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Anticoagulant activity of artificial biomedical materials with co-immobilized antithrombin III and heparin
BIOCHIMIE, 1980, 62, 595-601.
Anticoagulant activity of artificial biomedical materials with co-immobilized antithrombin III and heparin. Yoshiharu MJURA, Sadayoshi AOYAGI, Fumiaki IKEDA and Kazuhisa MIYAMOTO.
Department of Biochemical Engineering, Faculty o[ Pharmaceutical Sciences, Osaka University, Saita, Osaka, 565 Japan.
R6sum6.
c o a q u l a t i o n factors includinq Factor Xa a n d thrombin. Heparin stimulates the inactivation of Factor Xa a n d thrombin b y AT III. Thus AT III a n d h e p a r i n were co-immobilized on Sep h a r o s e 4B, p o l y v i n y l alcohol, p o l y h y d r o x y ethyl m e t h a c r y l a t e a n d silicone-coated n y l o n b y the c y a n o q e n bromide procedure. Those coimmobilized preparations, a b b r e v i a t e d a s I-AT III'Hep, actively neutralized both Factor Xa a n d thrombin. The activity of I-AT III'Hep w a s m u c h hiqher t h a n immobilized h e p a r i n a n d / o r immobilized AT III. I-AT III'Hep, like soluble AT III a n d heparin, i n s t a n t a n e o u s l y neutralized both thrombin a n d Factor Xa. W h e n two e n z y m e s , thrombin a n d Factor Xa, w e r e present, I.AT III'Hep neutralized Factor Xa in p r e f e r e n c e to thrombin : The neutralization of thrombin w a s inhibited b y the p r e s e n c e of Factor Xa, but neutralization of Factor Xa w a s i n d e p e n d e n t of the p r e s e n c e of thrombin. The a m o u n t of Factor Xa neutralized w a s hiqher t h a n that of thrombin.
Des m a t 6 r i a u x b i o m 6 d i c a u x antithromboq~nes ont 6t6 pr6par6s. L'antithrombine III (AT III) et l ' h 6 p a r i n e (Hep) ont 6t6 coimmobilis6es sur divers supports. Ces p r 6 p a r a t i o n s neutralisent i n s t a n t a n 6 m e n t le facteur Xa et la thrombine. Leur activit6 est plus q r a n d e que celles de I'AT III et de l ' h 6 p a r i n e immobilis6es s6par6ment. En pr6sence des deux enzymes, facteurs Xa et thrombine, le m a t 6 r i a u neutraBse pr6f6rentieUement le facteur Xa.
Summary. A n a p p r o a c h to providinq a n t i c o a q u l a n t activity to b i o m e d i c a l materials w a s presented, a p p l y i n q a n immobilization technique. Antithrombin III (AT III) inactivates the activated
Introduction. Blood coagulation is now somewhat elucidated at the molecular level. The coagulation is triggered by both intrinsic and extrinsic causes, and coagulation factors (also known as clotting factors) are successively activated. Factor X, one of the coagulation factors, is at the intersection of the two pathways, intrinsic and extrinsic. Activated Factor X, Factor Xa, converts prothromb,in to thrombin. At the last stage of blood coagulation, thrombin catalyzes the proteolysis of fibrinogen to fibrin, and fibrin clot results. Antithrombin III (A.T IlI) inactivates a number of the activated coagulation factors including Factor Xa and thrombin [1-4]. As a result, AT III plays an important role for keeping the liquidity of blood. The creation of biomedical materials capable of preventing the agglutinative thrombus has
been a very important subject in the development of artificial organs. Heparin, accelerating the neutralization rate of activaled coagulation factors by AT III [5-8], has been found to be an effective anticoagulant, and has been used to prevent thrombus in artificial organs. Administration of a large amount of heparin, however, causes bleeding, and platelet aggregation and adhesion [9, 1~]. Since A.T III is present in blood, it has been commonly considered that the AT III necessary for cooperation with heparin can be naturally supplied from blood. Thus, immobilization of heparin has been proposed for obtaining non thrombogenic biomaterials [ll-lg]. We observed that heparin covalently immobilized on biomaterials had an ability to bind thrombin and Factor Xa, but that these enzymes thus bound to the immobilized heparin were still active E321. This cooperative effect on the neutralization of Factor Xa and thrombin, i.e. the instantaneous
40
596
Y. Mittra and coll.
n e u t r a l i z a t i o n e x h i b i t e d b y AT III i n t h e p r e s e n c e of s o l u b l e h e p a r i n , w a s n o t o b s e r v e d w h e n h e p a r i n w a s c o v a l e n t l y i m m o b i l i z e d . It w a s f o u n d , however, that heparin, when co-immobilized with A T III, e x h i b i t e d m u c h h i g h e r a n t i c o a g u l a n t a c t i vity than immobilized heparin and/or immobil i z e d A T H I [20, 21]. E m p l o y i n g t h e c y a n o g e n b r o mide procedure, co-immobilization was successf u l l y a p p l i e d to s o m e b i o m a t e r i a l s s u c h as p o l y v i n y l al.cohol, p o l y h y d r o x y e t h y l methacrylate, a n d s i l i c o n e - c o a t e d n y l o n [20, 21]. H e r e , a m o r e detailed s t u d y on the a n t i c o a g u l a n t activity of t h e c o - i m m o b i l i z e d p r e p a r a t i o n o f AT III a n d h e p a r i n i s p r e s e n t e d , a n d t h e a c t i o n o f A T III i n t h e b l o o d c o a g u l a t i o n s y s t e m is d i s c u s s e d .
Materials and Methods. CNBr-activated Sepharose 4B was purchased f r o m P h a r m a c i a Fine Chemicals. Polyvinyl alcohol (PVA) whose fibers were 30 to 50 Ixm in diameter, p o l y h y d r o x y e t h y l m e t h a c r y l a t e (Poly HEMA) whose granules were 150 to 48 mesh, were used as carrier materials for the i m m o b i l i z a t i o n . Heparin, t h r o m b i n , bovine serum a l b u m i n and fibrinogen were purchased f r o m Sigma Chemical Company. Heparin (sodium salt) isolated f r o m porcine i n t e s t i n a l mucosa, had 170 USP units per mg protein. T h r o m b i n c o n t a i n i n g 600 NIH units per mg protein, isolated f r o m bovine plasma, was s u b s t a n t i a l l y free of Factors II, V, VII, IX, X, p l a s m i n and Factor Xa. F i b r i n o g e n isolated f r o m bovine blood was a b o u t 95 per cent clottable. Bovine a n t i t h r o m b i n III, AT III, and Factor X were purified as described previously [72-26]. Factor X w a s activated by Russell's Viper Venom in cephalin [271, purchased f r o m Sigma Chemical Company. F a c t o r VII and X deficient plasma, lyophilized f r o m bovine plasma, was purchased f r o m Sigma Chemical Company. Tert-butoxycarbonyl-L-Valyl-L-Prolyl-L-Arginine 4-M.ethyl-Coumarinyl-7-Amide (Boc-Val-Pro-Arg-MCA) which is a substrate for t h r o m b i n in the new a s s a y method, tertb u t o x y c a r b o n y l - L - Isoleucyl-L-Glutamyl-Glycyl-L-Arginine 4-Methyl-Coumarinyl-7-Amide (Boc-lle-Glu-GlyArg-MCA) for Factor Xa were purchased f r o m Peptide Institute Corporation. AT III a n d / o r h e p a r i n was coupled to CNBr-activated PVA, P.oly HEMA, silicone-coated n y l o n or Sepharose 4B as described previously [28, 20, 21] and abbreviated as follows : immobilized h e p a r i n (I-Hep), immobilized AT III p.rotected b y acetylated hepa~in (I-AT III) and co-immobilized AT III and h e p a r i u (I-AT I I I . Hep). As a control preparation, bovine s e r u m a l b u m i n was. i m m o b i l i z e d on these activated materials. The m e a s u r e m e n t of p l a s m a recalciflcation time (PRCT) was carried out in the following m a n n e r : the immobilized preparation, suspended in 10 mM Tris-HC1 buffer, pH 7.4 containing 0.145 M NaC1, was added to the 200 /xl of p r e w a r m e d eitrated bovine p l a s m a and preincubated for the defined t i m e at 37°C. 25 mM Ca2+ solution was added to the m i x t u r e and PRCT was m e a s u r e d w i t h gentle agitation.
BIOCHIMIE, 1980, 62, n ° 8-9.
