Studies of chemical and biologic properties of a fraction of sulodexide, a heparin-like glycosaminoglycan

A therosclerosrs, 60 (1986) 141-149 Elsevier Scientific Publishers Ireland,

141 Ltd

ATH 03768

Studies of Chemical and Biologic Properties of a Fraction of Sulodexide, a Heparin-like Glycosaminoglycan Bhandaru Radhakrishnamurthy I-2, Chakravarthi Sharma I, R. Rao Bhandaru Gerald S. Berenson’, Luciano Stanzani 3 and Roberto Mastacchi ’ Departments

of

‘,

’Medicrne and 2 Biochemistry, Louisrana State University Medical Center, New Orleans, LA (U.S.A.) and ’Alfa Furmaceuticl.

S.P.A.

Viu Ragam

del’99n5 40133 Bologna (Italy)

(Received 25 April, 1985) (Revised received 4 December, 1985) (Accepted 6 December, 1985)

Summary

The chemical composition and biologic properties of a fraction (f) of Sulodexide, a heparin-like GAG, were studied and compared with those of two sulfated GAG preparations and heparin from intestinal mucosa. f-Sulodexide and the sulfated GAG preparations were fractionated on a Dowex-1Cll column and subsequently on an antithrombin III affinity column. Low affinity and high affinity fractions had similar chemical composition and lipoprotein lipase releasing ability, but they varied in anticoagulant activity. Low affinity fractions from f-Sulodexide had negligible anticoagulant activity while high affinity fractions had one-half the activity of mucosal heparin. When compared to heparin, both fractions had one third amount of lipoprotein lipase releasing activity. The low anticoagulant activity of f-Sulodexide suggests a suitability for long-term use as an antiatherogenic agent.

Key words:

Anticoagulant activity - Antithrombin GAG - Lipoprotein lipase - Sulodexide

affinity

-

Glycosaminoglycans

(GAG)

-

Heparin-like

Introduction Supported by funds from the National Heart, Lung and Blood Institute of the U.S. Public Health Service (HL-02942 and HL-21649), and the National Research and Demonstration Center-Arteriosclerosis (HL15103) at Louisiana State University Medical Center in New Orleans and from ALfa Farmaceutici, Italy. Requests for reprints: Gerald S. Berenson, M.D., Department of Medicine, National Demonstration Center (NRDC-A), Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, LA 70112, U.S.A. 0021-9150/86/$03.50

0 1986 Elsevier Scientific

Publishers

Ireland,

Heparin, a highly sulfated glycosaminoglycan (GAG) is a potent anticoagulant and is used clinitally to prevent postoperative thrombosis. This GAG is also known to nromote nlasma clearing of postprandial lipemia and to inhibit the development of atherosclerotic lesions in experimental animals [l-3]. Heparin has also been used in the treatment of human coronary artery disease [4-61. Ltd

142

Because of complications like decalcification, thrombocytopenia, local hematoma at site of injections, heparin is not being recommended for long term therapy for atherosclerosis. Attempts are being made to isolate heparin-like compounds with low anticoagulant activity for prophylactic use for prevention of atherosclerosis [7]. We [S-lo] previously studied three heparin-like compounds Vessel@‘. Ateroid@, and a heparin analog for chemical and biological properties related to atherosclerosis and evaluated their potential for inhibition of experimental atherosclerotic lesions. f-Sulodexide has been marketed in Europe as a drug for alteration of blood lipid levels [11,12] and for peripheral atherosclerotic arteriopathy and as arterial antithrombotic agent [13]. Since the precise chemistry of f-Sulodexide is unknown we studied the GAG composition and biologic activity of the drug along with two crude preparations of sulfated GAG, probable sources of this subfraction. Materials and Methods f-Sulodexide and two crude preparations of sulfated GAG preparation A coded as LB/200 and preparation B coded as LB/400 were obtained through Alfa Farmaceutici, 40133 Bologna, Italy. Heparin standard (hexuronic acid 39.% hexosamine 27%, sulfate, 2.38 moles/mol hexosamine, molecular weight 12000: anticoagulant activity 160 units/mg) was donated by the Upjohn Company (Kalamazoo, MI) and other GAG standards were generous gifts from Drs. J.A. Cifonelli and M.B. Mathews, University of Chicago. Chondroitinase ABC from Miles Laboratories, Inc. (Elkhart, IN), glycerol tri[9:lO(n)3H]oleate from New England Nuclear Corp. (Boston, MA), triolein from Sigma Chemical Co. (St. Louis, MO), and thrombin from Parke-Davis (Detroit, MI) were purchased. Antithrombin III was a gift from Dr. M. Wickerhauser, American Red Cross, Bethesda, MD. Analytical methods We determined uranic acid by the method of Bitter and Muir [14] and hexosamine by the method of Boas [15] omitting the use of resin. Total sulfate was determined by the method of Terho and

