Die Angewandte Makromolekulare Chemie 166/167 (1989) 169-1 78 (2800)

Department of Pro te in & Molecular Biology & *Academic Department of Surgery, Royaf Free Hospital School of Medicine, Rowland H i l l S t r ee t , London NW3 2PF, U.K.

PEOSPHOLIPID POLYMERS C l4EH IUEHOCOMPATIBLE Ul'BlUALS +

B r e n d a Bal l , *Richard le B. B i r d , Dennis Chapman

Summary :

When blood comes i n t o contac t wi th an a r t i f i c i a l sur face , a number of events OCCUK which include p ro te in adsorption, p l a t e l e t ac t iva t ion and the ac t iva t ion of the i n t r i n s i c pathway of blood coagulation. With t h e increased appl ica t ion of blood containing a r t i f i c i a l devices, there is a g rea t demand t o develop new b i o m a t e r i a l s which r e t a r d thrombus format ion . Our new approach t o so lv ing t h i s problem is t o mimic the non-thrombogenic sur face of na tu ra l b io log ica l membranes a t l e a s t i n a s i m p l e form. We have developed a p o l y m e r i s a b l e phospholipid and polyes te rs based on the major phospholipid polar head group present on the erythrocyte outer membrane surface. The coagulation of blood exposed t o t h e s e polymers was examined by t h e t echn ique of M a t e r i a l Thrombelas tography, a r e l a t i v e l y s i m p l e t e s t f o r t h e i n v i t r o s c r e e n i n g of polymer thrombogenic i ty . We p r e s e n t r e s u l t s which i n d i c a t e t h a t t h e polymerised phospholipid and polyes te rs show reduced thrombogenicity, and may therefore have po ten t i a l f o r fu tu re biomaterials.

+Paper p r e s e n t e d a t t h e meet ing of t h e GDCh-Fachgruppe "Makromole k u l a r e Chemie" on "Polymers and b io logica l Functions" i n Bad Nauheim (West Germany) on April 18/19, 1988.

@ 1989 Hilthig & WcpiVerlag. Buel 0003-3146/89/$03.W

169

During o u r s t u d i e s of t h e b i o p h y s i c a l c h a r a c t e r i s t i c s o f t h e l i p i d s of biomembranes, eg t h e i r phase t r a n s i t i o n charac te r i s t ic , ’ t h e i r f l u i d i t y and i ts modu la t ion by c h o l e s t e r o l [Chapman e t a l l , Ladbrooke e t a 1 2 ] we began t o consider how the l i p i d mat r ix of biomembranes might be modulated by chemical processes eg. hydrogenation, ox ida t ion o r polymerisation. Because many of t he l i p i d s of biomembranes a r e unsaturated i n charac te r , we thought i t worthwhile t o a t t e m p t t o hydrogena te t h e doub le bonds which a r e p r e s e n t i n t h e l i p i d m a t r i x of biomembranes, i n o r d e r t o a r t i f i c a l l y modula te t h e biomembrane f l u i d i t y . To accomplish t h i s we developed a water so luble homogeneous c a t a l y s t [Chapman and Quinn3]. Using t h i s approach we were ab le t o s a t i s f a c t o r i l y hydrogenate the l i p i d s of biomembranes and a l s o serum l ipopro te ins [Katagi r i e t a14] . I n t h e c a s e of l i p o p r o t e i n s , we were a b l e t o show t h a t o n l y t h e ou te r phospholipid monolayer was hydrogenated.