The d e t e r m i n a t i o n of a n t i - t h r o m b i n and a n t i - F a c t o r Xa activities of i m m o b i l i z e d p r e p a r a t i o n s was carried out in the following m a n n e r : The immobilized prep a r a t i o n was allowed to react with t h r o m b i n a n d / o r Factor Xa u n d e r stirring at 37°C. After reacting for the defined time, the r e m a i n i n g enzyme activity was i m m e d i a t e l y measured by means of the clotting time method or the new assay method using the peptideM.'CA as a s~bstrate in two series of assays : In the first series, t h e i m m o b i l i z e d p r e p a r a t i o n was i m m e diately removed f r o m the reaction m i x t u r e after the reaction, and the r e m a i n i n g enzyme activity of reacted solution w i t h o u t i m m o b i l i z e d p r e p a r a t i o n w a s measured. In a n o t h e r a s s a y way, the immobilized prepar a t i o n was not removed a f t e r the reaction, and the enzyme activity of reaction m i x t u r e containing the i m m o b i l i z e d p r e p a r a t i o n was measured. All control e x p e r i m e n t s were carried out using the control preparations, on w h i c h bovine s e r u m a l b u m i n had been immobilized. No a n t i - t h r o m b i n and a n t i - F a c t o r Xa activities of the i m m o b i l i z e d a l b u m i n were observed. However, w h e n Sepharose 4B was used as a carrier, a ltg o r d e r a d s o r p t i o n of enzyme on Sepharose 4B gel was observed in the reaction of the immobilized preparation w i t h the enzyme. 1 per cent bovine s e r u m a l b u m i n was t h e n added to the reaction m i x t u r e to prevent the a d s o r p t i o n of e n z y m e protein. The a d s o r p t i o n of Factor Xa on the Sepharose 4B had consequently been eliminated, h o w e v e r t h a t of small a m o u n t s of t h r o m b i n still occurred. So, a n t i - e n z y m e activity of immobilized AT III a n d / o r h e p a r i n was obtained f r o m the difference between the r e m a i n i n g enzyme activity w h e n using i m m o b i l i z e d AT III a n d / o r h e p a r i n in the reaction and w h e n using the control preparation. Assay for t h r o m b i n activity by the clotting time m e t h o d was done using the previously described method and 1 per cent fibrinogen solution substrate [29]. Factor Xa activity was m e a s u r e d by a previously described m e t h o d [30] using Factor VII and X deficient p l a s m a as s ubstrate. T h r o m b i n activity and Factor Xa activity were also m e a s u r e d b y the new assay m e t h o d using peptide-MCA substrates. 50 ~1 to 101) ~xl of the enzyme solution were added to 1.0 ml of 0.1 mM peptide-MCA in 50 mM TrisHC1 buffer, pH 8.0, containing 100 mM NaC1 and 10 raM CaC12, previously p r e w a r m e d to 37~C. The reaction was carried out at 37°C for 3 to 10 min and stopped by adding 17 per cent acetic acid solution to give a final concentration of 10 per cent. The relative fluorescence of produced a m i n o - 4 - m e t h y l - c o u m a r i n , AMC, was measured, using excitation and emission wavelengths of 380 n m and 460 nm, respectively, in a fluorescence spectrophotometer, HITACHI model MPF-2A. The i n s t r u m e n t was standardized so t h a t a 10 ~M solution of AMC in 0.1 per cent d i m e t h y l s u l f o x i d e containing 10 per cent acetic acid gives 1.0 relative fluorescence [31].
R e s u l t s and Discussion. Various co-immobilized preparations or an i m m o b i l i z e d m i x e d p r e p a r a t i o n of A T I I I a n d heparin were examined for anticoagulant activity. R e s u l t s a r e s u m m a r i z e d i n t a b l e I. B o v i n e s e r u m a l b u m i n w a s i m m o b i l i z e d as a c o n t r o l p r e p a r a -
Anticoagulant activity of artificial biomedical materials.
597
A T III, o r w h e n I - A T I I I w a s i n c u b a t e d w i t h solub l e h e p a r i n ( P r e p a r a t i o n 3 a n d 4). T h e s a m e r e s u l t w a s o b t a i n e d w i t h P r e p a r a t i o n 5, w h i c h a c t u a l l y w a s a m i x t u r e of I - H e p a n d I - A T III. O n l y w h e n
t i o n , w h i c h s h o w e d n o a n t i c o a g u l a n t a c t i v i t y . As h a s b e e n r e c o g n i z e d , h e p a r i n h a s a n a f f i n i t y to c o m p l e x w i t h A T HI, a n d i n t h i s state, h e p a r i n s t i m u l a t e s t h e a n t i c o a g u l a n t a c t i v i t y of A T I U u p
TABLE 1.
Various preparations of immobilized heparin and antithrombin III, and their anticoagulant activity. Preparation no.
Immobilization procedure
Abbreviation
Anti coagulant activity
Schematic expression A
1
AT Ill and heparin were co-immobilized
I - - A T l l l . Hep
~--A g I11 5)f__Hep
++
~
--Hep
2
AT iii and excess h e p a r i n were co-immobilized
"d--AT II1
++
~-Hep 3
AT 111 was immobilized, h e p a r i n was b o u n d to AT III
~---AT l l l - - ( H e p )
+
4
Heparin was immobilized, AT I11 was bound to h e p a r i n
~ - - H e p - - ( A T 111)
+
5
AT Ill and heparin were immol)ilized separately
l - - A T Ill + l-Hep
~__
AT Ill +
~_
tlep
+
/I 6
Bovine serum a l b u m i n was immobilized as at control gel
Control
~--BSA
---
TABLE II.
Prolongation of plasma recalcification time by various immobilized preparations of heparin and/or anlithrombin IlL Plasma reealeifieation time (min) Immobilized preparation
Control I-Hep I-AT I I I I-AT IlI" Hep
Sepharose ~,B
PVA
Poly ItEMA
Carrier t.0 mg
25 mg
2.0 mg
2.0 mg
3.0 3.4 3.8 20
2.0 6.1 9.2 No clot
5.7 6.0 7.3 8.0
4.3 5.8 5.0 7.8
I m m o b i l i z e d p r e p a r a t i o n (Sepharose 4B : 1.0 mg in 20 Ixl buffer solution or 2.5 mg in 50 ~tl buffer solution, PVA : 2.0 rag, and Poly HEMA : 2.0 mg in 20 Ixl buffer solution) was mixed w i t h 200 ~1 p l a s m a p r e w a r m e d at 37°C. After one min incubation, 200 Ixl of 25 mM Ca2+ s o l u t i o n were added to the mixture, a n d p l a s m a recalcification time (PRCT) was measured. PRCT s h o w n here was the mean of a b o u t 10 me'~surements.
to 1 0 0 - f o l d [33-35]. H o w e v e r , t h i s c o o p e r a t i v e p h e n o m e n o n w a s n o t o b s e r v e d w h e n h e p a r i n w a s covalently immobilized and incubated with free
BIOCHIMIE, 1980, 62, n ° 8-9.
AT III and heparin were preincubated and then c o - i m m o b i l i z e d in a c o m p l e x f o r m , w a s tile s t i i n u l a t i v e e f f e c t p r e s e r v e d ( P r e p a r a t i o n 1 a n d 2).
598
Y. Miura
Effect on plasma r e c a l c i f i c a t i o n time (PRCT) of the c o - i m m o b i l i z e d p r e p a r a t i o n , I-AT I I I . Hep, was c o m p a r e d w i t h those of I-Hep and c o n t r o l p r e p a r a t i o n , as s h o w n in figure 1. Slight p r o l o n gation of P R C T was o b s e r v e d b o t h w i t h c o n t r o l and I-Hep, p r o b a b l y b e c a u s e of a d s o r p t i o n p h e n o m e n a of blood coagulation factors onto these preparations. I-AT I I I . Hep, upon i n c u b a t i o n in
I Carrier
500
I
I
: Sepharose
4B
--
.,-t.~ / ~ 0 •
I-AT
III. H e p mg)
and coll.
w e i g h t of c a r r i e r . W h i l e Sepharose 4B b o u n d 6.0 mg of h e p a r i n p e r gr d r y weight, PVA a n d Poly HEM, A i m m o b i l i z e d only 2.0 mg and 1.5 mg h e p a r i n p e r gr c a r r i e r , r e s p e c t i v e l y . Regardless of the b i n d i n g c a p a c i t y of carriers, c o - i m m o b i lized p r e p a r a t i o n s exhibited, in all cases, the best p r o l o n g a t i o n effect. T h r o m b i n plays an i m p o r t a n t role in fibrin clot f o r m a t i o n , c a t a l y z i n g the p r o t e o l y s i s of fibrinogen. A c c o r d i n g l y , a n t i t h r o m b i n a c t i v i t y of hepar i n a n d / o r AT III i m m o b i l i z e d on Poly HEMA w a s m e a s u r e d (fig. 2) : t h r o m b i n solution w a s i n c u b a t e d for 0 to 30 rain w i t h each i m m o b i l i z e d prep a r a t i o n , w h i c h w a s then r e m o v e d and the remaining thrombin activity was measured using fibrinogen as substrate. T h e n e u t r a l i z a t i o n of t h r o m b i n by I-AT I I I . Hep w a s almost instanta-
100
.o 50
I
2
Carrier
~
L~4 "":0 u~
io
m
100
i
~
I-Hep (2.5 ms)
I : Poly
I HEMA
Control
~--
--
.;
5
Control (2.5 mg)
,
I l0
0
Preincubation
l
20 Time
--
30
c i t r a t e d plasma, could s i g n i f i c a n t l y p r o l o n g the r e c a l c i f i c a t i o n time, i n d i c a t i n g that c a p t u r e of the a c t i v a t e d c o a g u l a t i o n factors was m u c h h i g h e r than in the case of s i m p l e a d s o r p t i o n . Table H s h o w s the effect of i m m o b i l i z a t i o n c a r r i e r . W h e n PVA or P o l y HEMA w a s used as a c a r r i e r f o r i m m o b i l i z a t i o n of h e p a r i n and AT III, the p r o l o n gation of P R C T w a s o b s e r v e d but to a s o m e w h a t lesser extent than S e p h a r o s e 4B w a s the c a r r i e r . This should be d u e to the d i f f e r e n c e in c a r r i e r , especially d i f f e r e n c e s in s u r f a c e area p e r unit 1980,
62, n ° 8-9.
