The Fate of Surface Bound Heparin - Advances in Chemistry (ACS

11 The Fate of Surface Bound Heparin

Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 4, 2018 | https://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch011

M. F. A. GOOSEN and M. V. SEFTON Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario, M5S 1A4, Canada

Displacement by plasma of radiolabeled thrombin and radio labeled thrombin-antithrombin III inactive complex from a heparinized surface was measured and found to be significant; for example, removing 63% of the thrombin and 90% of the complex that could not be removed by phosphate-buffered saline alone. Heparin-poly(vinyl alcohol) (PVA) gel beads with a very low heparin release rate, prepared by acetal coupling of the heparin to the PVA, adsorbed thrombin and potentiated the inactivation of thrombin by antithrombin III, as measured by both thrombin time and chromogenic substrate assays. These results indicate that surface bound heparin retains its biological activity after immobilization and does not become saturated with inactive thrombin-antithrombin III complex. These results support the argument that heparinization can be an important means of preparing the materials needed for the development of improved cardiocirculatory-assist devices and blood handling procedures.

T

he N a t i o n a l H e a r t , L u n g , a n d B l o o d Institute ( N H L B I ) Task F o r c e o n B i o m a t e r i a l s has reiterated t h e n e e d for the d e v e l o p m e n t o f b l o o d c o m p a t i b l e materials since "progress i n this f i e l d is a c o n d i t i o n for advances i n the a p p l i c a t i o n o f cardiocirculatory-assist devices a n d other p r o c e d u r e s w h i c h r e q u i r e c o n t i n u o u s or i n t e r m i t t e n t h a n d l i n g of b l o o d " (I). F o r exam p l e , t h e task force has specifically i d e n t i f i e d t h e d e v e l o p m e n t o f s m a l l d i a m e t e r b l o o d vessel prostheses a n d c h r o n i c b l o o d access catheters as p r i o r ity applications of b l o o d - c o m p a t i b l e materials. B o t h of these devices are u s e d i n low-flow situations w h e r e r e d t h r o m b u s f o r m a t i o n (i.e., i n t r i n s i c c l o t t i n g system activation) p r e d o m i n a t e s (2).

O n e of the t e c h n i q u e s u s e d w i t h v a r y i n g degrees of success to p r o d u c e n o n t h r o m b o g e n i c materials (and p a r t i c u l a r l y to prevent i n t r i n s i c c l o t t i n g system activation) has b e e n h e p a r i n i z a t i o n . 00^239^82/ρίβ9-0147$06.0(νθ » 1 9 W Q t ë î y K U W a r y n u l Society iiPr

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II

III

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


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BIOMATE RIALS: INTERFACIAL P H E N O M E N A A N D APPLICATIONS

N u m e r o u s methods have b e e n u s e d to c o u p l e h e p a r i n , i o n i c a l l y or covalently, to p o l y m e r surfaces. Ionic b o n d i n g , w h e t h e r b y d i r e c t quat e r n i z a t i o n of suitable f u n c t i o n a l groups o n the p o l y m e r surface (3, 4), b y the a d d i t i o n of quaternary a m m o n i u m - h e p a r i n complexes (5-7), or b y the cop o l y m e r i z a t i o n of cationic or tertiary a m i n e m o n o m e r s (8, 9), results i n materials w i t h excellent s h o r t - t e r m c o m p a t i b i l i t y . C o v a l e n t c o u p l i n g (10, II), o n the other h a n d , f r e q u e n t l y has inactivated the h e p a r i n , r e s u l t i n g i n o n l y v e r y l i m i t e d thromboresistance. H o w e v e r , the covalent c o u p l i n g of h e p a r i n to polyvinyl alcohol) (PVA) t h r o u g h an acetal b r i d g e has r e s u l t e d i n materials that appear to retain l o n g - t e r m b l o o d c o m p a t i b i l i t y i n v i t r o ( 1 2 , 1 3 ) a n d i n arteriovenous (AV) shunts i n dogs (14,15). S i m i l a r thromboresistance w i t h o u t apparent h e p a r i n e l u t i o n has b e e n r e p o r t e d for h e p a r i n i z e d p o l y m e t h y l aery late (16) a n d C e - i n i t i a t e d h e p a r i n - m e t h y l methacrylate graft c o p o l y m e r s (17). T h e r e p o r t e d n o n t h r o m b o g e n i c activity of cyanogen b r o m i d e - b o n d e d h e p a r i n (18) may, however, be c o m p l i c a t e d b y the c o n tinuous loss of h e p a r i n f r o m this surface (19). T h e covalent b i n d i n g of a m o d i f i e d h e p a r i n is r e p o r t e d elsewhere i n this v o l u m e (20). 4 +