Hartiala [16] and N-sulfate by the procedure 01 Lagunoff and Warren [17]. Gas chromatography techniques [18-201 as described previously were used for differential determination of glucuronic acid and iduronic acid, glucosamine and galactosamine, and to estimate N-acetyl groups. Total protein was determined by the Lowry et al. [21] method. Electrophoresis Electrophoresis of fractions of f-Sulodexide and sulfated GAG for additional identification was performed on cellulose acetate strips in pyridine/formic acid buffer or cadmium acetate buffer as described previously [22,23]. The GAG were localized by Alcian blue stain. Fractionation of f-Sulodexide and suijated GAG f-Sulodexide and both the samples of sulfated GAG were fractionated on a Dowex-1 Cl (200-400 mesh) column (50 x 1.9 cm) eluting with a stepwise increasing concentration of NaCl (1.0-4.0 M) [24]. The fractions were exhaustively dialyzed against distilled water, followed by demineralized water and then lyophilized. Digestion of f-Sulodexide and sulfated GAG b_b chondroitinase ABC f-Sulodexide and sulfated GAG samples were digested by chondroitinase-ABC by the procedure of Yamagata et al. [25] in Tris buffer, pH 8.0, at 37°C for 24 h. The course of hydrolysis was followed by measuring the absorbance 230 nm in a Beckman spectrophotometer. The digest was exhaustively digested against distilled water. lyophilized and analyzed. Gel filtration Gelfiltration of f-Sulodexide and sulfated GAG fractions was performed on Sepharose CL-6B column (70 x 1.5 cm) with continuous flow analysis of eluent by orcinol-H,SO, as described previously [26]. The column was calibrated with standard samples of GAG. Antithrombin Ill-affinity chromatography Antithrombin III was coupled to Sepharose and used as affinity matrix for further fractionation of f-Sulodexide and sulfated GAG fractions into high-

143

and low-affinity fractions. Antithrombin was coupled through amino groups to cyanogen bromide-activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) in the presence of excess amounts of heparin according to the procedure of Hook et al. [27]. Amino acid analysis of antithrombin-substituted Sepharose indicated a protein content of about 5 mg/ml of final gel suspension. Affinity chromatography of GAG fractions on immobilized antithrombin was carried out as follows: Five milligrams of GAG in 1.0 ml of 0.2 M NaCl in 0.1 M Tris/HCl, pH 7.4, was applied onto antithrombin-Sepharose gel column (3 ml) that was previously equilibrated with the buffer at 4°C. The column was washed with the buffer (24 ml) until the effluent was free from uranic acid. To elute low affinity GAG the column was then eluted with the Tris-HCl buffer containing 2.5 M NaCl (25 ml) to obtain high affinity GAG. The eluates were dialyzed against distilled water to remove salts and then lyophilized. Anticoagulant activity Anticoagulant activity of f-Sulodexide and sulfated GAG fractions was assayed by the thrombin time test in a BBL fibrosystem (Becton Dickinson, Cockeysville, MD) as described before [28]. Blood for this assay was drawn from healthy laboratory personnel and collected over 0.1 volume of trisodium citrate (3.2%). Plasma was separated from erythrocytes by centrifugation at 3 500 rpm for 20 min at 5°C and used in the assay. A solution of 0.1 ml of thrombin (1.0 unit/ml) was added to a mixture of 0.1 ml of plasma and 0.1 ml of GAG solution of appropriate concentration and the clotting time recorded. The system was standardized with heparin of known anticoagulant activity. Lipoprotein lipase and hepatic lipase activities in post-GAG plasma Lipoprotein lipase and hepatic lipase activities in post-GAG rabbit plasma were measured by the method of Neilsson-Ehle and Schotz [29]. GAG (360 pg uranic acid/kg body weight) were injected into New Zealand white rabbits weighing 1.0-1.5 kg through the ear vein. Exactly 10 min after the injection blood was drawn from the animals into