I
(min)
Fie. 1 . - The prolongation o~ p l a s m a reealeifiealion time by c o - i m m o b i l i z e d a n t i t h r o m b i n I I l and heparin. Control preparation or 2.5 mg of I-Hep suspended in 50 ~1 of 10 mM Tris-HC1 buffer, pH 7.4 containing 0.145 M NaC1 and 1 per cent serum albumin, or 1.0 mg of I-AT I I I . Hep in 20 pl buffer solution were mixed with 200 I~l of freshly citrated bovine plasma. The mixture was incubated at 37°C for the specified time. After incubation 200 ~t ot 25 mM Ca2+ solution were added to the mixture, and the plasma clotting time was measured.
BIOCHIMIE,
~ 5o
"
"-.._.,.__I-.~'r
II1.~p~
.5
o I
I
I
10
20
3O
Incubation
Time
(min)
Fit;. 2. Decrease of t h r o m b i n activity upon incubation mith i m m o b i l i z e d heparin a n d / o r a n t i t h r o m bin II1. 5 mg of immobilized preparation suspended in 200 tt] buffer solution were mixed with 200 td of thrombin solution (0.5 unit). After incubation for the specified time, the mixture was filtered and the throml)in remaining in the supernatant solution was assayed by the clotting time method. -
-
neous. In the m i x t u r e w i t h I-AT III or I-Hep, g r a d u a l decrease in t h r o m b i n a c t i v i t y was observed, but not w i t h the c o n t r o l p r e p a r a t i o n . Table III shows the n e u t r a l i z a t i o n of [hrombin by h e p a r i n a n d / o r AT III i m m o b i l i z e d on Sel)harosc 4B. In this e x p e r i m e n t , r e m a i n i n g Iln'ombin ~ctivity 'was
A n t i c o a g u l a n t activity m e a s u r e d iu I w o d i f f e r e n t w a y s : a f t e r 10 r a i n i n c u b a t i o n w i t h e a c h b i l i z e d p r e p a r a t i o n s , S e p h a r o s e 4B diately removed and thrombin was
In o n e of t h e gel w a s assayed
of
artificial biomedical materials.
series, immoimmein t h e
TABLE III.
Decrease of thrombin activity on incubation with immobilized preparations of heparin and~or antithrombin lII. Remaining ttlrombin activity (percent) Immobilized preparation
Control I-Hep I-AT III I-AT I I l ' H e p
Assay after gel removal
Assay without gel removal
100 56 67 56
100 100 73 63
Heparin a n d / o r a n t i t h r o m b i n III immobilized on Sepharose JzB gel was used. 10 mg of immobilized preoaration were alohved to react with t h r o m b i n (84 ~,g in 20 u! buffer solution) for 10 rain at 37°C. The r e m a i n i n g activity of t h r o m b i n was assayed by using a peptideMCA as substrate.
s u p e r n a t a n t s o l u t i o n . In t h e o t h e r s e r i e s , t h r o m b i n a c t i v i t y w a s m e a s u r e d in the i n c u b a t i o n m i x t u r e , thus both free and bound thrombin were subject e d to assay. TABLE IV.
Decrease of Factor Xa aclioily upon incubation with immobilized preparations of heparin and~or antithrombin IlL Remaining factor Xa activity (per cent) Immobilized preparation
Assay after gel removal
Assay without gel rem0va I
thrombin was inactivated when bound to I . A T I I I • H e p o r I - A T III ; n o i n a c t i v a t i o n o c c u r r e d o n t h r o m b i n b o u n d to I - H e p w h e n a p e p t i d e _ MCA w a s u s e d as s u b s t r a t e . T o evalua.te t h e a n t i - F a c t o r Xa a c t i v i l y of t h e immobilized preparations of h e p a r i n and/or A T III, F a c t o r Xa s o l u t i o n w a s i n c u b a t e d w i t h e a c h i m l n o b i l i z e d p r e p a r a t i o n f o r 5 n l i n at 37°C, and the remaining activity was then assayed by a c l o t t i n g t i m e m e t h o d u s i n g F a c t o r VII a n d X d e f i c i e n t p l a s m a as t e s t i n g s o l u t i o n ( t a b l e IV). T h o u g h b o t h I - H e p a n d I-AT III d e c r e a s e d F a c t o r X:/ a c t i v i t y , t h e r a t e of n e u t r a l i z a t i o n b y the c o - i m m o bilizer preparation was nmch higher than that by I - A T III a n d I - H e p . S i m i l a r o b s e r v a t i o n s w e r e obtained for thrombin neulralizatiun, ttowever, t h e r a t e of F a c t o r Xa n e u t r a l i z a t i o n b y I-AT I H • H e p w a s s o m e w h a t h i g h e r t h a n t h a t of t h r o m b i n n e u t r a l i z a t i o n , a n d t h e ~mmunl of F a c t o r Xa n e u t r a l i z e d b y I-AT I I I . H e p w a s m o r e t h a n 1hat of l h r o m b i n n e u t r a l i z e d .
i
00 ] ]
~
I O O
I
; Factor Xa + Thrombin ; Factor xa
100 87 89 36
i
FIG.
5 3. --
I
/'
i0 I-AT III,Hep
100 76 67 34
I
!I
0 Control I-Hep I-AT lII I - A T IIl" Hep
599
20 (mg)
N e t l l r a l i z a t i o n of F a c l o r X a in lhe r e a c t i o n
of co-immobilized antithrolnbin Ill and heparin with Factor Xa and~or thrombin.
Heparin a n d / o r a n t i t h r o m b i n III immobilized on Sepharose 4B gel w a s used. 10 mg of i m m o b i l i z e d prep a r a t i o n were allowed to react w i t h Factor Xa (117I ttg in 20 ul buffer solution) for 5 rain at 37°C. The r e m a i n i n g activity of Factor Xa was measured by the clotting time method, using Factor VII and X deficient p l a s m a as substrate.
The specified a m o u n t of I-AT III• Hep on Sepharose 4B was allowed to react with the enzyme m i x t u r e of Factor Xa 90 Ixg and t h r o m b i n 19 ttg, or with 70 ~g of Factor Xa alone at I-AT I I I . Hep 5 rag, Factor Xa 100 ~tg at I-AT I I I . Hep 10 and 20 rag, respectively. After reacting for 10 min, the I-AT llI • Hep was removed f r o m the reaction m i x t u r e and the r e m a i n i n g Factor Xa activity was immediately measured using the peptide-MCA substrate.
These experiments revealed interesting points : t h r o m b i n c o u l d b i n d to all t y p e s of i m m o b i l i z e d i n h i b i t o r s , I - H e p , I-AT III, a n d I-AT I I I . H e p ;
So f a r as t h e n e u t r a l i z a t i o n of a s i n g l e e n z y m e . t h r o m b i n o r F a c t o r Xa, w a s c o n c e r n e d . I-AT I I [ - H e p w a s f o u n d to b e v e r y e f f e c t i v e . In c a s e
BIOCHIMIE, 1980, 62, n ° 8-9.
