The Anticoagulant Activity of Derivatized and Immobilized Heparins

12 The Anticoagulant Activity of Derivatized and Immobilized Heparins 1

C. D. EBERT, E. S. LEE, J. DENERIS, and S. W. KIM

Downloaded by UNIV OF PITTSBURGH on February 21, 2016 | http://pubs.acs.org Publication Date: July 27, 1982 | doi: 10.1021/ba-1982-0199.ch012

University of Utah, Department of Pharmaceutics, Salt Lake City, UT 84112

Heparin anticoagulant activity decreases as the degree of carboxylic derivatization increases; however, partially derivatized heparin, both carboxylic and hydroxyl derivatives, retains anticoagulant activity. Heparin was immobilized via carboxylic groups to diaminoalkane agarose gels to provide coupling spacer groups of various lengths. Anticoagulant activity increased precipitously beginning with 10-carbon unit spacer groups, but heparin coupled with less than 10 spacer groups demonstrated only minimal anticoagulant activity.

T

he c o m p l e x interactions o f b l o o d u p o n contacting foreign surfaces r e s u l t i n g i n t h r o m b o s i s is a n i n h e r e n t p r o b l e m i n u s i n g blood-contacting

b i o m e d i c a l devices. A l t h o u g h t h e precise mechanisms b y w h i c h surfacei n d u c e d t h r o m b o g e n e s i s occurs are not f u l l y u n d e r s t o o d , b o t h c e l l u l a r a n d m o l e c u l a r b l o o d c o m p o n e n t s participate, perhaps synergistically, i n eventual t h r o m b u s f o r m a t i o n . B i o m e d i c a l d e v i c e technology has e x p e r i e n c e d d r a matic advances i n t h e past two decades; however, t h e c l i n i c a l means f o r t h r o m b u s p r e v e n t i o n remains unchanged—anticoagulants are a d m i n i s t e r e d systemically c o n c o m i t a n t w i t h t h e o p e r a t i o n o f the d e v i c e . T h e anticoagulant most c o m m o n l y u s e d today is h e p a r i n . It is a h i g h l y

p o l y d i s p e r s e d , a c i d i c carbohydrate w i t h a linear, h e l i c a l structure c o m p o s e d of alternating sulfoglucosamine a n d h e x u r o n i c acid molecules j o i n e d b y glyc o s i d e linkages (I ). T h e m o l e c u l a r w e i g h t of c o m m e r c i a l h e p a r i n ranges f r o m b e l o w 10,000 to w e l l over 20,000 (2), a n d as a general r u l e , h i g h m o l e c u l a r weight h e p a r i n has greater anticoagulant activity than l o w m o l e c u l a r w e i g h t h e p a r i n (3). I n a d d i t i o n to a w i d e range i n m o l e c u l a r w e i g h t , the c h e m i c a l structure of h e p a r i n varies c o n s i d e r a b l y . W h e n high-activity (360 IU/mg) a n d low-activity (12 IU/mg) h e p a r i n fractions w e r e analyzed c h e m i c a l l y , the h i g h a u t h o r to w h o m c o r r e s p o n d e n c e shall b e addressed. 0065-2393/82/0199-0161$06.00/0 ® 1982 American Chemical Society In Biomaterials: Interfacial Phenomena and Applications; Cooper, Stuart L., et al.; Advances in Chemistry; American Chemical Society: Washington, DC, 1982.

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

activity h e p a r i n was c o m p o s e d p r e d o m i n a n t l y of chains containing a u n i q u e tetrasaccharide w i t h the f o l l o w i n g s e q u e n c e : L - i d u r o n i c a c i d —> N - a c e t y l a t e d D-glucosamine-6-sulfate —> D - g l u c u r o n i c a c i d —> Ν-sulfate D-glucosamine-6-sulfate —> T h e l o w - a c t i v i t y fraction c o n t a i n e d o n l y 8 . 5 % of this tetrasaccharide

(4).


T h e r e f o r e , correlations b e t w e e n the c h e m i c a l a n d p h y s i c a l structure a n d the anticoagulant activity m u s t exist. T h e m e c h a n i s m s b y w h i c h h e p a r i n exerts an anticoagulant f u n c t i o n are controversial. W h e n h e p a r i n is a d d e d to a solution of a n t i t h r o m b i n III (ATIII), a naturally o c c u r r i n g i n h i b i t o r of the p r o t e o l y t i c e n z y m e t h r o m b i n , the rate of t h r o m b i n n e u t r a l i z a t i o n is essentially instantaneous as c o m p a r e d w i t h a gradual t h r o m b i n n e u t r a l i z a t i o n rate i n the absence of h e p a r i n (5). T h i s result has l e d to the g e n e r a l l y a c c e p t e d s c h e m e w h e r e h e p a r i n first binds to A T - I I I , greatly p o t e n t i a t i n g t h r o m b i n b i n d i n g to A T - H I b i n d i n g sites i n the heparin/AT-III c o m p l e x .

