t i d e n t i c a l a s s h o w n i n F i g u r e 4 B . To i d e n t i f y t h e c o m p o n e n t s i n each p e a k , t h e enzyme a n d h e p a r i n w e r e c h r o m a t o g r a g h e d s e p a r a t e l y b u t u n d e r t h e same e x p e r i m e n t a l c o n d i t i o n s ( F i g u r e s 1 a n d 3). The f i r s t peak was t h u s a t t r i b u t e d t o f r e e - h e p a r i n m o l e c u l e s a n d the second, eluted at a higher ionic s t r e n g t h , t o t h e enzyme alone. In contrast t o t h e Th-AT I I I c o m p l e x , t h e Th-Hep c o m p l e x was d i s s o c i a t e d b y b o t h s o l i d p h a s e s . D e s o r p t i o n o f B o u n d T h r o m b i n b y A n t i t h r o m b i n I I I , H e p a r i n o r Hep-AT I I I Complex. T h r o m b i n was i n j e c t e d o n t o PAOM o r P S S 0 chromatogra p h i c s u p p o r t s i n 0.1 M N a C l s o l u t i o n . To m i n i m i z e p o s s i b l e k i n e t i c effects, the flow-rate was r e d u c e d t o 0.2 m l / m i n . A f t e r w a s h i n g w i t h 0.1 M N a C l b u f f e r , a n e x c e s s o f e i t h e r AT I I I , H e p , o r AT I l l Hep c o m p l e x was injected. The f l o w was t h e n s t o p p e d f o r f i v e minutes i n order to obtain a b e t t e r exchange o f macromolecules b e t w e e n t h e m o b i l e a n d t h e s t a t i o n a r y p h a s e s . T h e o b j e c t i v e was t o determine whether AT I I I , Hep o r AT I l l - H e p c o m p l e x c o u l d a c t a s eluents f o r the "biospecific d e s o r p t i o n " o f t h e bound enzyme a t 0.1 M N a C l . The e l u t i o n curve obtained with PAOM r e s i n s ( F i g u r e 5A) d e m o n s t r a t e s t h a t n o n e o f t h e t h r e e s p e c i e s was a b l e t o d e s o r b t h e bound enzyme, w h i c h was o n l y eluted at 2 M ionic strength. In contrast, thrombin was t o t a l l y e l u t e d f r o m PSSO^ r e s i n s b y AT I I I a n d AT I l l - H e p c o m p l e x ( F i g u r e s 5B a n d 5 C ) . 0 n l y p a r t i a l d e s o r p t i o n was o b s e r v e d when h e p a r i n was u s e d a l o n e ( F i g u r e 5 D ) . The c o m p a r i s o n between t h e chromatogram o f Th-AT c o m p l e x ( F i g u r e 4A) a n d t h e d e s o r p t i o n o f bound t h r o m b i n by a n t i t h r o m b i n I I I from t h e PAOM resins ( F i g u r e 5A) i n d i c a t e s t h a t t h e a c t i v e seryl residue of thrombin and p o s s i b l y o t h e r b i n d i n g s i t e s a r e b l o c k e d b y t h e PAOM r e s i n s . H o w e v e r , t h e same a m i n o a c i d r e s i d u e s of bound t h r o m b i n o n t h e PSSO^ s u p p o r t a r e a l w a y s a v a i l a b l e t o AT I I I , i n t h e p r e s e n c e o r i n t h e absence o f h e p a r i n . These c o m p a r i sons o f t h e " b i o s p e c i f i c d e s o r p t i o n " b e h a v i o r o f PAOM a n d P S S 0 ~ r e s i n s demonstrate t h e d i f f e r e n t m e c h a n i s m s a c c o r d i n g t o w h i c h AT I l l - l i k e or heparin-like p r o p e r t i e s c a n b e a t t r i b u t e d t o PAOM o r PSSO^ m a t e r i a l s , r e s p e c t i v e l y . 3

F i n a l l y , when t h e same e x p e r i m e n t was p e r f o r m e d u n d e r h y d r o dynamic e l u t i o n conditions without any f l o w s t o p p a g e , t h e bound enzyme was n o t s u b s e q u e n t l y d e s o r b e d e i t h e r b y AT I I I o r h e p a r i n . In c o n s t r a s t , AT Ill-Hep complex was a b l e t o e f f e c t complete desorption of thrombin. I t i s concluded that the well-known c a t a l y t i c m e c h a n i s m o f h e p a r i n i n Th-AT I I I c o m p l e x f o r m a t i o n a l s o o c c u r s i n t h e Th/AT I l l / r e s i n s y s t e m u n d e r o u r e x p e r i m e n t a l c o n d i tions .