Y. M i u r a a n d coll.
600
of p r a c t i c a l a p p l i c a t i o n of b i o m a t e r i a l s to a n extracorporeal blood circulation system, the materials must be in contact with the blood in which 2O
I
%
I
.Q
10 o
el
Z
¥ 0
I
I
5
10
20
I-AT IIl*Hep (mg)
FIG. 4. Neutralization of thrombin in the reaction of co-immobilized antithrombin III and heparin with Factor Xa and~or thrombin. The specified a m o u n t of I-AT III .Hep, Sepharose 4B, was allowed to react w i t h t h e e n z y m e m i x t u r e of F a c t o r Xa 10 ILg and t h r o m b i n 20 ~g, or t h r o m b i n alone 20 Izg in the buffer solution. After r e a c t i n g for 3 min, the I-AT I I I . Hep was removed f r o m the reaction mixture a n d the r e m a i n i n g t h r o m b i n a c t i v i t y w a s i m m e diately m e a s u r e d u s i n g the peptide-MCA substrate. -
-
and consequently have been well studied. The a c t i v i t y of I~AT I I I - H e p w a s s t u d i e d i n t h e p r e s e n c e of t h e s e t w o e n z y m e s . F i g u r e 3 s h o w s t h e r e s u l t of s u c h a s t u d y , w h e r e b o t h F a c t o r X a a n d thrombin were allowed to react with the specified a m o u n t of I - A T I H - H e p , t h e a m o u n t of n e u t r a l ized Factor Xa was measured, and compared with t h e c a s e of I - A T I I I . H e p and Factor Xa alone. T h e a m o u n t of n e u t r a l i z e d F a c t o r X a w a s t h e s a m e i n b o t h cases. A c c o r d i n g l y , it w a s c o n c l u d e d t h a t t h e r e w a s n o e f f e c t of t h r o m b i n o n t h e n e u t r a l i z a t i o n of F a c t o r X a b y I - A T I I I . H e p . T h e n e u t r a l i z a t i o n of t h r o m b i n b y I-AT I I I . H e p w a s also e x a m i n e d i n t h e p r e s e n c e a n d a b s e n c e of F a c t o r X a (fig. 4). C o n t r a r y to t h e a b o v e c a s e , t h e n e u t r a l i z a t i o n of t h r o m b i n w a s d e c r e a s e d b y t h e p r e s e n c e of F a c t o r Xa. T a b l e ¥ c o m p a r e s t h e n e u t r a l i z e d a m o u n t s of t h e s e t w o e n z y m e s i n t h r e e d i f f e r e n t w a y s of i n c u b a t i o n w i t h e n z y m e s o l u tion. When thrombin or Factor Xa was incubated w i t h 5 m g of I - A T I I I " H e p , 9.0 ~g o r 15.3 e g of enzyme were neutralized, respectively. When a m i x t u r e of t h r o m b i n a n d F a c t o r X a w a s i n c u b a t e d w i t h t h e s a m e a m o u n t of I - A T I I I • H e p , 15.7 gg of Factor Xa were neutralized, but only a trace a m o u n t of t h r o m b i n w a s n e u t r a l i z e d . T h e n,eutral i z e d a m o u n t of F a c t o r X a b y I - A T H I • H e p w h e n t h r o m b i n w a s a b s e n t , w a s t h e s a m e as w h e n thrombin was present. An additional experiment in w h i c h m u c h l a r g e r a m o u n t s of t h r o m b i n w e r e
TABLE V. Neutralization of Factor Xa and~or l h r o m b i n upon incubation w i t h c o - i m m o b i l i z e d heparin and a n l i t h r o m b i n I l L Enzyme
1. 2. 3.
Factor Xa Thrombiu Factor Xa Thrombin
Initial amout (~tg)
Remaining activily after reaction tper cent)
Neutralized amout (ug)
41.5 15.5 43.4 18.7
62 99 65 52
15.7 0.2 15 3 9.0
Sepharose 4B was used as a crrier for i m m o b i l i z a t i o n . The specified a m o u n t of Factor Xa a n d / o r t h r o b i n was allowede to react w i t h 5 mg of I-AT I I I . Hep in 10 mM Tris-HC1 buffer, pH 7.4 c o n t a i n i n g 0.145 M NaC1 a n d 1 per cent b o v i n e serum a l b u m i n . After reacting for 10 win, I-AT I I I . Hep was removed f r o m the reaction m i x t u r e a n d the r e m a i n i n g enzyme activity was i m m e d i a t e l y m e a s u r e d using the peptide-MCA s n b s t r a t e s .
coagulation factors would be activated, and the c l o t t i n g b e j u s t r e a d y to p r o c e e d . T h u s , t h e e v a l u a t i o n of i n h i b i t o r y a c t i v i t y of I - A T } I I . H e p i n t h e p r e s e n c e o f t h e s e a c t i v a t e d f a c t o r s a p p e a r s to b e important. Among the activated factors in blood, t h r o m b i n a n d F a c t o r Xa a r e t h e m o s t i m p o r t a n t
BIOCHIMIE, 1980, 62, n ° 8-9.
mixed with Factor Xa and the mixture was allowe d to r e a c t w i t h I - A T I I I . H e p , reinforced the a b o v e o b s e r v a t i o n b e c a u s e n o d e c r e a s e of n e u t r a l i z e d a m o u n t of F a c t o r X a w a s r e c o g n i z e d , e v e n i n t h e p r e s e n c e of e x c e s s t h r o m b i n . O n t h e o t h e r h a n d , t h e n e u t r a l i z a t i o n of t h r o m b i n w a s n o t
Anticoagulant
activity
of artificial
o c c u r r e d b y t h e p r e s e n c e of s t o i c h i o m e t r i c a l a m o u n t of F a c t o r Xa. As a r e s u l t , it w a s c o n c l u d e d t h a t I - A T I l l . H e p w'as c a p a b l e of n e u t r a l i z i n g F a c t o r X a p r a c t i c a l l y i n p r e f e r e n c e to t h r o m bin. 1 ~g of A T I I l , b y n e u t r a l i z i n g 32 u n i t s of F a c t o r Xa, i n d i r e c t l y p r e v e n t s t h e g e n e r a t i o n of 16'00 N I H u n i t s of t h r o m b i n . T o d i r e c t l y n e u t r a lize t h i s a m o u n t of t h r o m b i n , 1000 ~g of A T I I I a r e r e q u i r e d . M u c h m o r e A T I I I is n e c e s s a r y to i n h i b i t t h e t h r o m b i n - f i b r i n o g e n r e a c t i o n t h a n is r e q u i r e d to n e u t r a l i z e F a c t o r Xa. F a c t o r X a is a more potent thrombogenic agent than thrombin itself. I n t h e p r e s e n t s t u d y , i t w a s s h o w n t h a t a g r e a t e r a m o u n t of F a c t o r X a t h a n o f t h r o m b i n w a s preferentially and rapidly neutralized by I-AT I I I . H e p . T h e r a p i d n e u t r a l i z , a t i o n of F a c t o r X a i n p r e f e r e n c e to t h r o m b i n is f a v o r a b l e f o r effect i v e p r e v e n t i o n of b l o o d c o a g u l a t i o n . T h e r e f o r e , t h e c o - i m m o b i l i z a t i o n of h e p a r i n a n d A T H I w o u l d b e a u s e f u l m e t h o d to o b t a i n a n t i - t h r o m b o genic artificial biomedical materials.
REFERENCES. 1. Jesty, J. (1979) J. Biol. Chem., 254, 1044-1049. 2. Abligaard, U. (1969) Scand. J. clin. Lab. Inves., 24, 23-27. 3. Binder, B. (1973) Thrombos. Diathes. haemorrh. (Stuttg.), 30, 280-283. 4. Jesty, J. (1978) Arch. Bioehem. Biophys., 185, 165173. 5. Bjork, I. ,~ N o r d e n m a n , B. (1976) Eur. J. Biochem., 68, 507-511. 6. 1)amus, P. S., Hicks, M. a Rosenberg, R. D. (1973) Nature, 246, 355-357. 7. Hotmer, E., Soderstrom, G. ~ Anderson, L.-O. (1979) Eur. J. Biochem., 93, 1-5. 8. Li, E. H. H., Fenton, II, J. W. ~ F e i n m a n , R. D. (1976) Arch. Biochem. Biophys., 175, 153-159.
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9. Joist, J. H. ~ Mustard, J. F. (1975 in <> (Bradshaw, R. A. a Wessler, S. ed.) pp. 255-262, P l e n u m Press, New York and London. 10. Mustard, J. F. (1970) Pharmacol. Rev., 22, 97-187. 11. Hoffmer, A. S., Schmer, G., Harris, C. ~ Kraft, W. G. (1972) Trans. Amer. Sac. Arlif. Int. Organs, 18, 10-16. 12. Largergren, H. a Eriksson, J. (1971) Trans. Amer. Sac. Artif. Int. Organs, 17, 10-15. 13. Yen, S. P. S. a R e m b a u m , A. (1971) J. Biomed. Materials Res. S y m p o s i u m , 1, 83-97. 14. R e m b a u m , A. (1969) NASA Tech. Brief., 69-10299. 15. Merker, R. L., Elyash, L. J. Mayhew, S. H. a Wang, 3. Y. C. (1969) AHP, 29-38. 16. Gott, V. L., Whiffen, J. D. ,~ Dutton, R. C. (1963) Science, 142, 1297-1298. 17. Goosen, M. F. A. ~ Sefton, M. V. (1979) J. Biomed. Mater. Res., 13, 347-364. 18. Schmer, G., Teng, L. N. L., Cok, J. J., Vizzo, J. E. Francisco, M. M. a Scrbner, B. H. (1976) Trans. Amer. Sac. Artif. Int. Organs, 22, 654-662. 19. Schmer, G. (1972) Trans. Amer. Sac. Artif. Int. Organs, 18, 321-324. 20. Miura, Y., Miyamoto, K., Aoyagi, S. ~ Kusada, Y. (1978) Artificial Organs, 7, 193-196. 21. Miura, Y., Aoyagi, S. ,~ Miyamoto, K. (1979) A r t i ficial Organs, 8, 288-291. 22. Andersson, M. M., Borg, H. a Andersson, L.-O. (1974) Thromb. Res., 5, 439-452. 23. Damns, P. S. a Wallace, G. A. (1974) Bioehem. Biophys. Res. Commun., 61, 1147-1153. 24. Rosenberg, R. D. a Damus, P. S. (1973) J. Biol. Chem., 248, 649046505. 25. Aronson, D. L., Mustafa, A. J. & Mushinski, J F. (1969) Biochim. Biophys. Acla, 188, 25-30. 26. Yin, E. T. a Wessler, S. (1968) J. Biol. Chem., 243, 112-117. 27. F u j i k a w a , K., Legaz, M. E. & Davie, E. W. (1972) Biochemistry, 11, 4892-4898. 28. Iverius, P. H. (1971) Bioehem. J., 124, 677-683. 29. Asai, K. (1976) Jap. J. Clin. Path., special issue, 27, 143-157. 30. Sakuragawa, N. (19'76) Jap. J. Clin. Path., special issue, 27, 130-142. 31. Morita, T., Kato, H., Iwanaga, S., Takada, K., Kimura, T. ~ S a k a k i b a r a , S. (1977) J. Biochem., 82, 1495-1498. 32. Miura, Y., Aoyagi, S. ~ Miyamoto, K. (1979) J. Bioreed. Mater. Res., snbmitted. "]3. E i n a r r s o n , R. • Andersson, L.-O. (1977) Biochiul. Biophys. Acta, 490, 104-111. 34. Andersson, L.-O., Engman, L. ,~ Henninsson. E. (1977) J. I m m u n o l . Methods, 14, 271-281. 35. H66h, M., Bj6rj, I., Hoopwood, .1. ~ Lindahl, U. (1976) FEBS Left., 66, 90-93.
