Separation of heparin into fractions with different anticoagulant activity by hydrophobic interaction chromatography

477

Biochimica et Biophysica Acta, 626 (1980) 477--485 © Elsevier/North-Holland Biomedical Press

BBA 38567

SEPARATION OF HEPARIN INTO FRACTIONS WITH DIFFERENT ANTICOAGULANT ACTIVITY BY HYDROPHOBIC INTERACTION CHROMATOGRAPHY

AKIRA OGAMO, HIDEKI UCHIYAMA and KINZO NAGASAWA

School of Pharmaceutical Sciences, Kitasato University, Minato-ku, Tokyo 108 (Japan) (Received June 4th, 1980)

Key words: Heparin; Anticoagulant; Hydrophobic interaction chromatography; Phenyl~epharose gel; (Porcine)

Summary Hog mucosal heparin purified on Sephadex G-100 (anticoagulant activity assayed by the method of the United States Pharmacopoeia, 179 units/mg) was separated by hydrophobic interaction chromatography on Phenyl-Sepharose CL-4B into two groups, one with high affinity and another with low affinity for the gels. The former group was further separated into three fractions differing in hydrophobicity. The anticoagulant activities of the fractions with higher hydrophobicity ranged from 210 to 254 units/mg, whereas that of the fraction with lower hydrophobicity was 100 units/mg. The difference in antithrombin III-activation potency was much more prominent. The data obtained from affinity chromatography of these fractions on antithrombin III-Sepharose also substantiated the observed difference in anticoagulant activity. Analytical data of the fractions revealed a characteristic difference in both N-acetyl content and molecular size. While the N-acetyl content (mol/mol of hexosamine) and Kay value (on Ultrogel AcA44) of the fraction with the lowest hydrophobicity were 0.12 mol and 0.48, those of the fractions with higher hydrophobicity were 0.15--0.17 mol and 0.35--0.23, respectively.

Introduction Many proteins show differences in their interaction with hydrophobic groups attached to a hydrophilic matrix. These differences in 'apparent hydrophobicity' have been used by several groups for fractionation of proteins by hydrophobic interaction chromatography [1--4]. Recently, we have succeeded in the preparation of biologically active fluorescent heparin consisting only of fluores-

478 cein-labelled species by using hydrophobic interaction chromatography with Octyl-Sepharose CL-4B gels to separate the said fluorescent heparin from the non-labelled heparin species (Uchiyama, H. and Nagasawa, K., unpublished results). The present report describes the separation of heparin into two distinct groups, one with high affinity and another with low affinity for agarose gels carrying phenyl-glycidyl ligands, and the striking differences in chemical and biological properties between them. Materials and Methods

Materials. Commercial hog-mucosal heparin (anticoagulant activity, 161 units/mg; Sigma Chemical Co., St. Louis, MO, U.S.A.) was purified by gel-chromatography according to the procedure of Laurent et al. [5] to obtain a heparin sample with reduced molecular weight heterogeneity. Fractions 3--5 of the seven fractions obtained by gel-chromatography of heparin (3 g) on a Sephadex G-100 column (5 X 87 cm) with 0.2 M NaC1 elution, were combined, dialyzed, and freeze-dried (yield 1.9 g; anticoagulant activity, 179 units/mg). Fractions 1 and 2 were excluded from the starting material because of their higher concentration of galactosamine (see Table II). Analytical and biological data of the product (named as 'starting heparin') are summarized in Tables II and III. Purified bovine antithrombin III was prepared as described by Damus and Rosenberg [6], and the antithrombin III was coupled to cyanogen bromideactivated Sepharose 4B according to the procedure of Cuatrecasas [ 7]. Analysis of the antithrombin III-substituted Sepharose obtained indicated a protein content of a b o u t 4.2 mg/ml gel. Analytical methods. Total sulfate content was analyzed by a turbidimetric method [8]. The N-sulfate content was determined by the previously reported method [9]. The N-acetyl content was determined after acid-hydrolysis of the samples by the GLC method [10]. Uronic acid content was determined by the carbazole method of Bitter and Muir [11]. The samples were hydrolysed in 3 M HC1 for 16 h at 100°C, and glucosamine and galactosamine were separated by ion-exchange chromatography on Dowex 50 resin [12], and determined by a modification of the Elson-Morgan procedure [12,13]. Serine content was analyzed by a JEOL automatic amino acid analyzer after hydrolysation in 6 M HC1 at a concentration of 2 mg/ml in evacuated sealed tubes for 20 h at 100°C [14]. Characterization of galactosaminoglycan contaminants in heparin preparations was carried out on cellulose acetate strips in 0.3 M calcium acetate before and after enzymic digestion with chondroitinases AC and ABC [15,16]. Hydrophobic interaction chromatography o f heparin on Phenyl-Sepharose CL-4B. The 'starting heparin' (400 mg) (dissolved in 80 ml of 3.8 M (NH4)2SO4 in 0.01 M HC1 (pH 3.3)) was loaded on a 2 X 43 cm Phenyl-Sepharose CL-4B column (Pharmacia Fine Chemicals, Uppsala, Sweden) prepared in the same solution, and was eluted stepwise with a flow-rate of 20 ml/h with 900 ml of the same solution, 600 m l of 3.4 M (NH4)2SO4 in 0.01 M HC1 (pH 3.35), 550 ml of 3.0 M (NH4)2SO4 in 0.01 M HC1 (pH 3.4), and 200 ml of 2.0 M (NH4)2SO4 in 0.01 M HCI (pH 3.5) at room temperature. The effluent was collected into 15-g fractions, 20 pl of which were directly analyzed for uronic acid