Ionically h e p a r i n i z e d materials or materials w i t h a steady leakage of h e p a r i n are suitable for a n d have b e e n u s e d w i t h success i n s h o r t - t e r m applications [e.g., t e m p o r a r y shunts d u r i n g vascular surgery (21 ) a n d others] (22, 23). H o w e v e r , the continuous loss of h e p a r i n f r o m the surface of a h e p a r i n i z e d m a t e r i a l w o u l d p r e c l u d e its use as a l o n g - t e r m i m p l a n t . U n fortunately, this statement has b e e n i n t e r p r e t e d to suggest that i n the absence of h e p a r i n loss a n d the c o r r e s p o n d i n g absence of a h e p a r i n m i c r o e n v i r o n m e n t at the b l o o d - m a t e r i a l interface, the surface-bound h e p a r i n s h o u l d not be effective i n p r e v e n t i n g thrombogenesis. O u r e x p e r i m e n t s w i t h i m m o b i l i z e d h e p a r i n ( 1 2 , 1 3 , 24) a n d the w o r k of others (17, 25) have shown that such a c o n c l u s i o n is i n v a l i d . S o m e covalently h e p a r i n i z e d materials, for example, h e p a r i n - P V A , can be effective i n p r e v e n t i n g thrombogenesis, like h e p a r i n i n s o l u t i o n , b y the accelerated inactivation of t h r o m b i n t h r o u g h the formation of surface-bound t h r o m b i n - a n t i t h r o m b i n III inactive c o m p l e x . C a r e f u l e x a m i n a t i o n of the possible fates of the b o u n d h e p a r i n a n d the b o u n d inactive c o m p l e x suggests h y p o t h e t i c a l mechanisms w h i c h , i f effective, c o u l d l i m i t the u t i l i t y of h e p a r i n i z e d materials (Table I). F o r example, e i t h e r the surface may b e c o m e saturated w i t h a h e p a r i n c o m p l e x that can no longer accelerate the inactivation of t h r o m b i n , or c o n s u m p t i o n of antit h r o m b i n , p r o t h r o m b i n , or other c l o t t i n g factors may result, l e a v i n g the b l o o d systematically hypocoagulable. I n this chapter w e p r e s e n t the results of an i n v i t r o investigation into the fate of surface b o u n d h e p a r i n a n d b o u n d inactive c o m p l e x to assess the concerns w i t h l o n g - t e r m use of h e p a r i n . H e p a r i n was i m m o b i l i z e d onto P V A u s i n g a covalent acetal c o u p l i n g p r o c e d u r e (12) to p r o d u c e a h y d r o g e l ( ~ 7 7 % w/w water) i n w h i c h the h e p a r i n appears to be b o u n d t h r o u g h the a m i n o a c i d t e r m i n u s of the m o l e c u l e (26). A l t h o u g h the d e t a i l e d structure of


11.

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149

Surface Bound Heparin

Table I. Conceivable Fates of Surface-Bound Heparin •

H e p a r i n is lost f r o m surface to create a h e p a r i n m i c r o e n v i r o n -

m e n t at the blood/material interface. •

H e p a r i n r e m a i n s o n the surface, b u t b e c o m e s saturated w i t h

inactive c o m p l e x . •

H e p a r i n acts catalytically and does not b e c o m e saturated, but


a high p r o d u c t i o n rate of inactive c o m p l e x causes a systemic hypocoagulability. •

H e p a r i n does not b e c o m e saturated, a n d inactive c o m p l e x for

m a t i o n is m a i n t a i n e d to w i t h i n tolerable levels. the pH

linkage can o n l y b e h y p o t h e s i z e d , like o t h e r acetal bonds, it is stable at 7.4 (12):

the e l u t i o n rate o f h e p a r i n f r o m h e p a r i n - P V A films into

p h o s p h a t e - b u f f e r e d saline ( P B S ) or 3 M N a C l was o n the o r d e r o f 10~

5

μg/cm · m i n (12, 27). T h e g e l has b e e n u s e d b y itself as a f i l m o r g r o u n d into 2

fine beads (27), o r m o r e i m p o r t a n t l y , as a coating o n an appropriate sub strate. T h e coating has b e e n a p p l i e d , for e x a m p l e , to the i n s i d e o f surfaceh y d r o x y l a t e d styrene-butadiene—styrene (SBS) block c o p o l y m e r t u b i n g , for the p r e p a r a t i o n o f s m a l l - d i a m e t e r prostheses (12), a n d to the inside o f u l t r a filtration

h o l l o w fibers u s e d i n an artificial e n d o c r i n e pancreas (24).