Anticoagulant activity of artificial biomedical materials with co-immobilized antithrombin III and heparin. Yoshiharu MJURA, Sadayoshi AOYAGI, Fumiaki IKEDA and Kazuhisa MIYAMOTO.
Department of Biochemical Engineering, Faculty o[ Pharmaceutical Sciences, Osaka University, Saita, Osaka, 565 Japan.
R6sum6.
c o a q u l a t i o n factors includinq Factor Xa a n d thrombin. Heparin stimulates the inactivation of Factor Xa a n d thrombin b y AT III. Thus AT III a n d h e p a r i n were co-immobilized on Sep h a r o s e 4B, p o l y v i n y l alcohol, p o l y h y d r o x y ethyl m e t h a c r y l a t e a n d silicone-coated n y l o n b y the c y a n o q e n bromide procedure. Those coimmobilized preparations, a b b r e v i a t e d a s I-AT III'Hep, actively neutralized both Factor Xa a n d thrombin. The activity of I-AT III'Hep w a s m u c h hiqher t h a n immobilized h e p a r i n a n d / o r immobilized AT III. I-AT III'Hep, like soluble AT III a n d heparin, i n s t a n t a n e o u s l y neutralized both thrombin a n d Factor Xa. W h e n two e n z y m e s , thrombin a n d Factor Xa, w e r e present, I.AT III'Hep neutralized Factor Xa in p r e f e r e n c e to thrombin : The neutralization of thrombin w a s inhibited b y the p r e s e n c e of Factor Xa, but neutralization of Factor Xa w a s i n d e p e n d e n t of the p r e s e n c e of thrombin. The a m o u n t of Factor Xa neutralized w a s hiqher t h a n that of thrombin.
Des m a t 6 r i a u x b i o m 6 d i c a u x antithromboq~nes ont 6t6 pr6par6s. L'antithrombine III (AT III) et l ' h 6 p a r i n e (Hep) ont 6t6 coimmobilis6es sur divers supports. Ces p r 6 p a r a t i o n s neutralisent i n s t a n t a n 6 m e n t le facteur Xa et la thrombine. Leur activit6 est plus q r a n d e que celles de I'AT III et de l ' h 6 p a r i n e immobilis6es s6par6ment. En pr6sence des deux enzymes, facteurs Xa et thrombine, le m a t 6 r i a u neutraBse pr6f6rentieUement le facteur Xa.
Summary. A n a p p r o a c h to providinq a n t i c o a q u l a n t activity to b i o m e d i c a l materials w a s presented, a p p l y i n q a n immobilization technique. Antithrombin III (AT III) inactivates the activated
Introduction. Blood coagulation is now somewhat elucidated at the molecular level. The coagulation is triggered by both intrinsic and extrinsic causes, and coagulation factors (also known as clotting factors) are successively activated. Factor X, one of the coagulation factors, is at the intersection of the two pathways, intrinsic and extrinsic. Activated Factor X, Factor Xa, converts prothromb,in to thrombin. At the last stage of blood coagulation, thrombin catalyzes the proteolysis of fibrinogen to fibrin, and fibrin clot results. Antithrombin III (A.T IlI) inactivates a number of the activated coagulation factors including Factor Xa and thrombin [1-4]. As a result, AT III plays an important role for keeping the liquidity of blood. The creation of biomedical materials capable of preventing the agglutinative thrombus has
been a very important subject in the development of artificial organs. Heparin, accelerating the neutralization rate of activaled coagulation factors by AT III [5-8], has been found to be an effective anticoagulant, and has been used to prevent thrombus in artificial organs. Administration of a large amount of heparin, however, causes bleeding, and platelet aggregation and adhesion [9, 1~]. Since A.T III is present in blood, it has been commonly considered that the AT III necessary for cooperation with heparin can be naturally supplied from blood. Thus, immobilization of heparin has been proposed for obtaining non thrombogenic biomaterials [ll-lg]. We observed that heparin covalently immobilized on biomaterials had an ability to bind thrombin and Factor Xa, but that these enzymes thus bound to the immobilized heparin were still active E321. This cooperative effect on the neutralization of Factor Xa and thrombin, i.e. the instantaneous
40
596
Y. Mittra and coll.
n e u t r a l i z a t i o n e x h i b i t e d b y AT III i n t h e p r e s e n c e of s o l u b l e h e p a r i n , w a s n o t o b s e r v e d w h e n h e p a r i n w a s c o v a l e n t l y i m m o b i l i z e d . It w a s f o u n d , however, that heparin, when co-immobilized with A T III, e x h i b i t e d m u c h h i g h e r a n t i c o a g u l a n t a c t i vity than immobilized heparin and/or immobil i z e d A T H I [20, 21]. E m p l o y i n g t h e c y a n o g e n b r o mide procedure, co-immobilization was successf u l l y a p p l i e d to s o m e b i o m a t e r i a l s s u c h as p o l y v i n y l al.cohol, p o l y h y d r o x y e t h y l methacrylate, a n d s i l i c o n e - c o a t e d n y l o n [20, 21]. H e r e , a m o r e detailed s t u d y on the a n t i c o a g u l a n t activity of t h e c o - i m m o b i l i z e d p r e p a r a t i o n o f AT III a n d h e p a r i n i s p r e s e n t e d , a n d t h e a c t i o n o f A T III i n t h e b l o o d c o a g u l a t i o n s y s t e m is d i s c u s s e d .
Materials and Methods. CNBr-activated Sepharose 4B was purchased f r o m P h a r m a c i a Fine Chemicals. Polyvinyl alcohol (PVA) whose fibers were 30 to 50 Ixm in diameter, p o l y h y d r o x y e t h y l m e t h a c r y l a t e (Poly HEMA) whose granules were 150 to 48 mesh, were used as carrier materials for the i m m o b i l i z a t i o n . Heparin, t h r o m b i n , bovine serum a l b u m i n and fibrinogen were purchased f r o m Sigma Chemical Company. Heparin (sodium salt) isolated f r o m porcine i n t e s t i n a l mucosa, had 170 USP units per mg protein. T h r o m b i n c o n t a i n i n g 600 NIH units per mg protein, isolated f r o m bovine plasma, was s u b s t a n t i a l l y free of Factors II, V, VII, IX, X, p l a s m i n and Factor Xa. F i b r i n o g e n isolated f r o m bovine blood was a b o u t 95 per cent clottable. Bovine a n t i t h r o m b i n III, AT III, and Factor X were purified as described previously [72-26]. Factor X w a s activated by Russell's Viper Venom in cephalin [271, purchased f r o m Sigma Chemical Company. F a c t o r VII and X deficient plasma, lyophilized f r o m bovine plasma, was purchased f r o m Sigma Chemical Company. Tert-butoxycarbonyl-L-Valyl-L-Prolyl-L-Arginine 4-M.ethyl-Coumarinyl-7-Amide (Boc-Val-Pro-Arg-MCA) which is a substrate for t h r o m b i n in the new a s s a y method, tertb u t o x y c a r b o n y l - L - Isoleucyl-L-Glutamyl-Glycyl-L-Arginine 4-Methyl-Coumarinyl-7-Amide (Boc-lle-Glu-GlyArg-MCA) for Factor Xa were purchased f r o m Peptide Institute Corporation. AT III a n d / o r h e p a r i n was coupled to CNBr-activated PVA, P.oly HEMA, silicone-coated n y l o n or Sepharose 4B as described previously [28, 20, 21] and abbreviated as follows : immobilized h e p a r i n (I-Hep), immobilized AT III p.rotected b y acetylated hepa~in (I-AT III) and co-immobilized AT III and h e p a r i u (I-AT I I I . Hep). As a control preparation, bovine s e r u m a l b u m i n was. i m m o b i l i z e d on these activated materials. The m e a s u r e m e n t of p l a s m a recalciflcation time (PRCT) was carried out in the following m a n n e r : the immobilized preparation, suspended in 10 mM Tris-HC1 buffer, pH 7.4 containing 0.145 M NaC1, was added to the 200 /xl of p r e w a r m e d eitrated bovine p l a s m a and preincubated for the defined t i m e at 37°C. 25 mM Ca2+ solution was added to the m i x t u r e and PRCT was m e a s u r e d w i t h gentle agitation.