479

without any undesirable effect due to high concentration of (NH4)2SO4 in the effluents. The fractions (3.8 M fraction, 217 ml; 3.4 M fraction, 170 ml; 3.0 M fraction, 136 ml; 2.0 M fraction, 63 ml) pooled as indicated in Fig. 1, were 10 X diluted with water and neutralized with 1 M NaOH. After a 10% solution of cetylpyridinium chloride was added to form a 0.2% cetylpyridinium chloride solution, the mixtures were kept at 37°C for 16 h. After centrifugation of the mixtures ( 1 6 0 0 0 X g , 20°C, 15 min), the precipitates were collected and washed with 0.1% cetylpyridinium chloride solution, and redissolved in 2.1 M NaC1 (50--100 ml). After mixing with cold ethanol (150--300 ml) under stirring, the mixtures were kept at 4°C for 16 h and the precipitates formed were collected b y centrifugation (700 X g, 20°C, 15 min), then washed with ethanol/ water (3 : 1, v/v). The precipitates obtained were dissolved in water (10 ml) and the solutions were desalted by passage through a column (2.6 × 92 cm) of Sephadex G-25 and freeze-dried. Yields of the heparin fractions obtained are shown in Table I.

Affinity-chromatography of heparin samples on antithrombin III-Sepharose. The heparin samples listed on Table III were separated into non-adsorbed, lowaffinity and high-affinity heparin fractions for antithrombin III by affinitychromatography on antithrombin III-Sepharose, essentially as described by Laurent et al. [5]. However, the column size was increased (7.6 ml, 1.8 X 3 cm) due to the lower protein content of the antithrombin III-Sepharose gels used, and the separation conditions were slightly modified. Approx. 2 mg of the heparin samples were applied to the column in 0.05 M Tris-HC1 buffer, pH 7.4, +0.05 M NaC1 at 4°C. The column was washed with the same buffer and eluted with a linear gradient (100 ml, 0.05--3 M NaC1, in Tris-HC1 buffer, pH 7.4). The flow rate was 25 ml/h and 3-ml fractions were collected. A sample of 500 /A was taken for the carbazole reaction and ionic strength measurement. The areas of absorbance at 530 nm of the non-adsorbed fraction, low-affinity fraction, and high-affinity fraction were measured, and the proportions of the three types of heparin were calculated. Gel filtration. Analytical gel-chromatography of heparin samples was performed on Ultrogel AcA44 agarose-acrylamide gel (LKB-Produkter AB, Bromma, Sweden) which was found to be favorable for the separation of the higher molecular weight region of heparin. The samples (approx. 2 mg) dissolved in 0.3 M NaC1 (1 ml), were applied on a 1.5 × 94 cm column of the gels in 0.3 M NaC1 and eluted with the same solvent at 20 ml/h according to the procedure used by Johnson and Mulloy [17]. The effluent was collected into 3.2-g fractions, each of which was analyzed for uronic acid. Assay of biological activity. Anticoagulant activity was assayed by the whole-blood assay method of the United States Pharmacopoeia [18], and the activity was expressed as units/mg. An antithrombin III-activation assay, based on the inactivation of thrombin in the presence of heparin and antithrombin III, was carried o u t according to the procedure of BjSrk and Nordenman [19] with some modification as described below. A hog-mucosal heparin with an anticoagulant activity of 164 units/mg (Cohelfred Laboratories, Inc., Chicago, IL) was used as a standard for the preparation of a calibration curve of residual thrombin activity versus heparin concentration. To a mixture of heparin (30-220 ng in 200/~1 of 0.2 M NaC1/0.1 M Tris-HC1 buffer, pH 8.0) and of a throm-