Experimental Heparin-PVA Beads. Heparin-PVA films were prepared (12) from an aqueous solution containing 10% (w/w) PVA (20% acetylated PVA, Gelvatol 20-60, Monsanto Canada Ltd.), 5% MgCl -6 H 0 , 0.5% glutaraldehyde, 3% formaldehyde, 4% glycer ol, and 1% sodium heparin (porcine mucosal, 176 USP U/mg, Canada Packers Ltd.). The films were ground at 77 Κ to form beads within a nominal particle diameter range of 105-250 μπι (27). Control beads were prepared without heparin. The elution rate of heparin from the beads suspended in 0.05M phosphate-buffered 0.15M NaCl (PBS) was measured by adding toluidine blue to samples of the used wash solution and quantifying the absorbance change at 600 nm (26). The detection limit of this assay is 2 ppm heparin, which means that heparin elution rates as low as 1 X 10~ μg/g wet gel*min can be determined conveniently. In Vitro Clotting Tests. The plasma recalcification time of citrated human plasma was determined in the presence of heparin-PVA beads and control PVA beads without heparin. Various amounts (10-200 mg) of gel were incubated with 0.5 mL of plasma at room temperature for 5 min. After the addition of 0.5 mL of0.025M CaCl , the time to clot was noted by tilting the test tube gently each minute, until the beads clumped together or were found to stick to the test tube wall. The thrombin time was determined similarly by incubation of 2 IU of crude bovine thrombin (10 IU/mL, Miles Laboratories) with the beads for 5 min at room temperature in an albumin-coated glass tube, followed by 0.2 mL of citrated human plasma. The time to clot was noted by tilting the test tube gently every few seconds. PBS, after incubation with heparin-PVA beads for 5-60 min, was analyzed for the presence of heparin using both toluidine blue and the thrombin time test (PBS in place of gel beads). 2

2

2

2



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Thrombin Affinity. The affinity of thrombin for heparin-PVA and PVA beads was analyzed by loading 62 IU of crude bovine thrombin in PBS (96 IU/mg, Park Davis Co.) on chromatography columns (1.6-cm diameter x 2.5 cm) packed with the beads. The columns were washed with 100 mL of PBS and/or 20% (w/w) bovine albumin in PBS (Miles Lab.). The residual thrombin activity bound to the columns was measured by loading 0.5 mg chromogenic substrate (0.5 mg/mL PBS; S2238, Ortho Diagnostics, or Chromozym T H , Boehringer-Mannheim) onto the column and measuring the change in effluent absorbance at 381 nm. In some experiments, 3 mg of crude antithrombin III (15 m L of citrated human plasma, heat defibrinated at 54°C for 5 min) was used to inactivate the bound thrombin. The effect of precoating the gel columns with 20 mL of 10% (w/w) bovine albumin in PBS on purified human α-thrombin binding (3.53 IU/mg, courtesy of J. W. Fenton II, N.Y. State Depart ment of Health, Albany) also was determined. The residual thrombin activity is reported as color yield: the ratio of color produced by enzymatic action to the total color possible from the load of chromogen. Bound Thrombin/Antithrombin III Exchange. To determine whether the surface bound heparin becomes saturated with inactive complex, the ability to dis place bound inactive complex and thereby regenerate the heparin was assessed by measuring the elution of the appropriate radiolabeled protein from the heparin-PVA column. Two sets of experiments were performed: one with only radiolabeled throm bin loaded onto a heparin-PVA column and one with unlabeled thrombin followed by radiolabeled antithrombin III loaded onto an identical column. Unbound protein was eluted with 20 mL of PBS. Flow rates of 97 mL/min were used throughout. Then, "loosely" bound protein was removed with another wash for 4r-6 h with PBS (400-600 mL), followed by the overnight recirculation of 20 mL of PBS (closed system) until no further increase in PBS radioactivity occurred. At that time, the system was reopened (i.e., the eluent was passed through the column in the conventional manner), the gel was washed with 50-60 mL of PBS followed by defibrinated plasma (containing antithrombin III), and the increase in radioactivity in the eluted plasma was mea sured. The system was closed again, and defibrinated plasma was recirculated over night to end the wash cycle. The amounts of thrombin, antithrombin III, and defi brinated plasma used in the two experiments are listed in Table II; no effect of the quantities used has been noted in subsequent experiments. Recirculation of eluent (closed system) was used to attain equilibrium. Then, 100 mL of PBS was passed through the column, and residual radioactivity on the beads was measured by adding the beads directly into 10 mL of Aquasol (New England Nuclear). For the liquid samples, 0.1-mL aliquots were taken and added to 10 mL of Aquasol for liquid scintillation counting (Beckman LS 8000 liquid scintillation spectrometer). Proteins were labeled with I using the standard Enzymobead method (Biorad Laboratories), and retained approximately 70% of their original biological activity after labeling. 125