BIOCHIMIE, 1980, 62, n ° 8-9.
The d e t e r m i n a t i o n of a n t i - t h r o m b i n and a n t i - F a c t o r Xa activities of i m m o b i l i z e d p r e p a r a t i o n s was carried out in the following m a n n e r : The immobilized prep a r a t i o n was allowed to react with t h r o m b i n a n d / o r Factor Xa u n d e r stirring at 37°C. After reacting for the defined time, the r e m a i n i n g enzyme activity was i m m e d i a t e l y measured by means of the clotting time method or the new assay method using the peptideM.'CA as a s~bstrate in two series of assays : In the first series, t h e i m m o b i l i z e d p r e p a r a t i o n was i m m e diately removed f r o m the reaction m i x t u r e after the reaction, and the r e m a i n i n g enzyme activity of reacted solution w i t h o u t i m m o b i l i z e d p r e p a r a t i o n w a s measured. In a n o t h e r a s s a y way, the immobilized prepar a t i o n was not removed a f t e r the reaction, and the enzyme activity of reaction m i x t u r e containing the i m m o b i l i z e d p r e p a r a t i o n was measured. All control e x p e r i m e n t s were carried out using the control preparations, on w h i c h bovine s e r u m a l b u m i n had been immobilized. No a n t i - t h r o m b i n and a n t i - F a c t o r Xa activities of the i m m o b i l i z e d a l b u m i n were observed. However, w h e n Sepharose 4B was used as a carrier, a ltg o r d e r a d s o r p t i o n of enzyme on Sepharose 4B gel was observed in the reaction of the immobilized preparation w i t h the enzyme. 1 per cent bovine s e r u m a l b u m i n was t h e n added to the reaction m i x t u r e to prevent the a d s o r p t i o n of e n z y m e protein. The a d s o r p t i o n of Factor Xa on the Sepharose 4B had consequently been eliminated, h o w e v e r t h a t of small a m o u n t s of t h r o m b i n still occurred. So, a n t i - e n z y m e activity of immobilized AT III a n d / o r h e p a r i n was obtained f r o m the difference between the r e m a i n i n g enzyme activity w h e n using i m m o b i l i z e d AT III a n d / o r h e p a r i n in the reaction and w h e n using the control preparation. Assay for t h r o m b i n activity by the clotting time m e t h o d was done using the previously described method and 1 per cent fibrinogen solution substrate [29]. Factor Xa activity was m e a s u r e d by a previously described m e t h o d [30] using Factor VII and X deficient p l a s m a as s ubstrate. T h r o m b i n activity and Factor Xa activity were also m e a s u r e d b y the new assay m e t h o d using peptide-MCA substrates. 50 ~1 to 101) ~xl of the enzyme solution were added to 1.0 ml of 0.1 mM peptide-MCA in 50 mM TrisHC1 buffer, pH 8.0, containing 100 mM NaC1 and 10 raM CaC12, previously p r e w a r m e d to 37~C. The reaction was carried out at 37°C for 3 to 10 min and stopped by adding 17 per cent acetic acid solution to give a final concentration of 10 per cent. The relative fluorescence of produced a m i n o - 4 - m e t h y l - c o u m a r i n , AMC, was measured, using excitation and emission wavelengths of 380 n m and 460 nm, respectively, in a fluorescence spectrophotometer, HITACHI model MPF-2A. The i n s t r u m e n t was standardized so t h a t a 10 ~M solution of AMC in 0.1 per cent d i m e t h y l s u l f o x i d e containing 10 per cent acetic acid gives 1.0 relative fluorescence [31].
R e s u l t s and Discussion. Various co-immobilized preparations or an i m m o b i l i z e d m i x e d p r e p a r a t i o n of A T I I I a n d heparin were examined for anticoagulant activity. R e s u l t s a r e s u m m a r i z e d i n t a b l e I. B o v i n e s e r u m a l b u m i n w a s i m m o b i l i z e d as a c o n t r o l p r e p a r a -
Anticoagulant activity of artificial biomedical materials.
597
A T III, o r w h e n I - A T I I I w a s i n c u b a t e d w i t h solub l e h e p a r i n ( P r e p a r a t i o n 3 a n d 4). T h e s a m e r e s u l t w a s o b t a i n e d w i t h P r e p a r a t i o n 5, w h i c h a c t u a l l y w a s a m i x t u r e of I - H e p a n d I - A T III. O n l y w h e n
t i o n , w h i c h s h o w e d n o a n t i c o a g u l a n t a c t i v i t y . As h a s b e e n r e c o g n i z e d , h e p a r i n h a s a n a f f i n i t y to c o m p l e x w i t h A T HI, a n d i n t h i s state, h e p a r i n s t i m u l a t e s t h e a n t i c o a g u l a n t a c t i v i t y of A T I U u p
TABLE 1.
Various preparations of immobilized heparin and antithrombin III, and their anticoagulant activity. Preparation no.
Immobilization procedure
Abbreviation
Anti coagulant activity
Schematic expression A
1
AT Ill and heparin were co-immobilized
I - - A T l l l . Hep
~--A g I11 5)f__Hep
++
~
--Hep
2
AT iii and excess h e p a r i n were co-immobilized
"d--AT II1
++
~-Hep 3
AT 111 was immobilized, h e p a r i n was b o u n d to AT III
~---AT l l l - - ( H e p )
+
4
Heparin was immobilized, AT I11 was bound to h e p a r i n
~ - - H e p - - ( A T 111)
+
5
AT Ill and heparin were immol)ilized separately
l - - A T Ill + l-Hep
~__
AT Ill +
~_
tlep
+
/I 6
Bovine serum a l b u m i n was immobilized as at control gel
Control
~--BSA
---
TABLE II.
Prolongation of plasma recalcification time by various immobilized preparations of heparin and/or anlithrombin IlL Plasma reealeifieation time (min) Immobilized preparation
Control I-Hep I-AT I I I I-AT IlI" Hep
Sepharose ~,B
PVA
Poly ItEMA
Carrier t.0 mg
25 mg
2.0 mg
2.0 mg
3.0 3.4 3.8 20
2.0 6.1 9.2 No clot
5.7 6.0 7.3 8.0
4.3 5.8 5.0 7.8
I m m o b i l i z e d p r e p a r a t i o n (Sepharose 4B : 1.0 mg in 20 Ixl buffer solution or 2.5 mg in 50 ~tl buffer solution, PVA : 2.0 rag, and Poly HEMA : 2.0 mg in 20 Ixl buffer solution) was mixed w i t h 200 ~1 p l a s m a p r e w a r m e d at 37°C. After one min incubation, 200 Ixl of 25 mM Ca2+ s o l u t i o n were added to the mixture, a n d p l a s m a recalcification time (PRCT) was measured. PRCT s h o w n here was the mean of a b o u t 10 me'~surements.
to 1 0 0 - f o l d [33-35]. H o w e v e r , t h i s c o o p e r a t i v e p h e n o m e n o n w a s n o t o b s e r v e d w h e n h e p a r i n w a s covalently immobilized and incubated with free
BIOCHIMIE, 1980, 62, n ° 8-9.
AT III and heparin were preincubated and then c o - i m m o b i l i z e d in a c o m p l e x f o r m , w a s tile s t i i n u l a t i v e e f f e c t p r e s e r v e d ( P r e p a r a t i o n 1 a n d 2).