0

I 20

I 40

I 60

L 100

ELUTION

I 80

I 0

TIME(min)

J

I 20

I 40

I 60

I 80

F i g u r e 5. D e s o r p t i o n of bound thrombin from PAOM (A) by AT I I I , Hep and AT I l l - H e p ; and from PSSO3 r e s i n s by a n t i t h r o m b i n I I I ( B ) , AT I l l - H e p complex (C) and h e p a r i n ( D ) , at 25 °C. E l u e n t : 0.05 M phosphate b u f f e r (pH = 7.4), NaCl 0.1 M and 2 M, f l o w r a t e 0.2 ml/min.

J


L_ 100


13.

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Literature Cited 1. Jozefowicz, M.; Jozefonvicz, J.; Fougnot, C. and Labarre. D., in Chemistry and Biology of Heparin, Lundblad, R.; Brown, W.V.; Mamm, K. GH. and Roberts, H.R. Editors), Elsevier, Amsterdam 1981, 475. 2. Fougnot, C.; Labarre, D.; Jozefonvicz, J.; and Jozefowicz, M. in Macromolecular Biomaterials ; Hastings, G.W. and Ducheyne, P. Editors) CRC Press, Inc., 1984, 215. 3. Jozefowicz, M.; and Jozefonvicz, J., Pure & Appl. Chem., 1984, 56, 1335-1344 4. Fougnot, C.; Jozefonvicz, J.; Bara, L. and Samama, Μ., Ann. Biomed. Eng., 1979, 7, 429. 5. Fougnot, C.; Jozefowicz, M.; Bara, L. and Samama, Μ., Anna. Biomed. Eng., 1979, 7, 441. 6. Fougnot, C.; Jozefowicz, M.; Bara, L., and Samama, Μ., Thromb. Res., 1982, 28, 37. 7. Boisson, C.; Gulino, D.; Jozefonvicz, J.; Fischer, A.M., and Tapon-Bretaudiere, J., Thromb. Res., 1984, 34, 269. 8. Boisson, C.; Gulino, D.; Jozefonvicz, J. in press. 9. Fougnot, C.; Jozefowicz, M. and Rosenberg, R.D., Biomaterials 1984, 5, 85. 10. Fischer, A.M.; Tapon-Bretaudiere, J.; Bros, Α.; Boisson, C.; Gulino, D. and Jozefonvicz, J. Thromb. Haemost., 1985, 54, 22. 11. Rosenberg, R.D. and Damus, P.S., J. Biol. Chem., 1973, 248. 12. Yu, X.J.; Fischer, A.M.; Muller, D.; Bros, Α.; Tapon -Bretaudiere, J. and Jozefonvicz, J., J. Chromatog., 1986, 376, 429. 13. Fischer, A.M.; Yu, X.J.; Tapon-bretaudiere, J.; Muller, D.; Bros, Α.; Jozefonvicz, J., J. Chromatog. 1986, 363, 95. 14. Muller, D.; Yu, X.J. Fischer, A.M.; Bros, A. and Jozefonvicz, J.; J. Chromatog. 1986, 359, 351-357. 15. Danielsson, A. and Bjork, I. Febs letters, 1980, 110, 241244. 16. Machovich, R.; Blasks, G. and Palos, L., Biochim. Biophys. Acta, 1975, 359, 153-200. 17. Petit, M.A. and Jozefonvicz, J., J. Appl. polym. Sci., 1977, 2589. 18. Fougnot, C.; Jozefowicz, M. and Rosenberg, R.D. Biomaterials, 1983 4, 294. 19. Boisson, C.; Brash, J.L. unpublished. 20. Bjork, I; Danielsson, Α.; Fenton II, J.W. and Jornavvall, Η., Febs Letter, 1981, 126, 257. 21. Machovich, R., Biochim. Biophys. Acta 1975, 412, 13-17. 22. Holmer, E.; Soderstrom, G. and Andersson, L-O., Eur. J. Biochem 1979, 33, 1-5. 23. Rosenberg, R.D. and Rosenberg J.S., J. Clin. Invest., 1984, 74, 1-6. 24. Li, E.H.M.; Orton, C. and Feimman, R.D., Biochem., 1974, 13, 5012. 25. Hatton, M.W.C. and Regoeezi, E.Thromb. Res., 1977, 10, 645 Received January 30, 1987


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