Results S t a b i l i t y o f I m m o b i l i z e d H e p a r i n . The amount of heparin eluting from the gel beads was calculated from the amount of heparin found in the wash solvent at any time (determined using the toluidine blue technique) and the amount of heparin initially present in the heparin-PVA solution. After the first 50 h of washing, the rate of elution decreased rapidly to a low, relatively constant value of 1.67 X 10~ μg/g wet gel-min. A similar elution rate was obtained in plasma using S-heparin (27). Assuming a particle diameter of 180 μπι and a gel density of 1.05 g/cm , this release rate corre sponds to 5.3 X 10~ μ g / c m · m i n . The rate is comparable to the release rate 2

35

3

5

2


Cooper et al.; Biomaterials: Interfacial Phenomena and Applications Advances in Chemistry; American Chemical Society: Washington, DC, 1982. l g g e l ; 7 m g heparin/g g e l h e a t - d e f i b r i n a t e d plasma, 7 m L (open) f o l l o w e d b y r e c i r c u l a t i o n o f 20 m L

125

18 I U o f c r u d e b o v i n e I-thrombin

1 g g e l ; 7 m g heparin/g g e l heat-defibrinated plasma, 350 m L (open) f o l l o w e d b y r e c i r c u l a t i o n o f 90 m L

125

III

100 I U o f c r u d e b o v i n e t h r o m b i n (unlabeled) f o l l o w e d b y 2000 I U of p u r e h u m a n I - a n t i t h r o m b i n I I I 23

Thrombin-antithrombin

Note: specific activities before labeling: crude bovine thrombin—96 IU/mg (Parke-Davis), pure human antithrombin III—1000 IU/mg (M. Wickerhauzer, American Red Cross, Bethesda, MD); "open" implies material used as eluent in conventional chromatographic mode; and ratio of loaded protein does not include antithrombin III content of defibrinated plasma.

H e p a r i n - P V A gel Displacement eluent

moles a n t i t h r o m b i n loaded/ moles t h r o m b i n l o a d e d

Loaded protein

Thrombin

Table II. Displacement of Bound Thrombin-Antithrombin III: Experimental Parameters


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

152

d e t e r m i n e d e a r l i e r u s i n g S - h e p a r i n for a h e p a r i n - P V A film on S B S 35

(12).

T h e s e release rates are a p p r o x i m a t e l y 1/1000 the 4 x 10~ μg/cm · m i n m i n i 2

2

m u m r e q u i r e d for thromboresistance of i o n i c a l l y h e p a r i n i z e d catheters (4). A f t e r w a s h i n g for 500 h , 3 0 % of the o r i g i n a l h e p a r i n was left i n the gel beads, g i v i n g a final h e p a r i n c o n t e n t of a p p r o x i m a t e l y 7 mg/g wet gel. In Vitro Activity of Bound Heparin.

T h e prolongation of the t h r o m b i n

t i m e caused b y the a d d i t i o n of h e p a r i n - P V A beads to plasma is contrasted Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 4, 2018 | https://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch011

w i t h the n e g l i g i b l e effect of P V A beads w i t h o u t h e p a r i n i n F i g u r e l a . A similar p r o l o n g a t i o n was o b s e r v e d for the p l a s m a recalcification t i m e ( F i g u r e l b ) . I n b o t h cases, c l u m p i n g or adhesion of the beads caused p r e s u m a b l y b y f i b r i n was the e n d p o i n t of the assay. F i g u r e 2 shows that t h r o m b i n b i n d s to b o t h h e p a r i n - P V A beads a n d P V A beads w i t h o u t h e p a r i n . A f t e r a load of c r u d e b o v i n e t h r o m b i n (62 I U ) followed b y P B S a n d c h r o m o g e n , color y i e l d s o f 8 9 % a n d 8 1 % w e r e o b t a i n e d for the h e p a r i n - P V A a n d P V A g e l , respectively, i n d i c a t i n g the presence of active t h r o m b i n o n the c o l u m n s . Passing 15 m L of 2 0 % (w/w) bovine a l b u m i n t h r o u g h the same c o l u m n s f o l l o w e d b y c h r o m o g e n l o w e r e d the color y i e l d for

140 120

f Ε

Ρ c

100

80

!Ω

Ι

60

I-

40 20

10

20

30

Amount of Gel

40

50

(mg)

Figure la. Thrombin time of plasma in the presence of PVA (o) and heparin-PVA (·) beads. Thrombin incubated with beads prior to plasma addition (2 IU of crude bovine thrombin, 0.2 mL of citrated human plasma, and 7 mg of heparin!g gel) (13).