The s u c c e s s of t h e s e s t u d i e s encouraged u s t o c o n s i d e r f u r t h e r chemica l m o d i f i c a t i o n s of biomembrane l i p i d s , i n p a r t i c u l a r whe the r i t would be poss ib le t o polymerise biomembrane s t ruc tures . Polymerising the mat r ix of na tu ra l biomembranes o r r econs t i t u t ed model membranes (liposomes) would enable us t o produce immobilized membrane enzyme systems. Liposomes (phospholipid v e s i c l e s ) c u r r e n t l y have many a p p l i c a t i o n s i n b io t echno logy as c a r r i e r v e s i c l e s i n t h e t r a n s p o r t of b i o l o g i c a l mo lecu le s , and i n medic ine as p o t e n t i a l d r u g d e l i v e r y s y s t e m s and r e d c e l l s u r r o g a t e s . However, c o n v e n t i o n a l l i posomes a r e u n s t a b l e and a r e a f f e c t e d by p r o c e s s e s such a s l y o p h i l i z a t i o n and f r e e z i n g ( v i t a l f o r long t e r m s t o r a g e of l iposomes) . Polymerised liposomes could have the p o t e n t i a l f o r s t a b i l i s i n g the liposome s t r u c t u r e s i n t h e s e v a r i o u s s i t u a t i o n s . Our main o b j e c t i v e t h e r e f o r e i n developing polymerisable phospholipids w a s t o make s y n t h e t i c p h o s p h o l i p i d s , which while r e t a in ing a s i m i l a r s t r u c t u r e t o conventional phospholipids, a r e capable of producing model membranes and n a t u r a l biomembranes of more s t a b l e s t ruc tu re .

W e s y n t h e s i z e d p o l y m e r i s a b l e p h o s p h o l i p i d s by t h e i n t r o d u c t i o n o f photosens i t ive d i ace ty l en ic groups i n t o one o r both acyl chains [Johnston e t a 1 5961. We a l s o i n t r o d u c e d d i a c e t y l e n e f a t t y a c i d s t o mutan t s which would i n c o r p o r a t e t h e s e f a t t y a c i d s b i o s y n t h e t i c a l l y i n t o t h e c e l l membrane s t r u c t u r e of t h e s e p a r t i c u l a r c e l l s [Leve r e t al’]. The p h o s p h o l i p i d s p o l y m e r i s e upon i r r a d i a t i o n w i t h UV-light fo rming a c r o s s - l i n k e d l i n e a r s t r u c t u r e c o n s i s t i n g of a l t e r n a t e conjugated double- and triple-bonds [Fig. 11. This was confirmed by 13C-NMR and Raman spectroscopic s t u d i e s [Pons e t a 1

D i a c e t y l e n i c p o l y m e r i s a t i o n is f a c i l i t a t e d when the s t r u c t u r e of the monomer most c l o s e l y r e s e m b l e s t h a t of t h e polymer l a t t i c e , i.e. i n t h e g e l s t a t e [Baugham and Yeel’]. UV-irradiation is performed a t temperatures wel l below the gel-to-liquid-crystall ine phase t r a n s i t i o n temperature (T,) of t he l i p i d , u s u a l l y a t O°C o r 4OC, i n a n oxygen-free envi ronment . The polymer a b s o r b s i n t h e v i s i b l e r e g i o n of t h e spec t rum and p o l y m e r i s a t i o n is accompanied by a c h a r a c t e r i s t i c red colouration, the i n t e n s i t y of which is an ind ica t ion of the amount of monomer t o polymer conversion [Johnston e t a 1 51. Diacetylenic polymerisation was demonstrated not only i n the s o l i d s t a t e and t h e aqueous s t a t e , i.e. as l i posomes , b u t a l s o i n monolayers a t a i r - w a t e r i n t e r f a c e s , i n m u l t i l a y e r s , and a f t e r i n c o r p o r a t i o n i n t o t h e membranes of

170

l i v i n g cells [Hayward and Chapman ‘‘1.

c.

Fig. 1. Formation of the polyconjugated, phospholipid polymer from the d i ace ty l en ic monomer. PC=phosphorylcholine.

Recent permeabi l i ty s tud ie s [Freeman e t all’] using 6-carboxyfluorescein (CF) and ( 3 H ) i n u l i n have demons t r a t ed t h a t monomeric C Z 5 i d e n t i c a l c h a i n d i ace ty l en ic liposomes a r e permeable t o small molecules such as CF (MW-350) but r e l a t i v e l y impermeable t o l a rge molecules such a s i n u l i n (MW=5200). After UV p o l y m e r i s a t i o n of t h e s e l i posomes a d e c r e a s e i n p e r m e a b i l i t y of t h e s e v e s i c l e s t o s m a l l mo lecu le s was observed. Po lymer i c C 2 5 i d e n t i c a l c h a i n d i ace ty l en ic PC liposomes were a l s o shown t o exh ib i t high r e s i s t ance t o the des t ruc t ive ac t ion of plasma high dens i ty l ipopro te ins (HDL).