598
Y. Miura
Effect on plasma r e c a l c i f i c a t i o n time (PRCT) of the c o - i m m o b i l i z e d p r e p a r a t i o n , I-AT I I I . Hep, was c o m p a r e d w i t h those of I-Hep and c o n t r o l p r e p a r a t i o n , as s h o w n in figure 1. Slight p r o l o n gation of P R C T was o b s e r v e d b o t h w i t h c o n t r o l and I-Hep, p r o b a b l y b e c a u s e of a d s o r p t i o n p h e n o m e n a of blood coagulation factors onto these preparations. I-AT I I I . Hep, upon i n c u b a t i o n in
I Carrier
500
I
I
: Sepharose
4B
--
.,-t.~ / ~ 0 •
I-AT
III. H e p mg)
and coll.
w e i g h t of c a r r i e r . W h i l e Sepharose 4B b o u n d 6.0 mg of h e p a r i n p e r gr d r y weight, PVA a n d Poly HEM, A i m m o b i l i z e d only 2.0 mg and 1.5 mg h e p a r i n p e r gr c a r r i e r , r e s p e c t i v e l y . Regardless of the b i n d i n g c a p a c i t y of carriers, c o - i m m o b i lized p r e p a r a t i o n s exhibited, in all cases, the best p r o l o n g a t i o n effect. T h r o m b i n plays an i m p o r t a n t role in fibrin clot f o r m a t i o n , c a t a l y z i n g the p r o t e o l y s i s of fibrinogen. A c c o r d i n g l y , a n t i t h r o m b i n a c t i v i t y of hepar i n a n d / o r AT III i m m o b i l i z e d on Poly HEMA w a s m e a s u r e d (fig. 2) : t h r o m b i n solution w a s i n c u b a t e d for 0 to 30 rain w i t h each i m m o b i l i z e d prep a r a t i o n , w h i c h w a s then r e m o v e d and the remaining thrombin activity was measured using fibrinogen as substrate. T h e n e u t r a l i z a t i o n of t h r o m b i n by I-AT I I I . Hep w a s almost instanta-
100
.o 50
I
2
Carrier
~
L~4 "":0 u~
io
m
100
i
~
I-Hep (2.5 ms)
I : Poly
I HEMA
Control
~--
--
.;
5
Control (2.5 mg)
,
I l0
0
Preincubation
l
20 Time
--
30
c i t r a t e d plasma, could s i g n i f i c a n t l y p r o l o n g the r e c a l c i f i c a t i o n time, i n d i c a t i n g that c a p t u r e of the a c t i v a t e d c o a g u l a t i o n factors was m u c h h i g h e r than in the case of s i m p l e a d s o r p t i o n . Table H s h o w s the effect of i m m o b i l i z a t i o n c a r r i e r . W h e n PVA or P o l y HEMA w a s used as a c a r r i e r f o r i m m o b i l i z a t i o n of h e p a r i n and AT III, the p r o l o n gation of P R C T w a s o b s e r v e d but to a s o m e w h a t lesser extent than S e p h a r o s e 4B w a s the c a r r i e r . This should be d u e to the d i f f e r e n c e in c a r r i e r , especially d i f f e r e n c e s in s u r f a c e area p e r unit 1980,
62, n ° 8-9.
I
(min)
Fie. 1 . - The prolongation o~ p l a s m a reealeifiealion time by c o - i m m o b i l i z e d a n t i t h r o m b i n I I l and heparin. Control preparation or 2.5 mg of I-Hep suspended in 50 ~1 of 10 mM Tris-HC1 buffer, pH 7.4 containing 0.145 M NaC1 and 1 per cent serum albumin, or 1.0 mg of I-AT I I I . Hep in 20 pl buffer solution were mixed with 200 I~l of freshly citrated bovine plasma. The mixture was incubated at 37°C for the specified time. After incubation 200 ~t ot 25 mM Ca2+ solution were added to the mixture, and the plasma clotting time was measured.
BIOCHIMIE,
~ 5o
"
"-.._.,.__I-.~'r
II1.~p~
.5
o I
I
I
10
20
3O
Incubation
Time
(min)
Fit;. 2. Decrease of t h r o m b i n activity upon incubation mith i m m o b i l i z e d heparin a n d / o r a n t i t h r o m bin II1. 5 mg of immobilized preparation suspended in 200 tt] buffer solution were mixed with 200 td of thrombin solution (0.5 unit). After incubation for the specified time, the mixture was filtered and the throml)in remaining in the supernatant solution was assayed by the clotting time method. -
-
neous. In the m i x t u r e w i t h I-AT III or I-Hep, g r a d u a l decrease in t h r o m b i n a c t i v i t y was observed, but not w i t h the c o n t r o l p r e p a r a t i o n . Table III shows the n e u t r a l i z a t i o n of [hrombin by h e p a r i n a n d / o r AT III i m m o b i l i z e d on Sel)harosc 4B. In this e x p e r i m e n t , r e m a i n i n g Iln'ombin ~ctivity 'was
A n t i c o a g u l a n t activity m e a s u r e d iu I w o d i f f e r e n t w a y s : a f t e r 10 r a i n i n c u b a t i o n w i t h e a c h b i l i z e d p r e p a r a t i o n s , S e p h a r o s e 4B diately removed and thrombin was
In o n e of t h e gel w a s assayed
of
artificial biomedical materials.
series, immoimmein t h e
TABLE III.
Decrease of thrombin activity on incubation with immobilized preparations of heparin and~or antithrombin lII. Remaining ttlrombin activity (percent) Immobilized preparation
Control I-Hep I-AT III I-AT I I l ' H e p
Assay after gel removal
Assay without gel removal
100 56 67 56
100 100 73 63
Heparin a n d / o r a n t i t h r o m b i n III immobilized on Sepharose JzB gel was used. 10 mg of immobilized preoaration were alohved to react with t h r o m b i n (84 ~,g in 20 u! buffer solution) for 10 rain at 37°C. The r e m a i n i n g activity of t h r o m b i n was assayed by using a peptideMCA as substrate.
s u p e r n a t a n t s o l u t i o n . In t h e o t h e r s e r i e s , t h r o m b i n a c t i v i t y w a s m e a s u r e d in the i n c u b a t i o n m i x t u r e , thus both free and bound thrombin were subject e d to assay. TABLE IV.
Decrease of Factor Xa aclioily upon incubation with immobilized preparations of heparin and~or antithrombin IlL Remaining factor Xa activity (per cent) Immobilized preparation
Assay after gel removal
Assay without gel rem0va I
thrombin was inactivated when bound to I . A T I I I • H e p o r I - A T III ; n o i n a c t i v a t i o n o c c u r r e d o n t h r o m b i n b o u n d to I - H e p w h e n a p e p t i d e _ MCA w a s u s e d as s u b s t r a t e . T o evalua.te t h e a n t i - F a c t o r Xa a c t i v i l y of t h e immobilized preparations of h e p a r i n and/or A T III, F a c t o r Xa s o l u t i o n w a s i n c u b a t e d w i t h e a c h i m l n o b i l i z e d p r e p a r a t i o n f o r 5 n l i n at 37°C, and the remaining activity was then assayed by a c l o t t i n g t i m e m e t h o d u s i n g F a c t o r VII a n d X d e f i c i e n t p l a s m a as t e s t i n g s o l u t i o n ( t a b l e IV). T h o u g h b o t h I - H e p a n d I-AT III d e c r e a s e d F a c t o r X:/ a c t i v i t y , t h e r a t e of n e u t r a l i z a t i o n b y the c o - i m m o bilizer preparation was nmch higher than that by I - A T III a n d I - H e p . S i m i l a r o b s e r v a t i o n s w e r e obtained for thrombin neulralizatiun, ttowever, t h e r a t e of F a c t o r Xa n e u t r a l i z a t i o n b y I-AT I H • H e p w a s s o m e w h a t h i g h e r t h a n t h a t of t h r o m b i n n e u t r a l i z a t i o n , a n d t h e ~mmunl of F a c t o r Xa n e u t r a l i z e d b y I-AT I I I . H e p w a s m o r e t h a n 1hat of l h r o m b i n n e u t r a l i z e d .
i
00 ] ]
~
I O O
I
; Factor Xa + Thrombin ; Factor xa
100 87 89 36
i
FIG.
5 3. --
I
/'
i0 I-AT III,Hep
100 76 67 34
I
!I
0 Control I-Hep I-AT lII I - A T IIl" Hep
599
20 (mg)
N e t l l r a l i z a t i o n of F a c l o r X a in lhe r e a c t i o n
of co-immobilized antithrolnbin Ill and heparin with Factor Xa and~or thrombin.
Heparin a n d / o r a n t i t h r o m b i n III immobilized on Sepharose 4B gel w a s used. 10 mg of i m m o b i l i z e d prep a r a t i o n were allowed to react w i t h Factor Xa (117I ttg in 20 ul buffer solution) for 5 rain at 37°C. The r e m a i n i n g activity of Factor Xa was measured by the clotting time method, using Factor VII and X deficient p l a s m a as substrate.
The specified a m o u n t of I-AT III• Hep on Sepharose 4B was allowed to react with the enzyme m i x t u r e of Factor Xa 90 Ixg and t h r o m b i n 19 ttg, or with 70 ~g of Factor Xa alone at I-AT I I I . Hep 5 rag, Factor Xa 100 ~tg at I-AT I I I . Hep 10 and 20 rag, respectively. After reacting for 10 min, the I-AT llI • Hep was removed f r o m the reaction m i x t u r e and the r e m a i n i n g Factor Xa activity was immediately measured using the peptide-MCA substrate.