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0

153


80 160 AMOUNT OF GEL (mg/ml)

240 Elsevier North Holland Inc.

Figure lb. Recalcification time of plasma incubated with PVA (o) and heparin-PVA (*) beads (0.5 mL of citrated human plasma, 0.5 mL of 0.025M CaCl , and 7 mg of heparin/g gel) (13). 2

P V A to 54%, i n d i c a t i n g that a substantial q u a n t i t y of t h r o m b i n b o u n d to P V A had

b e e n d e s o r b e d b y b o v i n e a l b u m i n . A s i m i l a r color y i e l d was o b t a i n e d

w h e n 15 m L o f d e f i b r i n a t e d p l a s m a (containing 3 m g o f a n t i t h r o m b i n III) w e r e u s e d i n place o f t h e b o v i n e a l b u m i n . I n contrast, c r u d e bovine t h r o m bin

b o u n d to h e p a r i n - P V A , w h i l e n o t b e i n g significantly inactivated (or

desorbed) b y 15 m L o f 2 0 % (w/w) b o v i n e a l b u m i n , was almost c o m p l e t e l y inactivated b y 3 m g o f c r u d e a n t i t h r o m b i n I I I , as shown b y the 2 4 % color yield. P r e c o a t i n g t h e g e l c o l u m n s w i t h 20 m L o f 10% (w/w) bovine a l b u m i n significantly r e d u c e d h u m a n t h r o m b i n b i n d i n g to P V A , relative to h e p a r i n P V A ( F i g u r e 2). L o a d i n g p r e c o a t e d c o l u m n s w i t h 1072 I U of p u r i f i e d h u m a n α-thrombin, f o l l o w e d b y c h r o m o g e n , gave a color y i e l d of 7 8 % w i t h h e p a r i n P V A a n d 3 8 % w i t h P V A alone. I n contrast, 62 I U of c r u d e bovine t h r o m b i n loaded o n an u n t r e a t e d h e p a r i n - P V A c o l u m n p r o d u c e d a slightly h i g h e r color y i e l d , 8 9 % , suggesting that p r e c o a t i n g the g e l c o l u m n w i t h a l b u m i n reduces the n u m b e r o f available t h r o m b i n b i n d i n g sites o n t h e h e p a r i n - P V A g e l .


154


PURIFIED H U M A N

C R U D E BOVINE THROMBIN

THROMBIN*


100

r

Thrombosis Research

Figure 2. Inactivation of thrombin (T) by antithrombin III (AT) on heparinized beads in a chromatography column (27). Thrombin (62 IU) loaded on the gel columns was followed by either 3 mg of crude antithrombin III or by 15 mL of 20% (w/w) bovine albumin (ALB). Key: * in the experiment with purified human thrombin (1072 IU), the gel columns were precoated with albumin; WE , PVA-heparin; B , PVA; andflowrate, 97 mL/h, 7 mg hepannlg gel, 3 g gel.

Exchange o f B o u n d Thrombin/Antithrombin III. Displacement of bound radiolabeled thrombin or bound thrombin-radiolabeled antithrombin III inactive c o m p l e x f r o m h e p a r i n - P V A b y defibrinated plasma containing u n l a b e l e d a n t i t h r o m b i n I I I , is shown i n F i g u r e 3. A p p r o x i m a t e l y 8 % o f the loaded I - t h r o m b i n a n d 1 4 % o f the l o a d e d I - a n t i t h r o m b i n III w e r e left on the c o l u m n after the i n i t i a l wash w i t h 20 m L o f P B S . T h e s e low percentages represent t h e l i m i t e d p u r i t y o f the r a d i o l a b e l e d proteins. Subsequent r e movals o f r a d i o l a b e l e d p r o t e i n f r o m the c o l u m n s are c o m p a r e d i n F i g u r e 3 on the same basis, b y setting the b o u n d fraction of p r o t e i n after this 2 0 - m L e l u t i o n e q u a l to 100%. 1 2 5