Many d i f f e r e n t po lymer i c l i p i d s have now been d e s c r i b e d i n t h e l i t e r a t u r e . These include methacryloylic-substituted l i p i d s [Regen e t a l l 3 , Kusumi e t a1 l 4 I , b u t a d i e n i c l i p i d s [Gros e t a l l 5 , Akimoto e t a 1 16 ] and v i n y l i n i c - subs t i tu ted l i p i d s [Tundo e t a 1 ”I . Polymerization i s i n i t i a t e d e i t h e r by a chemical agent, such a s azob i s i sobu ty ron i t r i l e (AIBN), o r by i r r a d i a t i o n wi th u l t r a v i o l e t (UV) l igh t . Most liposomes formed from these l i p i d s are prepared i n t h e i r monomeric s t a t e and subsequently polymerised.

Many po lymer i c b i o m a t e r i a l s a r e t h r o m b o g e n i c and t h e p r o d u c t i o n o f b i o m a t e r i a l s which a r e haemocompat ib le and non-immunogenic would be a new i n n o v a t i o n . Our a p p r o a c h t o t h i s p r o b l e m i s t h e m i m i c r y o f t h e thromboresistant nature of t he red blood c e l l sur face [Hayward and Chapman”]. Mammalian c e l l s conta in over 100 d i s t i n c t l y d i f f e r e n t l i p i d s due t o va r i a t ions i n the acyl chains and polar head groups. The asymmetric d i s t r i b u t i o n of the phospholipids i n the e ry throcyte and p l a t e l e t membranes has been w e l l reviewed [Zwaal e t al l8, Hayward and Chapman”]. In t he ou te r membrane of these c e l l s , phosphory lcho l ine (PC) i s t h e most prominent head group (88%), fo l lowed by phosphoryle thanolamine (PE). Phosphorylcholine i s present i n sphingomyelin and l e c i t h i n and c o n t r i b u t e s t o t h e i n t e r f a c i a l p r o p e r t i e s and

171

t h r o m b o r e s i s t a n t n a t u r e of t h e e r y t h r o c y t e o u t e r s u r f a c e [Hayward and Chapman'']. The i n n e r membrane s u r f a c e is main ly composed o f n e g a t i v e l y cha rged l i p i d s eg. p h o s p h o r y l s e r i n e (PS) and is thrombogenic. During t h e s t imu la t ion of p l a t e l e t s the nega t ive ly charged phospholipids become exposed, and i n c r e a s e t h e r a t e of t h rombin f o r m a t i o n [Beve r s e t a1 "1. Zwaal e t a12' demons t r a t ed a marked d i f f e r e n c e i n t h e blood c l o t t i n g a b i l i t y o f t h e i n n e r and o u t e r h a l v e s of t h e r e d c e l l membrane. R igh t - s ide o u t r ed c e l l g h o s t s do n o t r educe t h e b l ank c l o t t i n g t i m e o f t h e Stypven tes t , whereas , non-resea led g h o s t s ( i e t h e i n n e r s u r f a c e ) r educe t h e c l o t t i n g t i m e i n a concent ra t ion dependent manner.

To produce non-thrombogenic su r faces we synthesized and f u r t h e r inves t iga ted t h e d i a c e t y l e n i c p h o s p h o l i p i d s c o n t a i n i n g a phosphory lcho l ine head group [ J o h n s t o n e t a151. C l o t t i n g s t u d i e s u s i n g t h e Stypven c l o t t i n g a s s a y , which determines the procoagulant a c t i v i t y of l i p i d d ispers ions , demonstrated t h a t d i a c e t y l e n i c phospho l ip id l i p o s o m e s a r e non-thrombogenic i n b o t h t h e i r monomeric and polymeric s t a t e s [Hayward and Chapman'']. This prothrombinase assay involves the a c t i v a t i o n of c l o t t i n g f a c t o r s X and V by Russell 's v iper venom, and is s e n s i t i v e t o b o t h phospho l ip id s t r u c t u r e and c o n c e n t r a t i o n [Schiffman e t alZ21. The rate of coagulation was s i g n i f i c a n t l y increased i n t h e p re sence o f b r a i n l i p i d e x t r a c t ( an e x t r a c t c o n s i s t i n g l a r g e l y o f n e g a t i v e l y charged p h o s p h o l i p i d s ) and a l s o i n t h e p re sence of c e r t a i n c o n c e n t r a t i o n s of p h o s p h a t i d y l c h o l i n e : p h o s p h a t i d y l s e r i n e m i x t u r e s . D i a c e t y l e n i c m o n o m e r i c a n d p o l y m e r i c l i p o s o m e s , a l o n g w i t h dimyristoylphosphatidylcholine liposomes had neg l ig ib l e e f f e c t s on the r a t e of c l o t f o r m a t i o n , s u g g e s t i n g t h a t t h e s e p o l y m e r i s a b l e v e s i c l e s a r e t h r o m b o r e s i s t a n t , mimicking t h e t h r o m b o r e s i s t a n t s u r f a c e s of r ed c e l l membranes [Hayward and Chapman "1.