480 bin solution (100 #l, 1.0 N.I.H. unit/ml), was added a solution containing purified bovine antithrombin III (19.44 ng) and the synthetic fluorogenic thrombin substrate, Boc-Val-Pro-Arg-MCA [20] (4.0 #mol in 100 pl of 0.2 M NaC1/ 0.1 M Tris-HC1 buffer, pH 8.0). The mixture was incubated at room temperature (20°C) for 90 s. Increase in the fluorescence intensity per minute (,iF/ min) at 460 nm under excitation at 380 nm was measured on standard heparin with different concentrations. Each residual thrombin activity, which was expressed by the percentage of the ,iF/min value obtained in the absence of heparin, was plotted against log values of the corresponding heparin amounts. The assay of heparin samples was carried out as described above, and each of residual thrombin activities was compared with that caused by known amount of Cohelfred's heparin. Antithrombin III-activation potency of the samples was expressed as a percentage of Cohelfred's heparin activity. Results and Discussion Hydrophobic interaction chromatography of hog mucosal heparin was performed on agarose gels carrying hydrophobic ligands. Because Octyl-Sepharose CL-4B (Pharmacia Fine Chemicals) was slightly inferior to Phenyl-Sepharose CL-4B in the hydrophobic interaction with heparin, the latter was used in this study. The mucopolysaccharide was eluted stepwise with decreasing concentrations of (NH4)2SO4 (3.8 M-* 2 . 0 M ) in 0.01 M HC1 through a column of Phenyl-Sepharose CL-4B, and was separated into four fractions with different hydrophobicity (Fig. 1). From the chromatogram in Fig. 1, the distribution of these fractions in percentage of total applied heparin based on uronic acid assay were calculated to be 44.8% (3.8 M fraction), 30.2% (3.4 M fraction), 19.3% (3.0 M fraction), and 5.7% (2.0 M fraction), and their total recovery was 85.8%

0.3 3.8M(NH4)2SO~inO.01MHCI 3.8M fraction i

3.4M(NH4)2SO4inO.01MHCI ~0.2

~ 3.4Mfr,action 3.0M(NH4)2504in0.01MHCl ~ 3.0Mfraction 2.0H(NH4)2SO4in0.01MHCI I O M fraction

o

.c_ o.1

.<

\ 50

loo Tube number

150

. 200

Fig. 1. S e p a r a t i o n of h e p a r i n i n t o f r a c t i o n s w i t h d i f f e r e n t h y d r o p h o b i c i t y on P h e n y l - S e p h a r o s e CL-4B. The h o g m u c o s a l h e p a r i n ( 4 0 0 rag) p u r i f i e d o n S e p h a d e x G - 1 0 0 was c b x o m a t o g r a p h e d on a P h e n y l - S e p h a rose CL-4B c o l u m n (2 × 4 3 c m ) b y using s t e p w i s e e l u t i o n w i t h ( N H 4 ) 2 S O 4 ( 3 . 8 M--~ 2.0 M) in 0,01 M HC1. F r a c t i o n s (15 g p e r t u b e ) w e r e a n a l y z e d f o r u r o n i c acid c o n t e n t (20 pl w o r k i n g s o l u t i o n ) . E a c h of the p o o l e d f r a c t i o n s circled b y p a r e n t h e s e s was s u b j e c t e d to isolation.