125

A f t e r w a s h i n g t h e c o l u m n s i n an o p e n - l o o p system for 4 - 6 h w i t h P B S , and t h e n overnight i n a c l o s e d - l o o p system w i t h 20 m L o f P B S , a further 3 0 % of the i n i t i a l l y b o u n d p r o t e i n was r e m o v e d , u n t i l an e q u i l i b r i u m h a d b e e n established b e t w e e n the b o u n d a n d u n b o u n d proteins. O n changing the P B S eluent to h e a t - d e f i b r i n a t e d p l a s m a (containing a n t i t h r o m b i n III), the desorption o f b o t h I - t h r o m b i n a n d I - a n t i t h r o m b i n III f r o m the c o l u m n i n creased d r a m a t i c a l l y . I n t h e e x p e r i m e n t s w i t h radiolabeled a n t i t h r o m b i n III, the c o l u m n h a d b e e n exposed p r e v i o u s l y to t h r o m b i n , therefore, the disp l a c e d r a d i o l a b e l e d a n t i t h r o m b i n I I I s h o u l d m o r e p r o p e r l y be d e s c r i b e d as l a b e l e d inactive c o m p l e x . Because d e f i b r i n a t e d plasma contains anti1 2 5

125


11.

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t h r o m b i n I I I , t h e e l u t e d r a d i o l a b e l e d t h r o m b i n i n the t h r o m b i n - a l o n e exp e r i m e n t also m a y b e i n t h e f o r m o f inactive c o m p l e x . A l t h o u g h further investigation is r e q u i r e d to i d e n t i f y t h e f o r m o f the d e s o r b e d radioactivity (i.e., m o l e c u l a r w e i g h t , a n t i t h r o m b i n I I I content) a n d mechanisms o f exchange, b i n d i n g o f t h r o m b i n o r t h r o m b i n - a n t i t h r o m b i n I I I c o m p l e x to i m m o b i l i z e d h e p a r i n is n o t i r r e v e r s i b l e , a n d u n d e r some physiologically relevant circumstances

(presence o f plasma),

t h r o m b i n alone o r t h r o m b i n -


a n t i t h r o m b i n I I I inactive c o m p l e x c a n b e d i s p l a c e d f r o m t h e h e p a r i n i z e d surface. T h e s e results are consistent w i t h experiments i n rabbits that show that the h e p a r i n - t h r o m b i n - a n t i t h r o m b i n I I I c o m p l e x is readily dissociated i n vivo (28), a n d t h e w o r k o f G r i f f i t h w h o showed that t h e t h r o m b i n - a n t i t h r o m b i n I I I c o m p l e x has a weaker affinity for h e p a r i n than u n c o m p l e x e d t h r o m b i n o r a n t i t h r o m b i n I I I (29). F u r t h e r m o r e , O l s s o n et al. demonstrated that the s e q u e n c e i n v o l v i n g t h r o m b i n adsorption onto a h e p a r i n i z e d surface

J00

10

20 30 Time(hrs)

40

50

Figure 3. Displacement of bound radiolabeled protein from heparin-PVA. After loading the labeled proteins on the column and eluting unbound protein with 20 mL of PBS, the amount of radioactivity remaining on the columns was taken as 100% (Time —0). The columns were then washed with PBS, and at the indicated times, the eluent was changed to defibrinated plasma. Key: #, labeled thrombin; O , unlabeled crude bovine thrombin and labeled human antithrombin III.


156


and s u b s e q u e n t i n h i b i t i o n of the e n z y m e b y c i r c u l a t i n g a n t i t h r o m b i n III was regenerative (30).


Discussion T h e results r e p o r t e d h e r e , i n a d d i t i o n to those r e p o r t e d earlier b y ourselves a n d others, show that significant h e p a r i n e l u t i o n is not necessary for b o u n d h e p a r i n to retain b i o l o g i c a l activity. P r o l o n g e d partial t h r o m boplastin times of greater than 1200 s w e r e observed for plasma i n contact w i t h h e p a r i n - P V A - c o a t e d s t y r e n e - b u t a d i e n e - s t y r e n e block c o p o l y m e r films, despite a h e p a r i n e l u t i o n rate o n the o r d e r of 1 0 " μ g / c m · m i n (12). P r o l o n g e d t h r o m b i n times also w e r e observed for C e - i n i t i a t e d h e p a r i n m e t h y l methacrylate graft c o p o l y m e r s , w i t h o u t measurable h e p a r i n release (17). T h e a b i l i t y to inactive t h r o m b i n b y reaction w i t h a n t i t h r o m b i n III also has b e e n r e p o r t e d (25) for this latter m a t e r i a l a n d for h e p a r i n i z e d p o l y m e t h y l acrylate w i t h o u t h e p a r i n release (16). T h e passivating effect of a n t i t h r o m b i n III o n platelet aggregation i n S a l z m a n c o l u m n s was correlated w i t h this ability to inactive t h r o m b i n (25). " A r t i f i c i a l E n d o c r i n e Pancreas," made f r o m h e p a r i n - P V A - c o a t e d h o l l o w fibers (0.5 m m i . d . ) , r e m a i n e d patent for longer than c o n t r o l shunts w i t h o u t h e p a r i n , w h e n i m p l a n t e d as A V shunts i n monkeys (24). H e p a r i n i z e d devices w e r e patent for 2, 4, 5, or 6 days, w h i l e u n h e p a r i n i z e d shunts w e r e patent for less than 1 h . 5