These experiments revealed interesting points : t h r o m b i n c o u l d b i n d to all t y p e s of i m m o b i l i z e d i n h i b i t o r s , I - H e p , I-AT III, a n d I-AT I I I . H e p ;
So f a r as t h e n e u t r a l i z a t i o n of a s i n g l e e n z y m e . t h r o m b i n o r F a c t o r Xa, w a s c o n c e r n e d . I-AT I I [ - H e p w a s f o u n d to b e v e r y e f f e c t i v e . In c a s e
BIOCHIMIE, 1980, 62, n ° 8-9.
Y. M i u r a a n d coll.
600
of p r a c t i c a l a p p l i c a t i o n of b i o m a t e r i a l s to a n extracorporeal blood circulation system, the materials must be in contact with the blood in which 2O
I
%
I
.Q
10 o
el
Z
¥ 0
I
I
5
10
20
I-AT IIl*Hep (mg)
FIG. 4. Neutralization of thrombin in the reaction of co-immobilized antithrombin III and heparin with Factor Xa and~or thrombin. The specified a m o u n t of I-AT III .Hep, Sepharose 4B, was allowed to react w i t h t h e e n z y m e m i x t u r e of F a c t o r Xa 10 ILg and t h r o m b i n 20 ~g, or t h r o m b i n alone 20 Izg in the buffer solution. After r e a c t i n g for 3 min, the I-AT I I I . Hep was removed f r o m the reaction mixture a n d the r e m a i n i n g t h r o m b i n a c t i v i t y w a s i m m e diately m e a s u r e d u s i n g the peptide-MCA substrate. -
-
and consequently have been well studied. The a c t i v i t y of I~AT I I I - H e p w a s s t u d i e d i n t h e p r e s e n c e of t h e s e t w o e n z y m e s . F i g u r e 3 s h o w s t h e r e s u l t of s u c h a s t u d y , w h e r e b o t h F a c t o r X a a n d thrombin were allowed to react with the specified a m o u n t of I - A T I H - H e p , t h e a m o u n t of n e u t r a l ized Factor Xa was measured, and compared with t h e c a s e of I - A T I I I . H e p and Factor Xa alone. T h e a m o u n t of n e u t r a l i z e d F a c t o r X a w a s t h e s a m e i n b o t h cases. A c c o r d i n g l y , it w a s c o n c l u d e d t h a t t h e r e w a s n o e f f e c t of t h r o m b i n o n t h e n e u t r a l i z a t i o n of F a c t o r X a b y I - A T I I I . H e p . T h e n e u t r a l i z a t i o n of t h r o m b i n b y I-AT I I I . H e p w a s also e x a m i n e d i n t h e p r e s e n c e a n d a b s e n c e of F a c t o r X a (fig. 4). C o n t r a r y to t h e a b o v e c a s e , t h e n e u t r a l i z a t i o n of t h r o m b i n w a s d e c r e a s e d b y t h e p r e s e n c e of F a c t o r Xa. T a b l e ¥ c o m p a r e s t h e n e u t r a l i z e d a m o u n t s of t h e s e t w o e n z y m e s i n t h r e e d i f f e r e n t w a y s of i n c u b a t i o n w i t h e n z y m e s o l u tion. When thrombin or Factor Xa was incubated w i t h 5 m g of I - A T I I I " H e p , 9.0 ~g o r 15.3 e g of enzyme were neutralized, respectively. When a m i x t u r e of t h r o m b i n a n d F a c t o r X a w a s i n c u b a t e d w i t h t h e s a m e a m o u n t of I - A T I I I • H e p , 15.7 gg of Factor Xa were neutralized, but only a trace a m o u n t of t h r o m b i n w a s n e u t r a l i z e d . T h e n,eutral i z e d a m o u n t of F a c t o r X a b y I - A T H I • H e p w h e n t h r o m b i n w a s a b s e n t , w a s t h e s a m e as w h e n thrombin was present. An additional experiment in w h i c h m u c h l a r g e r a m o u n t s of t h r o m b i n w e r e
TABLE V. Neutralization of Factor Xa and~or l h r o m b i n upon incubation w i t h c o - i m m o b i l i z e d heparin and a n l i t h r o m b i n I l L Enzyme
1. 2. 3.
Factor Xa Thrombiu Factor Xa Thrombin
Initial amout (~tg)
Remaining activily after reaction tper cent)
Neutralized amout (ug)
41.5 15.5 43.4 18.7
62 99 65 52
15.7 0.2 15 3 9.0
Sepharose 4B was used as a crrier for i m m o b i l i z a t i o n . The specified a m o u n t of Factor Xa a n d / o r t h r o b i n was allowede to react w i t h 5 mg of I-AT I I I . Hep in 10 mM Tris-HC1 buffer, pH 7.4 c o n t a i n i n g 0.145 M NaC1 a n d 1 per cent b o v i n e serum a l b u m i n . After reacting for 10 win, I-AT I I I . Hep was removed f r o m the reaction m i x t u r e a n d the r e m a i n i n g enzyme activity was i m m e d i a t e l y m e a s u r e d using the peptide-MCA s n b s t r a t e s .
coagulation factors would be activated, and the c l o t t i n g b e j u s t r e a d y to p r o c e e d . T h u s , t h e e v a l u a t i o n of i n h i b i t o r y a c t i v i t y of I - A T } I I . H e p i n t h e p r e s e n c e o f t h e s e a c t i v a t e d f a c t o r s a p p e a r s to b e important. Among the activated factors in blood, t h r o m b i n a n d F a c t o r Xa a r e t h e m o s t i m p o r t a n t
BIOCHIMIE, 1980, 62, n ° 8-9.
mixed with Factor Xa and the mixture was allowe d to r e a c t w i t h I - A T I I I . H e p , reinforced the a b o v e o b s e r v a t i o n b e c a u s e n o d e c r e a s e of n e u t r a l i z e d a m o u n t of F a c t o r X a w a s r e c o g n i z e d , e v e n i n t h e p r e s e n c e of e x c e s s t h r o m b i n . O n t h e o t h e r h a n d , t h e n e u t r a l i z a t i o n of t h r o m b i n w a s n o t
Anticoagulant
activity
of artificial
o c c u r r e d b y t h e p r e s e n c e of s t o i c h i o m e t r i c a l a m o u n t of F a c t o r Xa. As a r e s u l t , it w a s c o n c l u d e d t h a t I - A T I l l . H e p w'as c a p a b l e of n e u t r a l i z i n g F a c t o r X a p r a c t i c a l l y i n p r e f e r e n c e to t h r o m bin. 1 ~g of A T I I l , b y n e u t r a l i z i n g 32 u n i t s of F a c t o r Xa, i n d i r e c t l y p r e v e n t s t h e g e n e r a t i o n of 16'00 N I H u n i t s of t h r o m b i n . T o d i r e c t l y n e u t r a lize t h i s a m o u n t of t h r o m b i n , 1000 ~g of A T I I I a r e r e q u i r e d . M u c h m o r e A T I I I is n e c e s s a r y to i n h i b i t t h e t h r o m b i n - f i b r i n o g e n r e a c t i o n t h a n is r e q u i r e d to n e u t r a l i z e F a c t o r Xa. F a c t o r X a is a more potent thrombogenic agent than thrombin itself. I n t h e p r e s e n t s t u d y , i t w a s s h o w n t h a t a g r e a t e r a m o u n t of F a c t o r X a t h a n o f t h r o m b i n w a s preferentially and rapidly neutralized by I-AT I I I . H e p . T h e r a p i d n e u t r a l i z , a t i o n of F a c t o r X a i n p r e f e r e n c e to t h r o m b i n is f a v o r a b l e f o r effect i v e p r e v e n t i o n of b l o o d c o a g u l a t i o n . T h e r e f o r e , t h e c o - i m m o b i l i z a t i o n of h e p a r i n a n d A T H I w o u l d b e a u s e f u l m e t h o d to o b t a i n a n t i - t h r o m b o genic artificial biomedical materials.
REFERENCES. 1. Jesty, J. (1979) J. Biol. Chem., 254, 1044-1049. 2. Abligaard, U. (1969) Scand. J. clin. Lab. Inves., 24, 23-27. 3. Binder, B. (1973) Thrombos. Diathes. haemorrh. (Stuttg.), 30, 280-283. 4. Jesty, J. (1978) Arch. Bioehem. Biophys., 185, 165173. 5. Bjork, I. ,~ N o r d e n m a n , B. (1976) Eur. J. Biochem., 68, 507-511. 6. 1)amus, P. S., Hicks, M. a Rosenberg, R. D. (1973) Nature, 246, 355-357. 7. Hotmer, E., Soderstrom, G. ~ Anderson, L.-O. (1979) Eur. J. Biochem., 93, 1-5. 8. Li, E. H. H., Fenton, II, J. W. ~ F e i n m a n , R. D. (1976) Arch. Biochem. Biophys., 175, 153-159.
BIOCHIMIE, 1980, 62, n" 8-9.
biomedical
materials.
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9. Joist, J. H. ~ Mustard, J. F. (1975 in <