481

TABLE I HEPARIN FRACTIONS SEPARATED ON PHENYL-SEPHAROSE

CL-4B

T h e v a l u e s p a r e n t h e s i z e d w e r e c a l c u l a t e d f r o m t h e a r e a s o f e a c h f r a c t i o n in t h e e l u t i o n d i a g r a m of F i g . 1. Heparin fraction (Molar c o n c e n t r a t i o n of (NH4)2 SO4)

Yield (mg)

3.8 3.4 3.0 2.0

132.9 (153.8) 84.6 (103.7) 47.0 (66.3) 16.4 (19.5)

M M M M

fraction fraction fraction fraction

Percentage of total material fractionated 47.3 30.1 16.7 5.9

Sum

280.9 (343.3)

Recovery

70.2 % (85.8%)

(44.8) (30.2) (19.3) (5.7)

100.0 (100.0)

(Table I). The pooled fractions as indicated in Fig. 1 were precipitated with cetylpyridinium chloride and the heparin preparations were isolated as sodium salts (yields shown in Table I). The chemical analysis of these preparations indicated that the N-acetyl content increases with increasing degree of interaction with Phenyl-Sepharose gels (Table II). Although there were small differences in the total sulfate content among these fractions (3.8 M, 3.4 M and 3.0 M fractions), the difference in N-acetyl content was prominent, together with the difference of molecular size as discussed later. A significant effect of N-acetyl content on the separation of heparin-like substances on Phenyl-Sepharose was also

T A B L E II CHEMICAL ANALYTICAL DATA OF HEPARIN PREPARATIONS G-100 OR PHENYL-SEPHAROSE CL-4B

FRACTIONATED

ON SEPHADEX

T o t a l s u l f a t e a n d N - s u l f a t e c o n t e n t s as s u l f u r . M o l a r n u m b e r s o f t o t a l s u l f a t e , N - s u l f a t e , a n d N - a c e t y l a r e g i v e n in p a r e n t h e s e s . T N P - N H 2 ( A 3 6 0 ) , a b s o r b a n c e o f 2 , 4 , 6 - t r i n i t r o p h e n y l a t e d a m i n o g r o u p s a t 3 6 0 n m p e r m g s a m p l e in 6 m l w o r k i n g s o l u t i o n . - - , n o t d e t e r m i n e d . - - . . . . , u n a b l e to b e c a l c u l a t e d b e c a u s e of a considerable c o n t a m i n a t i o n with derrnatan sulfate, n.d., not d e t e r m i n a b l e because of low concentrat i o n in g a l a c t o s a m i n e . Heparin preparation

Total sulfate (%)

N-Sulfate (%)

Fractions separated on Sephadex G-100 Fraction 1 --Fraction 2 12.11 (.... ) 4.24 Fraction 3 12.36 (2.32) 4.33 Fraction 4 12.50 (2.37) 4.38 Fraction 5 12.24 (2.29) 4.29 Fractions 3--5 (starting heparin) 12.25 (2.30) 4.32 Fractions 3.8 M 3.4 M 3.0 M 2.0 M

N-Acetyl (%)

TNP-NH 2 (A360)

(.... ) (0.81) (0.83) (0.80)

-1.76 1.61 1.43 1.31

(0.81)

1.56 (0.16)

0.0507

separated on PhenTI-Sepharose CL-4B fraction 12.61 (2.40) 4.46 (0.85) fraction 12.31 (2.32) 4.30 (0.81) fraction 12.17 (2.24) 4.37 (0.82) fraction 9.82 (.... ) 2.89 (. . . . )

1.17 (0.12) 1.53 (0.15) 1.70 (0.17) --

0.0570 0.0468 0.0436

Serine 0zmol/g)

GAIN] Total HexN (%)

23.2 5.2

(.... ) (0.16) (0.15) (0.13)