heparinized tubes and plasma separated by centrifugation at 4°C. The plasma was used as the enzyme source. Substrate for the assays was prepared by sonicating glycerol tri[9: lO( n)- 3Hloleate, triolein and lecithin in glycerol. This gave specific activity of 400 pCi/mmol triglyceride. Prior to assay one part of the emulsion was mixed with 4 parts of 3% bovine serum albumin in 0.35 M Tris/HCl buffer, pH 8.4. The assay system contained 100 ~1 ethylene glycol (1.0 M) in 0.05 M Tris buffer, pH 8.0, with or without 100 ~1 of protamine sulfate (5 mg/ml in ethylene, glycol/Tris buffer, pH 8.0) and 100 ~1 substrate emulsion. Enzyme reaction was initiated by adding 50 ~1 of post-GAG rabbit plasma. The reaction was stopped at the end of 15 min of incubation by rapid addition of 3.25 ml of a mixture containing heptane/chloroform/methanol (1.24 : 1 : l), followed by the addition of 1.0 ml of 2.0 M potassium carbonate/borate buffer, pH 10.5. The mixture was mixed and the upper phase was then counted in a Beckman LS-250 spectrometer using Instagel as scintillator. The enzyme activity was then calculated. Protamine sulfate-resistant activity was considered as hepatic lipase activity and the protamine sulfate-sensitive lipoprotein lipase activity was calculated by subtracting hepatic lipase activity from the total activity (activity without protamine sulfate in the assay system). Results Table 1 reports total uranic acid and hexosamine contents of f-Sulodexide and the two sulfated GAG preparations. The molar preparation of uranic acid to hexosamine was greater in f-Sulodexide than in the sulfated GAG. Small amounts of protein material were present in the three samples. Electrophoresis of the samples in pyridine/formate buffer indicated the presence of more than one component in the materials. Preliminary fractionation of these GAG preparations was achieved on a Dowex-1 Cl- column eluting with a stepwise increasing concentration of NaCl (Table 2). In the three preparations most of the GAG eluted from the column at NaCl concentration of 1.5 M and 2.0 M but the amounts varied. In f-Sulodexide almost equal amounts of uronate material eluted from the column at 1.5 M

144

TABLE

1

ANALYSES

OF f-SULODEXIDE

AND

SULFATED

GAG

PREPARATIONS

Component

f-Sulodexide

Sulfated

GAG

Total uranic acid (% of dry weight)

38.5

30.5

26.7

Total hexosamine (% of dry weight)

24.0

30.0

26.8

Preparation

: 1.0

Uranic acid/hexosamine (molar proportion)

1.5

Protein (% of dry weight)

0.55

Cellulose acetate electrophoresis (pyridine/formate buffer)

0.94

Preparation

: 1.o

Two components

’

0.60

Four components (HS, DS, CS, Hep)

(Hep, CS ‘)

and 2.0 M NaCl. In sulfated GAG preparations about 30-40% eluted at 1.5 M NaCl and 60-70% eluted at 2.0 M NaCl. Electrophoresis of the GAG

B

0.89: 1.0

0.25

a Alcian blue positive; mobility similar to standard GAG. h Faintly stained spot. Ahbreoiations: Hep = heparin, HS = heparin sulfate, CS = chondroitin

TABLE

A

sulfate,

DS = dermatan

Four components (HS, DS, CS, Hep)

sulfate

fractions indicated that they were mixtures. Heparin-like material was the most predominant GAG in both f-Sulodexide fractions while consid-

2

ANALYSES

OF GAG

GAG Preparation

f-Sulodexide

Sulfated GAG Preparation A

Preparation

B

FRACTIONS

FROM

DOWEX-1

COLUMNS

% of total uronate eluted

Fraction eluted at M NaCl

0.5

0

1.0 1.5 2.0 4.0

0 52.9 41.9 .__ c

0.5 1.0 1.5 2.0 4.0 0.5 1.0 1.5 2.0 4.0

___

a Electrophoresis in pyridine/formate b = not determined. c --- = trace. Abbreviations: HA = hyaluronic

Cl-

Mole uronate Mole hexosamine

1.61 1.47

mobility

1.15 0.96

28.4 61.2 ___

0.99 0.93

similar

acid. Other abbreviations

CS, Hep DS. Hep Hep

29.0 69.8 ___ ___ ___

HA HS, CS HS. CS, Hep DS, Hep Hep HA HS, CS. Hep DS, Hep Hep

to GAG

standards.

are same as in Table 1

-___ ‘I ______

h

___

buffer;

Eiectrophoresis

145

erable amounts of dermatan sulfate and chondroitin sulfate-like materials were present in the sulfated GAG fractions. Uranic acid and hexosamine analyses of the fractions indicated that proportions of uranic acid to hexosamine were greater in f-Sulodexide fractions than in the sulfated GAG fractions. Higher proportions of uranic acid to hexosamine are generally noted in heparin and heparin-sulfated preparations. In an effort to obtain pure heparin-like GAG from the preparations, the Dowex-1 Cl- column fractions were hydrolyzed by chondroitinase ABC and the enzyme resistant GAG materials were obtained and analyzed (Table 3). The fractions moved in electrophoresis as single Alcian bluepostive spots with mobility similar to heparin standard. Their molecular weights ranged from 2 900 to 13 800 as estimated by gel filtration. The 2.0 M NaCl GAG fractions had higher molecular weights than 1.5 M NaCl fractions. The GAG eluted from gel filtration column as single peaks and the peaks were sharp. All fractions had both glucuronic and iduronic acids in varied proportions. Higher proportions of iduronic acid were noted in 2.0 M NaCl fractions than in 1.5 M NaCl