2

4 +

T h i s c h a p t e r r e p o r t e d that t h r o m b i n times a n d plasma recalcification times w e r e p r o l o n g e d i n the p r e s e n c e of h e p a r i n - P V A beads, despite the very stable b i n d i n g of the h e p a r i n (elution rate of 1.67 Χ 1 0 " μg/g wet gel* min). A l s o , c o m p a r i s o n of the c h r o m o g e n i c substrate activity of t h r o m b i n b o u n d to h e p a r i n - P V A c o l u m n s a n d P V A columns w i t h o u t h e p a r i n after loading a n t i t h r o m b i n III ( F i g u r e 2) suggests that t h r o m b i n b i n d i n g to h e p a r i n - P V A activates the e n z y m e , m a k i n g it m o r e receptive to anti t h r o m b i n III. If, for e x a m p l e , b i n d i n g of t h r o m b i n to h e p a r i n h a d not i n creased the affinity of e n z y m e for i n h i b i t o r , t h e n the passage of c r u d e anti t h r o m b i n III t h r o u g h the c o l u m n s f o l l o w e d later b y c h r o m o g e n i c substrate w o u l d have p r o d u c e d s i m i l a r color yields for b o t h h e p a r i n - P V A a n d P V A . H o w e v e r , this result was not o b s e r v e d i n these experiments, l e a d i n g to the conclusion that the b o u n d h e p a r i n was biologically active. F u r t h e r m o r e , the inactivation of t h r o m b i n o b s e r v e d h e r e cannot be c o n s i d e r e d as a slow progressive inactivation of the t h r o m b i n b y a n t i t h r o m b i n III, because of the difference i n b e h a v i o r o n h e p a r i n - P V A a n d P V A . 2

W h i l e d i r e c t c o m p a r i s o n w i t h the m i n i m u m e l u t i o n rate suggested for ionically h e p a r i n i z e d catheters may be m i s l e a d i n g due to the different g e o m etries i n v o l v e d , the m e a s u r e d e l u t i o n rate is l o w e n o u g h to conclude that the h e p a r i n - P V A linkage is stable a n d that the observed biological activity cannot s i m p l y be a t t r i b u t e d to the p r e s e n c e of a m i c r o e n v i r o n m e n t of h e p a r i n


11.

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157

a r o u n d t h e beads. T h u s , this p r i m a r y c o n c l u s i o n o f p r e v i o u s investigators w i t h b o u n d h e p a r i n is n o t necessarily v a l i d . W h i l e this m o d e of action m i g h t account for the activity of loosely b o u n d h e p a r i n , it is not sufficient to e x p l a i n the activity of w e l l - b o u n d h e p a r i n such as h e p a r i n b o u n d b y acetal c o u p l i n g . T h e r e f o r e , b o u n d h e p a r i n m u s t b e assumed to b e effective i n p r e v e n t i n g thrombogenesis b y the f o r m a t i o n o f a surface b o u n d t h r o m b i n - a n t i t h r o m b i n III inactive c o m p l e x . Downloaded by UNIV OF MASSACHUSETTS AMHERST on June 4, 2018 | https://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch011

C o n c e r n over t h e fate o f the b o u n d c o m p l e x appears unnecessary,

since

r a d i o l a b e l e d t h r o m b i n o r t h r o m b i n - a n t i t h r o m b i n III complexes w e r e r e a d i l y d i s p l a c e d f r o m the surfaces b y d e f i b r i n a t e d p l a s m a c o n t a i n i n g c r u d e a n t i t h r o m b i n I I I . T h e r e f o r e , the b o u n d h e p a r i n apparently does not b e c o m e saturated w i t h inactive c o m p l e x , e n a b l i n g the b o u n d h e p a r i n , i f i t remains active, to act catalytically to potentiate the inactivation o f t h r o m b i n as it is generated.