Ju l i ano e t a lZ3 examined the e f f e c t s of conventional phospholipid liposomes and polymerised methacryolic liposomes containing PC head groups on p l a t e l e t aggrega t ion . These p r e l i m i n a r y s t u d i e s ind ica ted t h a t both non polymerised and polymerised PC liposomes had n e g l i g i b l e e f f e c t on p l a t e l e t agg rega t ion . However charged ves i c l e s can i n t e r a c t e i t h e r d i r e c t l y or i n d i r e c t l y t o reduce t h e p l a t e l e t r e sponse t o a g o n i s t s such a s ADP and thrombin. Bonte e t a l Z 4 a l s o recent ly demonstrated t h a t liposomes made from a conventional l i p i d and a d i l i p o y l l i p i d which c o n t a i n e d t h e PC p o l a r head group bound l e s s serum p ro te ins than liposomes wi th nega t ive ly charged l i p ids .

To i n v e s t i g a t e t h e th rombogen ic i ty of phospho l ip id s u r f a c e s w e r e c e n t l y e x p l o r e d t h e t e c h n i q u e o f m a t e r i a l t h r o m b e l a s t o g r a p h y (MTEG). The th rombe las tog raph is a mechan ica l ly o p e r a t e d sys t em which p r o v i d e s a continuous dynamic measure of blood coagulation. This technique w a s designed and developed by Hartert i n t h e l a t e 1940's [ H a r t e r t Z 5 ] . S ince then i t h a s been w i d e l y used i n many c l i n i c a l s e t t i n g s f o r t h e d i a g n o s i s of blood d i so rde r s [Franz and Coetzee'']. The thrombelastographic technique was l a t e r developed t o test the inf luence of ma te r i a l s on blood coagulation - Mater ia l Thrombelastography (MTEG) [Affeld e t alZ7, Carr e t a1 Bird e t alZ9, Hall e t a 1 "I. We have shown t h e v a l u e of d e t a i l e d s t a t i s t i c a l a n a l y s i s of t h e MTEG technique [Bird e t a 1 291.

172

The thrombelastograph measures the e l a s t i c p rope r t i e s of whole blood c l o t s as t h e y a r e formed. It u s e s a s t e e l (V2A) p i s t o n p l aced i n a c u v e t t e (hav ing a l m m c l e a r a n c e be tween t h e s u r f a c e s ) which c o n t a i n s t h e blood. The c u v e t t e o s c i l l a t e s t h rough 4' 45' (1 /12 r a d i a n ) i n a 10 second cyc le . Th i s g i v e s a s h e a r ra te of 0.28 p e r second. The p i s t o n is suspended by a t o r s i o n w i r e which is l i n k e d t o a r e c o r d i n g c h a r t . The t o r q u e o f t h e c u v e t t e is t r a n s m i t t e d t o t h e p i s t o n v i a t h e f i b r i n s t r a n d s i n t h e blood c l o t as coagulation proceeds. The r e su l t i ng t r ace , the thrombelastogram, records the e l a s t i c i t y of t h e blood c l o t . A normal TEG is shown i n f i g . 2 , w i t h t h e standard TEG parameters [ N i ~ o l a ~ ~ ] . Hypocoagulation produces a prolonged r and k t i m e , and a reduced ma, e and A'. Each pa rame te r r e f l e c t s a d i f f e r e n t component of whole blood c o a g u l a t i o n , and t h e TEG c o n t a i n s a d d i t i o n a l i n f o r m a t i o n compared t o t h e s t a n d a r d l a b o r a t o r y t es t s [Zuckerman e t a13']. The advantage o f t h e TEG is t h a t i t keeps t h e blood a t 37OC, and due t o t h e pa ra f f in f i l m covering the blood, the sample is not exposed t o the atmosphere and r e s u l t a n t changes o f pH. It is a dynamic t e s t i n which t h e m a t e r i a l sur faces a r e i n in t ima te contact wi th blood.