T h e heparin/AT-III c o m p l e x not o n l y b i n d s to

t h r o m b i n , b u t also to e v e r y active serine protease i n the i n t r i n s i c coagulation pathway (6). O n l y o n e - t h i r d of a c o m m e r c i a l h e p a r i n p r e p a r a t i o n possesses anti coagulant activity as d e m o n s t r a t e d by A T - I I I affinity c h r o m a t o g r a p h y (7). I n v i e w of the w i d e c h e m i c a l - s t r u c t u r a l variance of h e p a r i n a n d the a b i l i t y of h e p a r i n to b i n d w i t h m a n y e n z y m e s a n d proteins, these observations are not overly s u r p r i s i n g . H e p a r i n has m a n y pharmacological effects, of w h i c h anti coagulation is one. M a n y of these other functions manifest themselves as u n d e s i r e d side effects w h e n h e p a r i n is a d m i n i s t e r e d systemically for anti coagulation purposes i n c l u d i n g : respiratory i m p a i r m e n t s , platelet c y t o p e n i a , increased fatty acid transport across b i o m e m b r a n e s , i n h i b i t i o n of osteoblasts, and increased β - l y m p h o c y t e m i g r a t i o n (1 ). T h e s e u n w a n t e d effects, i n a d d i tion to the h i g h danger of i n t e r n a l h e m o r r h a g i n g f r o m p r o l o n g e d c l o t t i n g times, w h i c h is especially c r i t i c a l to patients w i t h h e m o p h i l i c disorders and/ or i m p a i r e d h e m a t o p o i e t i c f u n c t i o n (such as k i d n e y dialysis patients),

make

c o n v e n t i o n a l h e p a r i n therapy a h i g h risk to the great n u m b e r of i n d i v i d u a l s w h o u n d e r g o daily t r e a t m e n t w i t h b l o o d - c o n t a c t i n g b i o m e d i c a l devices. To c i r c u m v e n t m a n y of these u n d e s i r e d side effects associated

with

systemic h e p a r i n a d m i n i s t r a t i o n , m a n y investigators have endeavored i m m o b i l i z e h e p a r i n to b l o o d - c o n t a c t i n g p o l y m e r s to f o r m

to

thromboresistant

surfaces. C o n s i d e r i n g that h e p a r i n b i n d s to the e n d o t h e l i u m f o l l o w i n g sys t e m i c injection (I), this approach appears

attractive.

S a l z m a n et al. covalently c o u p l e d h e p a r i n to h y d r o x y l - b e a r i n g surfaces v i a an e t h y l e n e i m i d e i n t e r m e d i a t e .

B l o o d exposed to these surfaces ex

h i b i t e d p r o l o n g e d c l o t t i n g times, b u t platelet adhesion was

substantially

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

12.

EBERT ET A L .

Derivatized and Immobilized Heparins

163

h i g h e r than u n t r e a t e d , c o n t r o l l e d surfaces (8). T h e presence of h e p a r i n o n the surface i n f l u e n c e d the adsorption of plasma proteins, w h i c h subsequently caused platelet adhesion. O t h e r researchers (9, JO) i m m o b i l i z e d h e p a r i n to p o l y e t h y l e n e surfaces by p r i o r adsorption of a cationic surfactant. H e p a r i n was t h e n i o n i c a l l y b o u n d to the adsorbed surfactant a n d c h e m i c a l l y stabilized by subsequent glutarald e h y d e fixation. T h e s e surfaces p r o v i d e approximately a 3 % release of the total affixed h e p a r i n over a 10-h i n t e r v a l a n d essentially no release after that


i n i t i a l p e r i o d . B l o o d exposed to such surfaces demonstrated variable p r o l o n g e d c l o t t i n g times d e p e n d e n t on the amount of h e p a r i n that c o u l d migrate t h r o u g h the resultant a d s o r b e d p r o t e i n layer, as revealed b y A u g e r spectrosopy of the p r o t e i n adsorbate surface. D u r i n g the initial contact b e t w e e n the h e p a r i n - g l u t a r a l d e h y d e - s t a b i l i z e d surface a n d b l o o d , the h e p a r i n was released f r o m the surface and m i g r a t e d t h r o u g h the adsorbed p r o t e i n layer to the surface of the p r o t e i n adsorbate w h e r e it was available for interaction w i t h appropriate b l o o d factors. If insufficient amounts of h e p a r i n penetrated the adsorbed p r o t e i n layer, e i t h e r d u e to inadequate amounts of surface h e p a r i n or to too strong of a surface/heparin interaction, the h e p a r i n i z e d surfaces w e r e ineffective i n anticoagulation. Platelet adhesion was r e d u c e d dramatically for h e p a r i n i z e d - g l u t a r a l d e h y d e - s t a b i l i z e d surfaces relative to untreated c o n t r o l p o l y e t h y l e n e . T h i s result was a t t r i b u t e d to changes i n the p h y s i o c h e m i c a l nature of the h e p a r i n i z e d surface i n f l u e n c i n g the adsorption of a p l a t e l e t - c o m p a t i b l e p r o t e i n layer. A surface c o u l d be platelet c o m p a t i b l e a n d t h r o m b o g e n i c i f insufficient amounts of h e p a r i n p e n e t r a t e d the adsorbed layer (i.e., h e p a r i n t i g h t l y associated w i t h the surface was ineffective). M i u r a et al. (11) i m m o b i l i z e d h e p a r i n o n a variety of cyanogen b r o m i d e activated surfaces. A l t h o u g h p r o l o n g e d c l o t t i n g times w e r e o b s e r v e d , t h r o m b i n n e u t r a l i z a t i o n b y i m m o b i l i z e d h e p a r i n was i n d i s t i n g u i s h a b l e i n the presence or absence of A T - I I I , h i g h l y u n l i k e solution h e p a r i n behavior. A T - I I I c o u l d not interact p r o p e r l y w i t h surface-associated