15.9

2.7 n.d. n.d. n.d. 32.7

482

suggested by the elution profile of semi-synthetic heparins with various N-acetyl content (unpublished work). The heparin sample prepared for the application on Phenyl-Sepharose gels ('starting heparin') was a combination of fractions 3--5 of the seven fractions obtained by gel chromatography on Sephadex G-100 of commercial heparin. As is shown in Table II, all of the minute amount of galactosaminoglycan present in 'starting heparin' was concentrated into the 2.0 M fraction of the fractions separated on Phenyl-Sepharose, and it was characterized to be dermatan sulfate by electrophoretic analysis before and after enzymic procedure. According to the data in Table II, 67% of the 2.0 M fraction consisted of heparin, based on hexosamine determination. As can be seen in Table III, anticoagulant activities of these preparations (determined by both a whole-blood clotting procedure and an antithrombin III-activation assay) increase markedly with increasing degree of hydrophobic interaction with Phenyl-Sepharose gels. On the other hand, Kay values determined on Ultrogel A c A 4 4 gels indicate that an increase in hydrophobicity of these preparations is related with an increase in the molecular size of heparin. When polydisperse heparin molecules are fractionated according to molecular size, the anticoagulant activity of the fractions increases with increasing degree of polymerization of heparin [5,21] (see also the references given in Ref. 5), and a possible reason for this finding has been reported [5]. To estimate the dependence of the anticoagulant activity of the heparin preparations on the molecular size, assays for anticoagulant activity and Kav value on Ultrogel AcA44 were carried out on each of the fractions separated on Sephadex G-100. The data obtained showed that there was no marked difference in the anticoagulant

TABLE III BIOLOGICAL PROPERTIES OF HEPARIN G-100 OR P H E N Y L - S E P H A R O S E CL-4B

PREPARATIONS

FRACTIONATED

ON

SEPHADEX

K a y v a l u e s w e r e b a s e d o n p e a k p o s i t i o n s o b t a i n e d in a n a l y t i c a l gel cb_romatogram o n U l t r o g e l A c A 4 4 . A n t i t h r o m b i n III a c t i v a t i o n assay b a s e d o n t h e i n a c t i v a t i o n o f t h r o m b i n in t h e p r e s e n c e o f h e p a r i n a n d a n t i t h r o m b i n III, a n d its p o t e n c y w a s e x p r e s s e d as a p e r c e n t a g e o f C o h e l f r e d ' s h e p a r i n ( 1 6 4 u n i t s / r a g ) . Heparin preparation

Anticoagulant activity (units/rag)

Kay on Ultrogel AcA44

F r a c t i o n size s e p a r a t e d o n antit h r o m b i n I I I - S e p h a r o s e c o l u m n (%) Nonadsorbed

Lowaffinity

Highaffinity

0.8 5.2 3.2 11.6

37.8 43.6 48.5 42.7

61.5 51.1 48.3 45.7

136

6.6

42.6

50.8

separated on Phenyl-Sepharose CL-4B fraction 100 0.48 50 fraction 214 0.35 221 fraction 254 0.32 286 fraction 210 0.23 297

37.8 1.0 0.4 5.4

38.7 45.7 26.3 21.1

24.0 53.8 73.3 73.5

Fractions separated on Sephadex G-100 Fraction 2 186 0.26 Fraction 3 176 0.33 Fraction 4 175 0.35 Fraction 5 171 0.40 Fractions 3--5 (starting heparin) 179 0.35 Fractions 3.8 M 3.4 M 3.0 M 2.0 M