- 30 min 1

.

Fig. 2. A typ ica l thrombelastogram. S l s t a r t , r-reaction time, k= c l o t t i n g speed, mawiaximum amplitude, e- lOOma/ lOO-ma, - Ao=rate of c l o t formation.

I f t h e b lood samples a r e k e p t c o n s t a n t t hen t h e c o a t i n g o f t h e p i s t o n and c u v e t t e on one channe l of t h e TEG can be v a r i e d and i t s th rombogen ic i ty s tud ied - the technique of ma te r i a l thrombelastography MTEG [Affeld e t a127, Car r e t a l Z 8 , B i rd e t a l Z 9 , H a l l e t a130]. The r a t i o of t h e TEG p a r a m e t e r s from t h e t r e a t e d and t h e u n t r e a t e d channe l s p r o v i d e s t h e b a s i s f o r t h e compar ison of t h e d i f f e r e n t m a t e r i a l s . An i d e a l non-thrombogenic s u r f a c e would cause minimal ac t iva t ion of c l o t t i n g shown as an extended r and k t ime

173

w i t h a low A', ma and e. The r a t i o s a r e expres sed a s t e s t ( t ) s u b s t a n c e ove r the con t ro l ( 0 ) and such a sur face would have an rt/ro and kt/ko approaching i n f i n i t y while Aot/Aoo, mat/mao, and et/eo approach zero. The changes i n the t e s t channe l TEG shape i n d i c a t e which component h a s been a c t i v a t e d o r l e f t u n a f f e c t e d . The use o f t h e r a t i o e x c l u d e s e x t r a n e o u s e f f e c t s , and i t ' s d e v i a t i o n away from u n i t y i n d i c a t e s b o t h t h e component of whole b lood coagulation a f f ec t ed and the ex ten t of ac t iva t ion .

Fig. 3. Thrombelastography of biomembrane l i p ids .

We used t h e MTEG t echn ique t o d e m o n s t r a t e t h e r e l a t i o n s h i p be tween l i p i d asymmetry and haemocompatibility (Fig. 3).

Our s tud ie s ind ica t e t h a t t he predominant po lar head group on the erythrocyte o u t e r membrane s u r f a c e , PC when c o a t e d o n t o t h e s u r f a c e of t h e p i s t o n and cuvet te shows good haemocompatibility. The observations f o r DPPC can not be

174

a t t r i b u t e d t o t h e d e s o r p t i o n o f t h e l i p i d c o a t i n g f rom t h e s u r f a c e i n t o t h e b lood i n t h e form of l i posomes , and t h e r e b y a c t i n g on t h e b lood d i r e c t l y t o r educe t h e c l o t s t r e n g t h , s i n c e t h e a d d i t i o n of DPPC l iposomes t o t h e blood d i r e c t l y d id not s i g n i f i c a n t l y r e t a rd thrombus formation. However, t o check t h a t w e were obtaining the des i red o r i en ta t ion of t he po la r head groups wi th r e s p e c t t o t h e s u r f a c e ( i e w i t h t h e p o l a r g roups i n c o n t a c t w i t h t h e b lood , and the hydrocarbon chains i n contac t wi th the TEG sur faces and so hidden from t h e b lood) , we examined t h e e f f e c t o f c o a t i n g t h e p i s t o n and c u v e t t e w i t h oc t acosane ( a hydrocarbon). T h i s was thrombogenic [B i rd e t a12']. The reduction i n thrombogenicity observed wi th DPPC i s much l a rge r than t h a t seen w i t h t h e hydrophobic s u r f a c e s ( t h e p l a s t i c and oc tacosane) . Sphingomyelin (Sm) t e s t e d i n d i v i d u a l l y , o r when mixed i n t h e same l i p i d molar r a t i o s a s those present on the ou te r e ry throcyte membrane eg d ipa lmi toyl phosphatidyl- choline (DPPC):dipalmitoylphosphatidylethanolamine (DPPE): Sm (44: 12: 4 4 ) , a l s o show low thrombogenicity l i k e DPPC. DPPC and Sm show an increase i n the M r and Mk t imes ind ica t ing a reduced c l o t formation and a decrease i n the Me, MAo and M m a v a l u e s , i n d i c a t i n g a r e d u c t i o n i n p l a t e l e t a c t i v i t y . By c o n t r a s t t h e ne ga t i ve 1 y c ha r g e d 1 i p i d s d i p a 1 m i t o y 1 p h o s p h a t i d y 1 s e r i n e ( DP P S ) and dimyristoylphosphatidylglycerol (DMPG) which represent l i p i d s found on the e ry throcyte inner membrane sur face showed a increased r a t e of c l o t formation and p l a t e l e t a c t i v a t i o n a s compared t o the outer membrane l i p i d s ( ie lower M r and Mk and g r e a t e r Me, MAo and M m a va lues) . The r e s u l t s ob ta ined f rom o u r MTEG s t u d i e s c o r r e l a t e w e l l w i t h t h e i d e a t h a t once a c t i v a t e d , p l a t e l e t s expose t h e i r negatively charged inner membrane sur face t o the outer sur face and thus increase the r a t e of thrombin formation.