h e p a r i n or the h e p a r i n

was i m m o b i l i z e d v i a the A T - I I I b i n d i n g sites. Perhaps h e p a r i n acts b y first b i n d i n g to t h r o m b i n , p o t e n t i a t i n g A T - I I I b i n d i n g to a heparin/thrombin complex. T h e s e studies indicate that h e p a r i n d i r e c t l y affixed to a surface does not p r o v i d e o p t i m a l , s o l u t i o n - l i k e , anticoagulant behavior. T h e i m m o b i l i z a t i o n of h e p a r i n d i r e c t l y to the p o l y m e r surface r e s u l t e d i n alterations of the surface properties relative to c o n t r o l surfaces, w h i c h greatly i n f l u e n c e d the plasma p r o t e i n adsorption characteristics, and overall b l o o d c o m p a t i b i l i t y

a c o n t r o l l i n g factor i n platelet adhesion

(12).

F u r t h e r m o r e , the effects on the anticoagulant activity caused b y covalent c o u p l i n g v i a specific functional groups o n the h e p a r i n m o l e c u l e w e r e not investigated. W h e n these aspects are c o n s i d e r e d , the i m p r o v e d t h r o m b o resistance of the h e p a r i n i z e d materials is not necessarily d u e to the b i o c h e m ical interactions of h e p a r i n w i t h the appropriate b l o o d factors.


164


The

strategy w e adopted

i n d e v e l o p i n g thromboresistant,

heparin-

i m m o b i l i z e d surfaces is:


1. To elucidate h e p a r i n anticoagulant effects caused b y covalent c o u p l i n g v i a specific functional groups, those h e p a r i n groups i n v o l v e d i n i m m o b i l i z a t i o n reactions are first d e r i v a t i z e d , a n d the effect of derivatization o n anticoagulant activity is deter mined. 2. To c i r c u m v e n t alterations i n the p h y s i o c h e m i c a l properties o f the surface caused b y d i r e c t h e p a r i n c o u p l i n g , h e p a r i n w i l l be i m m o b i l i z e d v i a a spacer i n t e r m e d i a t e g r o u p , thus r e m o v i n g i m m o b i l i z e d h e p a r i n f r o m the surface p e r se a n d locating the heparin i n a more " b u l k - l i k e " plasma e n v i r o n m e n t . T h e use of the spacer g r o u p not o n l y aids i n retaining p h y s i o c h e m i c a l properties more similar to the original surface, but also places the heparin i n a b u l k solution phase w h e r e it can interact m o r e w i t h plasma factors as though it were i n true solution (i.e., surface effects are reduced). 3. H a v i n g p r e v i o u s l y q u a l i f i e d the functional group or groups that least affect anticoagulant activity f o l l o w i n g derivatization, heparin is i m m o b i l i z e d v i a spacer groups of various lengths u t i l i z i n g reactions specific for noncritical h e p a r i n functional groups. T h e spacer g r o u p distance p r o v i d i n g o p t i m a l h e p a r i n activity is then d e t e r m i n e d , a n d surfaces are i m m o b i l i z e d us ing that specific reaction scheme. This chapter focuses on the effects of anticoagulant activity caused by specific functional group derivatization of h e p a r i n , a n d on p r e l i m i n a r y i m m o bilization spacer group evaluations. T h e functional groups selected for i m m o bilization are h y d r o x y ! a n d carboxylic h e p a r i n groups.

Experimental Free amine groups on porcine intestinal heparin (Sigma) were first blocked with acetic anhydride to form N-acetylated heparin (13). This blocking reaction was per formed to insure against intermolecular or intramolecular cross-linking during hepa rin carboxylic activation reactions. The iV-acetylated heparin was then dialyzed exten sively and freeze-dried. This heparin preparation was used in all further derivatization and immobilization reactions. Functional Group Derivatization. C A R B O X Y L I C DERIVATIZATION. Two grams of iV-acetylated heparin and 2 mL of n-butylamine were dissolved in 40 mL of water, and the pH was adjusted to 4.75. A total of 0.8 g of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride was added to the heparin/n-butylamine solution in approximately 10-mg portions over a 6-h period while the reaction was maintained at 4°C and pH 4.75. Periodic samples were withdrawn from the reaction vessel, dia lyzed, and freeze-dried. In a separate reaction, 0.5 g of Ν -acetylated heparin and 1.0 g of 2-aminoethyl hydrogen sulfate were dissolved in 10 mL of water and adjusted to pH 4.75. A total of 0.2 g of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro chloride was added to the reaction vessel in approximately 10-mg portions over a 4-h


165


EBERT E T AL.