Antithxombin III activation potency

483

activity among heparin preparations with different Kay values (Fractions 2--5 in Table III). As is shown in Table II, the N-acetyl content of the fractions separated on Sephadex G-100 gels increased apparently with increasing degree of the molecular size. However, a secure relationship between them was left to be uncertain because of a contamination of these fractions with the small amount of dermatan sulfate (Fraction 2, 5.2%; Fractions 3--5, average 2.7%, based on hexosamine analysis). In contrast, the dependency of N-acetyl content on molecular size seemed to be evident among the fractions (3.8 M, 3.4 M and 3.0 M fractions) separated on Phenyl-Sepharose CL-4B, since these fractions were free from dermatan sulfate and the difference in N-acetyl content was prominent. In view of the data in Tables II and III, it was suggested that a distinct difference in both the N-acetyl content and the molecular size among the preparations obtained by hydrophobic interaction chromatography should result from the separation depended on total hydrophobicity of heparin molecules, and the heparin species with larger molecular size was higher in N-acetyl content. The heparin preparations fractionated on Sephadex G-100 or Phenyl-Sepharose CL-4B were separated on antithrombin III-Sepharose into non-adsorbed, low-affinity and high-affinity heparin fractions, and the proportions of the three types of heparin are given in Table III. Affinity-chromatograms of the heparin preparations with different hydrophobicity are also shown in Fig. 2. The result of affinity-chromatography in Table III shows a distinct difference in the ratio of high-affinity heparin among the heparin preparations obtained by fractionation on Phenyl-Sepharose. It is noticeable that non-adsorbed heparin is localized exclusively in the 3.8 M fraction, and that both the 3.0 M and the 2.0 M fractions consist of remarkably high ratio of high-affinity heparin. All of the non-adsorbed fraction and a part of the low-affinity fraction in the 2.0 M fraction were proved to be coexistent dermatan sulfate from the result of electrophoresis of each fraction before and after enzymic digestion. Therefore, the heparin species in the 2.0 M fraction is expected to show the most potent activity if the dermatan sulfate could be removed. Several investigations aimed to separate heparin into fractions with different anticoagulant activity using ion-exchange matrices, Dowex-1 [22], ECTEOLAcellulose [23,24], DEAE-cellulose [25] and DEAE-Sephadex [21], have been reported. In the present report, the fractionation of heparin was performed based on another principle, hydrophobic interaction. The difference of hydrophobicity of each heparin species is presumed to be prominent in the acidic medium containing (NH4)2SO4 in high concentration (NaC1 was found to be unsuitable), although heparin is a typical substance carrying extremely high density of anionic groups. As suggested from the work of Rosenberg et al. [26] and Lindahl and coworkers [ 5,27], heparin species with larger molecular size and higher N-acetyl content may have a larger probability of finding the sequence required for binding to antithrombin III in the heparin molecule. The results obtained above indicate that chromatography of heparin based on hydrophobic interaction with Phenyl-Sepharose gels is effective to capture heparin species with both larger molecular size and higher N-acetyl content, resulting in the separation of heparin species with potent anticoagulant activity.

484

(a) 3.8M fraction

0.3

Low-affinity

Non-adsorbed

~,0.2

I....~

High-affinity

(b

/

/2 //

¢

3

z

._c0.1

.1o

11

10

20 Tube

30 number

~.0

(b) 3.4M fraction

0.3

Low-affinity o ~

~0.2 ®

<

Non-adsorbed

1

HIcjh-affinity

>~r-,i),<

t'"

J/I/I// )

£

11

._c0.1

10

III

30 Tube number

40

5'o

c30Mracon

0.3

~0.2

20

z 1

NonLow
) <

1:

High-affinity

o

A

o COj

.......

t 10

2'0

Jo

Tube number

2o

s'o

Fig. 2. A f f i n i t y c h r o m a t o g r a p h y o f h e p a r i n f r a c t i o n s w i t h d i f f e r e n t h y d r o p h o b i c i t y o n a n t i t h r o m b i n IllS e p h a r o s e . S o l u t i o n s (2 m l e a c h ) o f (a) t h e 3 . 8 M f r a c t i o n ( 1 . 9 6 r a g ) , (b) t h e 3 . 4 M f r a c t i o n ( 2 . 0 0 r a g ) , a n d (c) t h e 3 . 0 M f r a c t i o n ( 1 . 8 0 m g ) , w h i c h h a d b e e n o b t a i n e d b y s e p a r a t i o n o f h e p a x i n o n P h e n y l Sepharosc CL-4B, were separately chromatographed on a column (1.8 X 3 cm) of antithromhin III-Sephar o s e . T h e c o l u m n w a s first w a s h e d w i t h 0 . 0 5 M Tris-HC1, p H 7 . 4 , c o n t a i n i n g 0 . 0 5 M NaC1 a n d t h e n e l u t e d w i t h a l i n e a r g r a d i e n t o f NaCI c o n t a i n i n g 0 . 0 5 M T r i s - H C l b u f f e r . F r a c t i o n s (3 g p e r t u b e ) w e r e a n a l y z e d f o r u r o n i c a c i d c o n t e n t ( 5 0 0 ~1 w o r k i n g s o l u t i o n ) . T h e m a t e r i a l w a s g r o u p e d i n t o n o n - a d s o r b e d , l o w a f f i n i t y , a n d h i g h - a f f i n i t y f r a c t i o n s as s h o w n i n t h e figures.