We next ' cons ide red t h e p o t e n t i a l of c o a t i n g p l a s t i c s u r f a c e s w i t h t h e s e a c e t y l e n i c p h o s p h o l i p i d s and p o l y m e r i s i n g them i n s i t u u s i n g UV r a d i a t i o n [Albrecht e t a133 1 and examining t h e i r biocompatibil i ty. We reported [Hall e t a130] a reduced thrombogenicity observed with the p i s ton and cuvet te coated w i t h DPPC ( c o n v e n t i o n a l p h o s p h o l i p i d ) (F ig . 3) and C Z 5 i d e n t i c a l c h a i n DAPC (photopolymerised phospholipid) when compared with the untreated sur face (Fig. 4 ) .

Both DPPC and DAPC showed an extended M r and Mk times and reduced M e , MAo and Mma values. A l l the r e s u l t s c l e a r l y h ighl ight the importance of the PC head group a s t h e e f f e c t i v e p a r t of t h e phospho l ip id molecule i n p r e v e n t i n g t h e a c t i v a t i o n o f c o a g u l a t i o n , r a t h e r t han t h e l i p o p h i l i c s i d e cha in . The TEG p a r a m e t e r s r and k r e f l e c t t h e deg ree of a c t i v a t i o n of c l o t t i n g f a c t o r s and the prolongation of M r and Mk impl ies reduced ac t iva t ion and a reduced rate of thrombin formation by the t e s t surface. DPPC almost doubles the M r , and the Mk v a l u e i s i n f i n i t e i n d i c a t i n g low l e v e l s of a c t i v a t i o n . These r e s u l t s support our e a r l i e r observations t h a t PC containing phospholipid ves i c l e s do n o t a c t i v a t e c l o t t i n g f a c t o r s i n t h e Stypven tes t [Hayward and Chapman'']. S i m i l a r l y t h e r e d u c t i o n of Mma f o r DPPC t o 6 % of normal i s p a r t i c u l a r l y s i g n i f i c a n t s i n c e ma i s l a r g e l y de t e rmined by p l a t e l e t s . Th i s r e s u l t i n d i c a t e s t h a t f e w o f t h e p l a t e l e t s p r e s e n t a r e a c t i v a t e d by t h e phosphory lcho l ine l i p i d coa t ing . A s can be s e e n i n Fig. 3 and Fig. 4 , DPPC and the polymerised l i p i d (DAPC) a r e more blood compatible than the polymer mater ia l Dacron, which i s commonly used f o r vascular prothesis.

175

DACRON

OAPC

POLYESTER C

POLYESTER D

POLYESTER G

Fig. 4. Thrombelastography of phospholipid polymers and Dacron coating.