12.

period while maintaining the pH at 4.75 and the temperature at 4°C. The carboxylicderivatized heparins were then dialyzed and freeze-dried. Carboxylic reaction schemes are illustrated in Figure 1. HYDROXYL

GROUP

DERIVATIZATION.

2

2


One-half

Epichlorohydrin Activation.

gram of Ν -acetylated heparin was dissolved in 10 mL of 1.0M N a C 0 solution. One milliliter of epichlorohydrin and either 1 mL of n-butylamine or 1 g of glycine or 2-aminoethyl hydrogen sulfate were added to the heparin solutions and allowed to react at 40°C for 5 h. Reaction schemes are illustrated in Figure 2. The hydroxylderivatized heparins were then dialyzed and freeze-dried. Divinylsulfone Activation. One-half gram of Ν-acetylated heparin was dis solved in 10 mL of 1.0M N a C 0 solution. One milliliter of divinylsulfone and either 1 mL of n-butylamine or 1 g of glycine or 2-aminoethyl hydrogen sulfate were added to the heparin solutions which were allowed to react at 22°C for 3 h. The reaction schemes are illustrated in Figure 3. The hydroxyl-derivatized heparins were then dialyzed and freeze-dried. Nuclear Magnetic Resonance (NMR) Spectrometry. Spectral verification of derivatization was provided by proton NMR of Ν-acetylated and carboxylic- and hydroxyl-derivatized heparins using a 300-MHz Varian SC 300 NMR spectrometer. Forty milligrams/milliliter of the respective heparin was dissolved in deuterium oxide with 1% 2,2-dimethyl-2-silapentane-5-sulfonate(DSS) and transferred to 5-mm NMR tubes. Spectra were obtained at room temperature with a spin rate of 20 rps and a gas flow rate of 13 ft /h. 3

3

3

C=N-C H » N-(CH )3-N-ChL ι 2

HEP-COOH

+

5

+ Η

2

EDC

t 0

HN-C H 2

5

II

HEP-C-0-C-NH-(CH ) -N-CH ι CH, 2

2

HoN-R

+ 2

(CH ) ι Ν 2

H C 3

/

N

5

3

HN-C H

HEP-COOH HN-C H ι C= 0 ι HN

3

0 Μ

II

HEP-C-N-R ι Η

+

HN-C H ι 2

5

C=0

ι HN

3

3

CH

(CH ) ι Ν / \ CH„ H C 2

3

c =o

ι HEP-C-N ι (CH?) 2'3 ι Ν / \ CH, H C

0

3

H N-R 2

3

Figure 1. The l-ethyl-3-(3-dimethylaminopropyl)carbodiimide-mediated N-acetylated heparin carboxylic group derivatization reaction scheme.


5

166


HEP-OH

+

C - C - C

EPICHLOROHYDRIN H E P - O - C - C - C

+

V


HEP-O-C

H N-R 2

- C - C - N - R ι OH

Figure 2. Epichlorohydrin-mediated N-acetylated heparin hydroxyl group derivatization reaction scheme. Heparin Immobilization. Tritium-labeled heparin (New England Nuclear) was iV-acetylated as described previously. All immobilization reactions were conducted with ( H)N-acetylated heparin. Diaminoalkane-derivatized agaroses (1,2-diaminoethane agarose, 1,4-diaminobutane agarose, 1,8-diaminooctane agarose, 1,10-diaminodecane agarose, and 1,12-diaminododecane agarose) were purchased (Sigma) and used for immobilization substrates. Tritium-labeled, N-acetylated heparin was carboxylic-activated at 4°C in phosphate-buffered saline (pH 7.4) with N-ethyl-5phenylisoxazolium-3'-sulfonate (Woodward's Reagent K) for 8 h. The Woodward's Reagent Κ was added in amounts to provide a maximum of 20% activation of the total carboxylic groups based on conductimetric titrations of the N-acetylated heparin (i.e., a minimum of 80% of the total carboxylic groups remains unmodified after immobilization on surfaces). A 10-mL aliquot of the activated heparin solution (8.0 mg activated heparin/mL solution) was added to 4 mL of each diaminoalkane-derivatized gel, the resultant slurry being gently stirred at 4°C for 24 h. Woodward's Reagent Κ 3

0 HEP-OH

+

C = C - S - C =C II

0 0 H E P - 0 - C - C - S - C =C II

+

H N-R 2

•

0

H E P - 0 - C - C - S - C - C - N - R n Figure 3. Divinylsulfone-mediated N-acetylated heparin hydroxyl group derivatization reaction scheme.



12.