485

Acknowledgements The authors thank Dr. G. Oshima, School of Pharmaceutical Sciences, Kitasato University, for his help in antithrombin III-activation assay. References 1 Shaltiel, S. (1974) Methods Enzymol. 34, 126--140 2 Ochoa, J.-L. (1978) Biochimie 60, 1--15 o 3 Hjert~n, S., Pahlman, S. and Rosengren, J. (1978) in C hroma t ogra phy of Synthetic and Biological Polymers (Epton, R., ed.), Vol. 2, pp. 53--59, Ellis H o r w o o d Ltd., Chichester oo 4 Janson, J.C. and Laas, T. (1976) in Chromatography of Synthetic and Biological Polymers (Epton, R., ed.), Vol. 2, pp. 60--66, Ellis Horwood Ltd., Chichester 5 Laurent, T.C., Tengblad, A., Thunberg, L., HS~k, M. and Lindahl, U. (1976) Biochem. J. 175, 691--701 6 Damus, P.S. and Rosenberg, R.D. (1976) Methods Enzymol. 45B, 653--669 7 Cuatreeasas, P. ( 1 9 7 0 ) J. Biol. Chem. 245, 3 0 5 9 - - 3 0 6 5 8 Dodgson, K.S. and Price, R.G. (1962) Biochem. J. 6 4 , 1 0 6 - - 1 1 0 9 Inoue, Y. and Nagasawa, K. (1976) Anal. Biochem. 71, 46--52 10 Nagasawa, K., Inoue, Y. and Kamata, T. (1977) Carbohydr. Res. 58, 47--55 11 Bitter, T. and Muir, H.M. (1962) Anal. Biochem. 4, 330--334 12 Gaxdell, S. (1953) Acta Chem. Scand. 7, 207--215 13 Blix, G. (1948) Acta Chem. Scand. 2 , 4 6 7 - - 4 7 3 14 Lindahl, U., Cifonelli, J.A., Lindah], B. and R o d i n , L. (1965) J. Biol. Chem. 240, 2 8 1 7 - - 2 8 2 0 15 Seno, N., Anno, K., Kondo, K., Nagase, S. and Saito, S. (1970) Anal. Bioehem. 37, 197--201 16 Yamagata, T., Saito, H., Habuchi, O. and Suzuki, S. (1968) J. Biol. Chem. 243, 1523--1535 17 Johnson, E.A. and Mulloy, B. (1976) Carbohydx. Res. 5 1 , 1 1 9 - - 1 2 7 18 The United States Pharmacopoeia, 16th Revision (1970) pp. 629---630 19 BjSrk, I. and Nord enman, B. (1976) Eur. J. Biochem. 66, 507--511 20 Morita, T., Ko to, H., Iwanaga, S., Takada, K., Kimura, T. and Sakakibara, S. (1977) J. Biochem. (Tokyo) 82, 1 4 9 5 - - 1 4 9 8 21 Piepkorn, M.W., Schmer, G. and Lagunoff, D. (1978) Thromb. Res. 13, 1077--1087 22 Di Ferrante, N. and Popenoe, E.A. (1970) Carbohydr. Res. 1 3 , 3 0 6 - - 3 1 0 23 Laurent, T.C. (1961) Arch. Biochem. Biophys, 9 2 , 2 2 4 - - 2 3 1 24 Lasker, S.E. and Stivala, S.S. (1966) Arch. Biochem. Biophys. 115, 360---372 25 Yue, R.H., Starr, T. and Gertler, M.M. (1979) Thromb. Haemostasis 42, 1452--1459 26 Rosenberg, R.D., Armand, G. and Lain, L. (1978) Proe. Natl. Aead. Sc~. U,S.A. 75, 3065--3069 27 Lindah], U., B~'ckstr~rn, G., H~6k, M., Thunberg, L., Fransson, L.-A. and Linker, A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3198---3202