Whether

the c o n s u m p t i o n rate o f a n t i t h r o m b i n III o r p r o -

t h r o m b i n , o n t h e o t h e r h a n d , can b e c o n t r o l l e d , o r w h e t h e r it w o u l d result in a systemic b l o o d defect, remains to b e e x a m i n e d . O t h e r c o n c e r n s r e g a r d i n g t h e practicality o f surface b o u n d h e p a r i n f o r the p r e p a r a t i o n o f materials w i t h l o n g - t e r m thromboresistance r e m a i n . B e cause the i n t e r a c t i o n o f h e p a r i n w i t h platelets is unclear (31 ), w h e t h e r the i m m o b i l i z e d h e p a r i n causes greater thrombosis u n d e r conditions

where

platelet d e p o s i t i o n is m o r e i m p o r t a n t than f i b r i n f o r m a t i o n remains to b e s h o w n . T h e passivating effect o f a n t i t h r o m b i n I I I o n platelet c o n s u m p t i o n caused b y surface-bound h e p a r i n is a significant observation i n this context (32,

33).

Conclusions T h e results r e p o r t e d here, i n conjunction w i t h earlier results, indicate that i m m o b i l i z e d h e p a r i n n e e d not necessarily be lost f r o m a surface i n o r d e r to i m p a r t thromboresistance to that surface. F o r h e p a r i n - P V A , and perhaps for other covalent reactions that d o not inactivate the h e p a r i n , the i r r e v e r s i b l y b o u n d h e p a r i n can accelerate the formation of a surface-bound inactive t h r o m b i n - a n t i t h r o m b i n III c o m p l e x . F u r t h e r m o r e , o u r results suggest that the inactive c o m p l e x is not itself p e r m a n e n t l y b o u n d to the surface, b u t rather can be d i s p l a c e d b y a c o m p o n e n t o r components i n plasma. W h i l e materials that lose h e p a r i n at a c o n t r o l l e d rate can be c l i n i c a l l y acceptable i n s h o r t - t e r m applications, the l o n g - t e r m use o f h e p a r i n i z e d m a terials requires materials that d o not lose h e p a r i n , yet retain the biological function o f the h e p a r i n . T h i s investigation a n d others indicate that these r e q u i r e m e n t s are not m u t u a l l y exclusive, a n d that b o u n d h e p a r i n can p o t e n tially retain its b i o l o g i c a l activity over the l o n g t e r m . H e p a r i n i z a t i o n can be an i m p o r t a n t means o f p r e p a r i n g the materials n e e d e d for the d e v e l o p m e n t of i m p r o v e d cardiocirculatory-assist devices a n d b l o o d h a n d l i n g procedures.


158


Acknowledgments T h e authors a c k n o w l e d g e the support of the N a t i o n a l H e a r t , L u n g , a n d B l o o d Institute, N I H , D H E W u n d e r G r a n t N o . 5 R 0 1 H L 2 4 0 2 0 ; the guidance of M . W . C . H a t t o n ; a n d t h e technical assistance o f B . A . Ramsoomair. M . F . A . G o o s e n thanks t h e C h e m i c a l Institute of C a n a d a for having awarded


him the Ogilvie F l o u r M i l l s - K e n n e t h Armstrong M e m o r i a l Fellowship.

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25. Salzman, E. W.; Silane, M.; Lindon, J.; Brier-Russell, E.; Dincer, Α.; Rosen berg, R.; Merrill, E. W. In "Chemistry and Biology of Heparin"; Lundblad, R. L.; Brown, W. V.; Mann, Κ. M.; Roberts, H. R., Eds.; Elsevier North Holland: Amsterdam, 1981; pp. 435-448. 26. Goosen, M. F. Α.; Ramsoomair, Β. Α.; Sefton, M. V., unpublished data. 27. Goosen, M. F. Α.; Sefton, M. V.; Hatton, M. W. C. Thromb. Res. 1980, 20, 543-554. 28. Lam, L. S. L.; Regoeczi, E.; Hatton, M. W. C. Br. J. Exp. Path. 1979, 60, 151-160. 29. Griffith, M. J. In "Chemistry and Biology of Heparin"; Lundblad, R. L.; Brown, W. V.; Mann, K. G.; Roberts, H. R., Eds.; Elsevier North Holland: Am sterdam, 1981; pp. 237-248. 30. Olsson, P.; Larsson, R.; Lins, L.-E.; Nilsson, E. Abstracts, VIIIth International Congress on Thrombosis and Haemostasis, 1981, 46(1), 323. 31. Zucker, M. B. Fed. Proc. 1977, 36, 47. 32. Lindon, J.; Rosenberg, R.; Merrill, E. W.; Salzman, E.J. Lab. Clin. Med. 1978, 91, 47. 33. Salzman, E. W.; Rosenberg, R. D.; Smith, M. H.; Lindon, J. N.; Fabveau, L. J. Clin. Invest. 1980, 65, 65-73. RECEIVED for review January 16, 1981. ACCEPTED October 15, 1981.


The Fate of Surface Bound Heparin - Advances in Chemistry (ACS

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