167


EBERT ET AL.

was used as the condensation agent during immobilization reactions because this reaction proceeds efficiently at pH 7.4. With carbodiimide reactions, acidic condi tions must be used which, over a 24-h period, can lead to substantial desulfonation of the heparin. The reaction scheme is illustrated in Figure 4. Each gel was then collected by suction filtration with sintered-glass funnels and washed with phosphatebuffered saline (pH 7.4) in 50-mL increments, collecting each washing for heparin quantitation via liquid scintillation counting. A 10-mL aliquot of 2 M 2-aminoethanol in phosphate-buffered saline (pH 7.4) was added to each gel to block all possible remaining activated carboxylic groups on immobilized heparin. The gels were then filtered and washed as described pre viously. Heparin was not detectable in any of these washings. Carboxyl and Sulfate Group Determinations. Conductimetric titrations of heparin (14) provided a convenient measure of sulfate and carboxylic groups. Sodium heparin was first converted into the acid form by elution of a known quantity of heparin, dissolved in neutral deionized distilled water, through a column of pre viously washed Amberlite IR-120 (H ) or bio-Rad AG 50W-X2 resin (i.e., 50-100 mg of heparin through a 7-mL column and diluted to a final volume of 150 mL to give a final concentration of 0.33 to 0.67 mg/mL heparin). The effluent was collected until onlv neutral water exited the column, and this then was diluted to a final volume of 150 mL. +

WOODWARDS REAGENT Κ

HEP-COO

^ c —0 II

H"

I +

^C H

X'

2

5

• 11

HEP

J

+

-C-O-C II

0

H N-(CH ) -b 2

2

n

II

C-C-NH-C H 11 * ° 2

5

0

0 II

HEP-C-N-(CH ) 2

n

+

C-OH 11 CH-C-NH-C2H5 II

0 Figure 4. Immobilization of N-acetulated diaminoalkane agarose gels using Ν-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's Reagent K) activated heparin carboxylic groups.


168


The solution was titrated with 0. IN NaOH while measuring solution conductivity vs. volume of NaOH solution added. The conductance of the sample solution, initially high mainly due to the contribution of mobile protons on the - S 0 H groups from both Ν-sulfates and Ο-sulfates (specific conductance λ+ = 350), decreased linearly as - S 0 H protons were replaced by N a ions (λ+ = 50). After all of the - S 0 H protons were neutralized, the curve leveled off. This plateau region corresponded to carbox ylic group proton dissociation. Conductance barely changed in this region due to the compensatory effects from carboxylic proton neutralization and increased Na ion concentration. After dissociation and neutralization of all carboxylic protons, conduc tance increased sharply primarily due to O H " ion contributions (λ_ = 198). Extrapo lation of the three branches of the conductimetric curves gave two intersection points, the first corresponding to the number of — S 0 H groups while the second correspond ed to the number of - C O O H groups (see Figure 5). Changes in the number of titratable carboxylic groups on derivatized heparins, relative to the N-acetylated heparin starting material, provide an accurate measure of the degree of carboxylic derivatization. 3

+

3

3


3

Anticoagulant Activity Assay. The anticoagulant activities of N-acetylated heparin and all further derivatized heparins were determined based on activated partial thromboplastin time (APTT) assay methods (15). Bovine blood was collected in 3.8% sodium-citrated (9 parts blood to 1 part citrate solution) and centrifuged at 5000 x g for 15 min. The supernatant plasma was collected and pooled for subsequent APTT testing. Prior to testing plasma was kept refrigerated no longer than 6 h after

0

2.0

4.0

6.0 0.1 Ν NaOH

9.0

11.0

13.0

(mis)

Figure 5. Conductimetric titration curve for epichlorohydrin-mediated 2-aminoethyl hydrogen sulfate N-acetylated heparin derivative (R =Ncoo-/N = V -V /V ). Key: Ν coo-, and N -,': ;•'·";:. s03

2

1

1

so



12.

169


EBERT E T A L .

30 h

0'

0

·

•

1

'

'

1

0.15 0.35 P L A S M A HEPARIN (UNITS/ml

'

'

0.5 PLASMA)

1

Figure 6. APTT heparin activity assay curve. Key: ·, control heparinized plasma (units/mL plasma) calibration APTT and O , derivatized heparin (at known weight concentrations, mg/mL plasma) APTT.

collection. Bovine plasma was heparinized in 0.1 unit/mL plasma increments, and APTT was determined for each plasma heparin concentration using activated thromboplastin reagent (Ortho). A linear relationship exists between 0 and 0.5 unit/mL plasma (see Figure 6). After determining the control heparin-APTT response curve, derivatized heparin was added to unheparinized bovine plasma (the same plasma used for preparing the control response curve) at known weight concentrations, and APTT was determined. Based on the interpolated heparin activity (units/mL plasma) of the derivatized heparin at known weight concentration (mg/mL plasma), the anticoagulant activity (units/mg) of the N-acetylated and hydroxyl- and carboxylic-derivatized heparins was determined. Activated partial thromboplastin time assays also were used to evaluate heparin activity as a function of the spacer group distance with the heparin immobilized gels. The APTT vs. plasma heparin activity calibration curve was first prepared as described above. A 0.5-mL aliquot of each heparinized gel was added to 10 mL of the same plasma used in preparing the calibration curve. The gel and plasma were then gently rotated for 10 min at room temperature and centrifuged at 2500 X g for 5 min. The supernatant plasma was collected, and APTT was determined for each plasma. APTT was similarly determined for plasma exposed to substrate gels without heparin immobilization (i.e., 1,2-diaminoethane agarose, 1,4-diaminobutane agarose, etc.). Tritium-labeled free heparin levels in all gel-exposed plasma samples were determined by liquid scintillation counting using a Beckman LS 9000 scintillation counter.