We have recent ly begun t o develop new haemocompatible polymers having good r h e o l o g i c a l and p h y s i c a l c h a r a c t e r i s t i c s . T h i s approach i n v o l v e s t h e incorpora t ion of PC groups i n t o the s t r u c t u r e of a polyes te r o r polyurethane polymer. The new p o l y e s t e r s we have developed are s y n t h e s i s e d f rom glycerophosphorylcholine (GPC), e t h y l e n e g l y c o l and p h t h a l i c a c i d l i n k e d randomly v i a e s t e r bonds. The MTEG r e s u l t s of t h e s e p o l y e s t e r s show a n improved h a e m o c o m p a t i b i l i t y compared w i t h t h e r e s u l t s o b t a i n e d w i t h t h e u n t r e a t e d s u r f a c e (F ig . 4). P o l y e s t e r s C and D va ry on ly i n t h e i r GPC conten t , 22.1% and 39.8% respectively. Although polyes te r C has a longer M r ,

176

i t has a l a r g e r Mma and MAo t h a n p o l y e s t e r D , and t h i s may be r e l a t e d t o t h e h igh GPC c o n t e n t o f p o l y e s t e r D. Note t h a t t h e v a r i a t i o n i n GPC c o n t e n t changes t h e MTEG p a r a m e t e r s a f f e c t e d . For p o l y e s t e r C, c l o t t i n g f a c t o r a c t i v a t i o n i s reduced (Mr = 1.24). For p o l y e s t e r D, p l a t e l e t a c t i v a t i o n i s reduced (Mma = 0.12). while Mr is unchanged.

The r e s u l t s f o r po lyes te r G show low ac t iva t ion of both c l o t t i n g f a c t o r s ( M r =

1.291) and p l a t e l e t s (Mma = 0.139). P o l y e s t e r G has 31.4% GPC and i s s i m i l a r i n s t r u c t u r e t o p o l y e s t e r s C and D, b u t a l s o c o n t a i n s po lyu re thane l i n k s t o form a polyester-polyurethane complex. Clearly f u r t h e r development is needed t o o p t i m i s e t h e r a t i o s o f p o l y e s t e r t o po lyu re thane , and t h e GPC con ten t . Polyes te rs and polyurethanes t h a t have a high molecular weight and incorpora te PC groups may be use fu l e i t h e r f o r sur face coa t ing of ex i s t ing b iomater ia l s o r f o r t he formation of novel b iomater ia l s wi th improved blood compatibil i ty.

Future developments i n t h i s f i e l d should see the production of polymers with v a r y i n g cha rge d i s t r i b u t i o n and of d i f f e r i n g rheologica l c h a r a c t e r i s t i c s t o p rov ide t h e optimum c o n d i t i o n s f o r new b i o m a t e r i a l a p p l i c a t i o n s . C l e a r l y fu r the r i n v i t r o t e s t such a s p ro te in adhesion p l a t e l e t ac t iva t iodaggrega t ion and c e l l adhes ion and a number of i n v i v o a n i m a l s t u d i e s need t o be made. Our concept of mimicking the p rope r t i e s of na tu ra l biomernbranes t o produce new haemocompatible b iomater ia l coa t ings and polymers may prove use fu l f o r various medical and health-care s i t ua t ions .

BH g r a t e f u l l y acknowledges the support of the SERC a s a BB case student. RB g ra t e fu l ly acknowledges the support of the Stanley Thomas Johnson Foundation. DC acknowledges the Wellcome Trus t f o r support. We thank D r L A . Durrani f o r supplying the DAPC phospholipid and D r M. Kojima f o r supplying the polyes te rs f o r t h i s work.

1.

2.

3. 4.

5.

6.

7.

8.

D. Chapman, R.M. W i l l i a m s , B.D. Ladbrooke, Chem. Phys. L i p i d s L ( 1 9 6 7 ) 445-475. B.D. Ladbrooke, R.M. W i l l i a m s , D. Chapman, Biochimica e t b i o p h y s i c a Acta (1968) 333 - 340. D. Chapman, P.J. Quinn , Chem. Phys. L i p i d s C. K a t a g i r i , J.S. Owen, P.J. Qu inn , D. Chapman, Eurpoean J o u r n a l of Biochemistry 118 (1981) 335 - 338. D.S. Johns ton , S . Sanghera , M. Pons, D. Chapman, Biochim. Biophys. Acta - 602 (1980) 57-69. D.S. Johns ton , L.R. McLean, M.A. Whittam, A.D. C la rk , D. Chapman, Biochemistry 2 (1983) 3194-3202. J.Lever, A. Alonso, A. Dur ran i , D. Chapman, Biochemica e t Biophys ica Acta 727 (1983) 327 - 335. M. Pons, D.S. Johns ton , D. Chapman, J. Polym. Sci.Polym. L e t t . Ed. 20

(1976) 363 - 372.