170


Results and Discussion Heparin Derivatization.

Results f r o m A P T T activity assays a n d c o n -

d u c t i m e t r i c titrations for c a r b o x y l i c - d e r i v a t i z e d heparins are p r e s e n t e d i n Table I. H y d r o x y l - d e r i v a t i z e d h e p a r i n results are listed i n Table II. A l l c o n d u c t i m e t r i c titration results are expressed as the ratio of the n u m b e r

of

carboxylic groups over the n u m b e r of sulfate groups (R = N _ O O H / N - S O H ) C

3

A c t i v a t e d partial t h r o m b o p l a s t i n t i m e results for h e p a r i n derivatives

are

expressed as the m e a n activity (units/mg) ± 9 5 % confidence l i m i t s (N greater


than 10 for all derivatives). N - A c e t y l a t e d h e p a r i n shows no significant alkane p r o t o n s p l i t t i n g patterns f r o m 0 to 5 p p m ( F i g u r e 7), w h i l e d e r i v a t i z e d heparins show characteristic alkane p r o t o n s p l i t t i n g patterns i n that range (see F i g u r e 8 for N M R s p e c t r u m of d i v i n y l s u l f o n e - a c t i v a t e d , 2 - a m i n o e t h y l derivative). T h e carboxylic d e r i v a t i z a t i o n e x p e r i m e n t s

c o n f i r m previous findings

(16), that derivatization leads to loss i n anticoagulant activity. In that study, however, o n l y f u l l y d e r i v a t i z e d c o m p o u n d s w e r e evaluated. T h e degree of derivatization was evaluated i n the n-butylamine/carboxylic derivatization series. Partial d e r i v a t i z a t i o n c o u l d be o b t a i n e d (up to 4 5 %

derivatization)

w i t h m i n i m a l losses i n anticoagulant activity. C o n s i d e r i n g that the average weight p e r h e p a r i n m o l e c u l e h a d to increase after derivatization w i t h the p r i m a r y amines, the activity p e r initial a m o u n t of h e p a r i n r e m a i n e d nearly u n c h a n g e d after partial d e r i v a t i z a t i o n . F u r t h e r derivatization l e d to decreases i n titratable - S 0 H groups, as d e t e r m i n e d by c o n d u c t i m e t r i c t i 3

trations, p r o b a b l y d u e to losses of 2 - 0 - s u l f a t e s i n L - i d u r o n i c acid residues u n d e r acidic conditions (17).

W h e t h e r the loss i n activity was strictly d u e to

derivatization or to desulfonation cannot be d e t e r m i n e d at this t i m e . A s w i t h the carboxylic derivatives, h y d r o x y l derivatives showed m i n i m a l losses i n activity u n d e r partial d e r i v a t i z a t i o n . In these e x p e r i m e n t s the substituted l i g a n d e n d g r o u p , e i t h e r a sulfate g r o u p (2-aminoethyl

hydro-

Table I. Carboxylic Derivatives ( H e p a r i n - C - N - R ) and Their Anticoagulant Activities R (Alkyl Group)

% Derivatization

Control (N-acetylated) n-Butylamine n-Butylamine n-Butylamine n-Butylamine 2-Aminoethyl hydrogen sulfate

Activity

0

(units/mg) COOH

0 17.4 24.4 45.9 100

157 132 126 128

± ± ± ± 0

14 8 15 8

0.55 0.50 0.44 0.30 0

34.3

111

± 10

0.40

~/S0 ~ 3

"Not taking into account the weight increase above the initial amount of heparin due to derivatization.


12.

EBERT ET AL.

171


Table Π. Hydroxyl Derivatives (Heparin-O-Cross-linking Agent-R) and Their Anticoagulant Activities

/so;

Cross-linking -R Estimated Activity coo Agent (Alkyl Group) Derivatization (units/mg) b


0

Epichlorohydrin n-butylamine Epichlorohydrin 2-aminoethyl h y d r o g e n sulfate E p i c h l o r o h y d r i n glycine n -butylamine Divinylsulfone Divinylsulfone 2-aminoethyl h y d r o g e n sulfate glycine Divinylsulfone

0.58

10-20

124

±11

10-20 10-20 10-20

132 ± 121 ± 113 ±

5 8 5

0.47 0.43 0.55

10-20

133 ± 10 180 ± 1 4

0.52 0.47

"Based on comparison of NMR spectra with carboxylic derivatized heparins where the % derivatization was measured. Not taking into account the weight increase above the initial amount of heparin due to derivatization. fc

gen sulfate), a c a r b o x y l i c g r o u p (glycine), or a neutral m e t h y l group ( n butylamine) h a d l i t t l e to no effect o n the activity of the d e r i v a t i z e d h e p a r i n . This result was substantiated

f u r t h e r b y carboxylic-derivatization

experi

ments w h e r e the 2 - a m i n o e t h y l h y d r o g e n sulfate derivative (34.3% d e r i vatized) d e m o n s t r a t e d

anticoagulant activity comparable to

n-butylamine

dérivâtes at a s i m i l a r d e g r e e of derivatization. T h e s e results indicate that, u n d e r partial derivatization, the c h e m i c a l nature of the d e r i v a t i z e d e n d g r o u p is not critical to anticoagulant activity w i t h i n the l i m i t e d n u m b e r of d e r i v a t i z i n g agents tested; however, the degree of derivatization is c r i t i c a l . F u r t h e r m o r e , b o t h carboxylic a n d h y d r o x y l hepar i n groups can be u t i l i z e d i n h e p a r i n surface c o u p l i n g reactions. Heparin Immobilization.

T h e amount of i m m o b i l i z e d h e p a r i n

per

m i l l i l i t e r of each respective g e l is l i s t e d i n Table III. Results f r o m A P T T evaluations o n p l a s m a exposed to h e p a r i n i z e d gels are presented i n F i g u r e 9. A l l h e p a r i n i z e d surfaces p r o d u c e p r o l o n g e d clotting times relative to control surfaces a n d the baseline A P T T . H o w e v e r , clotting times increase p r e c i p i t o u s l y i n an e x p o n e n t i a l fashion w h e n at a spacer distance of 10 carbon atoms. N o n e of the plasmas heparin

exposed

via scintillation counting

to h e p a r i n i z e d gels revealed free

(specific activity of

( H)N-acetylated 3

h e p a r i n = 43,114 dpm/mg) at levels w h i c h c o u l d account for the

increased

clotting t i m e . F r e e t r i t i u m - l a b e l e d h e p a r i n levels i n plasma exposed to 10and 12-carbon atom spacer gels w e r e 15.05 a n d 22.52 d p m / m L , respectively. This corresponds to p l a s m a h e p a r i n concentrations of 0.35 a n d 0.52

μg/mL,

w h i c h are e q u i v a l e n t to 0.054 u n i t s / m L a n d 0.080 units/mL, respectively. H e p a r i n i z e d gels w i t h 2-, 4- a n d 8-carbon spacer groups p r o d u c e d slightly p r o l o n g e d c l o t t i n g times relative to controls, w h i c h is insignificant c o m p a r e d




172



1

9

1

10

1

8

7

1

6

1

5

1

4

1

3

I

2

•

1

•

I

0

•

ΡΡΜ

Figure 8. Proton NMR spectrum of divinylsulfone-mediated 2-aminoethyl hydrogen sulfate heparin derivative.

«


CO

H

m

to

174


to 10- a n d 12-carbon spacer h e p a r i n i z e d gels. T h e s e results w o u l d indicate that as t h e spacer g r o u p distance increases,

t h e availability o f surface-

i m m o b i l i z e d h e p a r i n to the b i n d i n g sites o n specific p a r t i c i p a t i n g coagulation factors increases; however, because p r o l o n g e d c l o t t i n g times w e r e observed for l o w spacer g r o u p distances, these h e p a r i n i z e d surfaces are also b i n d i n g coagulation factors (perhaps nonspecifically). B e r g et a l . (18) r e p o r t e d that t h r o m b i n , i n i t i a l l y a n i o n i c i n the p r o t h r o m b i n z y m o g e n f o r m , b e c o m e s cationic at n o r m a l b l o o d p H f o l l o w i n g activation. T h e negatively charged hepa-


rin surfaces c o u l d r e a d i l y adsorb t h r o m b i n nonspecifically f r o m plasma. T h r o m b i n a d s o r p t i o n f r o m t h e test p l a s m a c o u l d explain w h y o n l y slightly elevated c l o t t i n g times w e r e o b s e r v e d for 8 o r less carbon u n i t spacer groups, k n o w i n g that relatively f e w p r o t h r o m b i n e n z y m e s w o u l d b e activated into t h r o m b i n i n the test p l a s m a at that p o i n t . T h e greatly elevated h e p a r i n activity associated w i t h 10 o r m o r e carbon unit spacer groups may b e d u e to specific h e p a r i n interactions w i t h coagulation factors (i.e., specific b i n d i n g of A T - I I I ) m o r e s i m i l a r to solution h e p a r i n behavior. T h e mass transport o f h e p a r i n b i n d i n g factors to t h e h e p a r i n i z e d surface is an i m p o r t a n t factor that must b e c o n s i d e r e d i n evaluating the efficacy o f t h e i m m o b i l i z e d anticoagulant. U t i l i z i n g the methods r e p o r t e d here for t h e i m m o b i l i z e d h e p a r i n evaluations, this aspect cannot b e a d dressed adequately at present, a n d f u r t h e r testing is necessary to resolve this question. Irrespective

o f t h e anticoagulant mechanisms

involved with immo-

b i l i z e d h e p a r i n s , t h e anticoagulant activity greatly increases as t h e i m m o b i l i z e d h e p a r i n m o l e c u l e s are r e m o v e d f r o m the surface e n v i r o n m e n t to a b u l k - l i k e p l a s m a e n v i r o n m e n t v i a spacer groups. T h e relative a m o u n t o f u n m o d i f i e d carboxylic groups o n i m m o b i l i z e d h e p a r i n available f o r interaction w i t h coagulation factors appears critical based o n carboxylic d e r i v atization results; therefore, t h e n u m b e r o f i m m o b i l i z a t i o n points p e r h e p a r i n m o l e c u l e must b e m i n i m i z e d for carboxylic i m m o b i l i z a t i o n reactions. T h e average n u m b e r o f i m m o b i l i z a t i o n points p e r h e p a r i n m o l e c u l e d u e to the n o r m a l variation i n c h e m i c a l structure a n d m o l e c u l a r w e i g h t o f c o m m e r c i a l Table III. Heparin Immobilization onto Agarose Gels Gel