177

(1982) 513-520. M. Pons, D.S. J o h n s t o n D. Chapman, Biochim. Biophys. Acta 693 (1982) 461-465. R.H. Baugham and K.C. Yee, Macromol. Rev. 12 (1978) 219-239. J.A. Hayward, D. Chapman, Biomater ia l s 2 (1984) 135-142. F.J. Freeman, J.A. Hayward, D. Chapman, Biochim. Biophys. Acta 924 (1987) 341-351.

9.

10. 11. 12.

13.

14.

15.

16.

17.

18.

19.

20.

21. 22. 23.

24.

25. 26. 27.

28.

29.

30.

31.

32.

33.

S.L. Regen, A. S ingh , G. Oehme, M. S ingh , J. Am. Chem. SOC. 104 (1982) 791-795 A. Kusumi, M. S ingh , D.A. T i r e l l , G. Oehme, A. S ingh , N.K.P. Samuel, J.S. Hyde, S.L. Regen, 3. Am. Chem. SOC. 105 (1983) 2975-2980. L. Gros, H. R ingsdor f , H. Schupp, Angew. Chem. I n t . Ed. Engl. 20 (1981) 305. A. Akimoto, K. Dorn, L. Gros , H. R i n g s d o r f , H. Schupp, Angew. Chem. 20 (1981) 90-91. P. Tundo, D.J. K ippenbe rge r , P.L. Klahn , N.E. P r i e t o , T.C. J a o , J.H. F e n d l e r , R.F.A. Zwaal, E.M. Bevers , L i p i d s and Membranes: p a s t , p r e s e n t and future. Amsterdam, Elsevier 1986, p. 231-257. J.A. Hayward, D. Chapman, ' B i o c o m p a t i b i l i t y o f t i s s u e ana logues ' . CRC Press , Boca Raton Flor ida , 1985, Vol. 2, p 119-132. E.M. Beve r s , P. Comfur ius , R.F.A. Zwaal, Biochim. Biophys. Acta 736 (1983) 57-66. R.F.A. Zwaal , P.Comfuris, U.M. Van Deenen, Na tu re 268 (1977) 358-360. S. Schiffman, I. Theodor, S.I. Rapaport Biochemistry S (1969) 1397-1405 R.L. J u l i a n o , M.J. HSU, D. P e t e r s o n , S.L. Regen, A. S ingh , Exp. C e l l . Res. 146 (1983) 422-427. F. Bonte, M.J. Hsu, A. Papp, K. Wu, S.L. Regan, R.L. J u l i a n o , Biochim. e t Biophys. Acta 900 (1987) 1-9. H. Har te r t , Klin Wochenscher 26 (1948) 577-583 R.C. Franz , W.J. Coe tzee , Surg. Ann. 13 (1981) 75-107. K. A f f e l d , J. Berge r , R. M u l l e r , E.S. Bucher l , Proc. Eur. SOC. A r t i f . Organs 1. (1974) 26-29. S. Carr, L. Zuckerman, J. C a p r i n i , Res. Comm. Chem. Path. & Pharm. 12 (1976) 507-519. R. l e R. B i rd , B. H a l l , D. Chapman, K.E.F. Hobbs, Throm. R e s . 1988 ( I n press ) . B. H a l l , R. l e R. B i rd M. Kojima and D. Chapman, B i o m a t e r i a l s 1988 ( i n press). P. de Nicola, Thrombelastography, Charles Thomas, Spr ingf ie ld , I l l i n o i s , 1957 L. Zuckerman, E. Cohen, J.P. Vagher, E. Woodward, J.A. C a p r i n i , Thromb. Haemostas. (S tu t tga r t ) (1981) 752-756. 0. A l b r e c h t , D.S. Johns ton , C. V i l l a v e r d e and D. Chapman, B ioch imica e t Biophysica Acta 687 (1982) 165 - 169.

J. Am. Chem. SOC. 104 (1982) 456-461.

178