Spacer Distance

mg Immobilized/

(Carbon Number)

mL Gel

1 , 2 - D i a m i n o e t h a n e agarose 1 , 4 - D i a m i n o b u t a n e agarose 1 , 8 - D i a m i n o o c t a n e agarose 1 , 1 0 - D i a m i n o d e c a n e agarose 1 , 1 2 - D i a m i n o d o d e c a n e agarose

2 4 8 10 12

0.50 1.00 3.14 2.33 0.95


12.

175


EBERT E T A L .

200 180 160

_

140

Ο Lu


CO

120

r-

α. ιοο < 80 60 40 20 0

4

8

12

16

SPACER CARBON NUMBER Figure 9. APTT vs. spacer unit carbon number results for heparin immobilized via carboxylic groups to diaminoalkane agarose gels. Key: —, the baseline APTT (i.e., unheparinized plasma); ·, respective control substrate APTT (i.e., untreated diaminoalkane agarose gels); and O , respective heparin immobilized gels. h e p a r i n is difficult to estimate. H o w e v e r , a d i s t r i b u t i o n o f i m m o b i l i z a t i o n points per h e p a r i n m o l e c u l e must exist, w i t h t h e lowest m o l e c u l a r w e i g h t molecules p r o b a b l y b e i n g i m m o b i l i z e d b y a single b o n d a n d t h e h i g h e r m o l e c u l a r w e i g h t m o l e c u l e s b e i n g i m m o b i l i z e d b y several bonds.

Acknowledgments T h e authors gratefully a c k n o w l e d g e the invaluable contributions to this work b y Jan F e i j e n a n d D e n n i s C o l e m a n . T h i s w o r k was s u p p o r t e d b y N I H Grant # H L - 2 0 2 5 1 .

S. W . K i m is a n N I H Research C a r e e r D e v e l o p m e n t

Awardee ( H L - 0 0 2 7 2 ) .

Literature Cited 1. Jaques, L. B. Science 1979, 206, 528. 2. Horner, A. A. "Heparin Chemistry and Clinical Usage"; Kakkar, V. V.; Thomas, D. P., Eds.; Academic: New York, 1976; p. 37. 3. Barrowcliffe, T. W.; Johnson, Ε. Α.; Eggleton, C. Α.; Kemball-Cook, G.; Thom as, D. P. Br. J. Haematol. 1979, 41, 573. 4. Rosenberg, R. D.; Lam, L. Proc. Natl. Acad. Sci. (U.S.A.) 1979, 76, 1218.



176


5. Seegers, W.; Warner, E. D.; Brinkhous, Κ. M.; Smith, H. P. Science 1942, 96, 300. 6. Rosenberg, R. D. "Heparin Chemistry and Clinical Usage"; Kakkar, V. V.; Thomas, D. P., Eds.; Academic: New York, 1976; p. 101. 7. Rosenberg, R. D. Semin. Hematol. 1977, 14, 427. 8. Salzman, E. W.; Merrill, E. W.; Binder, Α.; Wolf, C. F. W.; Ashford, T. P. J. Biomed. Mater. Res. 1969, 3, 64. 9. Lagergren, H.; Eriksson, J. C. Trans. Am. Soc. Artif. Intern. Organs 1971, 17, 10. 10. Larsson, R.; Eriksson, J. C.; Lagergren, H.; Olsson, P. Thromb. Res. 1979, 15, 157. 11. Miura, M.; Sadayoshi, Α.; Kusada, Y.; Miyamoto, K. J. Biomed. Mater. Res. 1980, 14, 619. 12. Lee, E. S.; Kim, S. W. Trans. Am. Soc. Artif. Intern. Organs 1979, 25, 124. 13. Danishefsky, I.; Steiner, H. Biochim. Biophys. Acta 1965, 101, 37. 14. Casu, B.; Gennaro, U. Carbohyd. Res. 1975, 39, 168. 15. Cifonelli, T. A. Carbohydr. Res. 1974, 37, 145. 16. Danishefsky, I.; Siskovic, R. Thromb. Res. 1972, 1, 173. 17. Kosakai, M.; Yosizawa, Z. J. Biochem. 1979, 86, 147. 18. Berg, W.; Hillvarn, B.; Arwin, H.; Stenberg, M.; Lundstrom, I. Thromb. Haemostas. 1979, 42, 972. RECEIVED for review January 16, 1981. ACCEPTED March 28, 1981.


The Anticoagulant Activity of Derivatized and Immobilized Heparins

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