TABLE

fractions. Glucosamine was the only hexosamine in the fractions. The sulfated content of the fractions ranged from 1.5 to 2.2 moles/mole hexosamine. The 2.0 M NaCl fractions had greater amount of N-SO4 and lesser amount of N-acetyl groups than the 1.5 M NaCl fractions. Further fractionation of chondroitin sulfate-free GAG was achieved on antithrombin III-affinity columns. The proportion of low-affinity fraction to high-affinity fraction varied in preparations (Table 4). The low-affinity fractions constituted 80-90% of the total enzyme-resistant materials. Under the same conditions of affinity chromatography heparin standard resolved into low-affinity and high-affinity fractions in a proportion of 7 : 3. Detailed chemical analyses of the fractions are reported in the table. Although there were no differences in the molar proportion of uronate to hexosamine between low-affinity and high-affinity fractions the low affinity fractions had greater total sulfate contents. The differences in N-sulfate and N-acetyl groups between the fractions were not consistent. The proportion of glucuronic acid to iduronic acid was consistently greater in low-affinity fractions than in high-affinity fractions.

3

ANALYSES

OF CHONDROITINASE

ABC DIGESTED

% Recovered after enzyme digestion a

Electrophoresis

f-Sulodexide 1.5 M NaCl 2.0 M NaCl

78 84

Hep Hep

Sulfated GAG Preparation A 1.5 M NaCl 2.0 M NaCl

42 74

Hep Hep

Preparation B 1.5 M NaCl 2.0 M NaCl

36 61

Hep Hep

GAG fraction

Molecular b weight ’

GAG FRACTIONS

Total UA

Total HexNH,

ISOLATED

d GlcUA:

(‘% of dry weight) ’

FROM

DOWEX-I

Cll

IdUA

Total-SO,

N-SO,

(mole/mole

COLUMN

N-Acetyl

HexNH,)

4 700 8 700

38.3 39.1

27.9 28.8

45 : 55 36:64

1.9 2.2

0.81 0.98

0.11 0.08

’

2 900 13800

29.6 34.8

27.5 28.4

67:33 52:48

1.6 1.9

0.78 0.80

0.24 0.16

f

2900 4700

28.4 36.2

26.9 29.3

74126 39:61

1.5 2.0

0.78 0.82

0.28 0.18

a ’ ’ d e

Based on uranic acid value. Cadmium acetate buffer, electrophoretic mobility similar to standard GAG. As estimated by gel filtration. Glucosamine was the only hexosamine in all sample as determined by gas-liquid chromatography. Lyophylized weight. ’ Had additional spot faintly stained by Alcian blue and migrated similar to heparan sulfate standard. Abbreoiations: UA = uranic acid; HexNH, = hexosamine; GlcUA = glucuronic acid, IdUA = iduronic

acid.

146

slightly greater post-plasma total lipoprotein lipase activity than high-affinity fractions. In f-Sulodexide no difference was noted in 2.0 M NaCl GAG fractions between low- and high-affinity fractions while in 1.5 M NaCl GAG fraction high-affinity fraction had greater activity than low-affinity fraction. All GAG fractions had post-plasma hepatic lipase activity. The proportion of lipoprotein lipase activity to hepatic lipase in GAG post-plasma varied among preparations and there was no consistent trend. With the exception of one fraction all the fractions had greater hepatic

As expected low-affinity fractions had little or no anticoagulant activity. The fractions from GAG eluted from Dowex-1 column at 1.5 M were less active than the fractions from GAG eluted from the column at 2.0 M NaCl. High-affinity fractions from f-Sulodexide had about one-half of the activity of heparin standard. While similar fractions from sulfated GAG, 2.0 M NaCl fractions had 90% and 120% of heparin activity. All fractions had post-plasma lipoprotein lipase activity ranging from 25% to 62% heparin activity. Low-affinity fractions from sulfated GAG preparations had

TABLE

4

CHEMICAL

AND BIOLOGICAL

GAG fraction

PROPERTIES

OF GAG

Analyses

FRACTIONS

FROM

ANTITHROMBIN

% Distribution of GAG in AT column fractions a

Uranic acid

86.3 13.7

1.19 1.21

2.02 1.86

0.84 0.80

0.14 0.21

3X:62 49:51

5 42

82.9 17.1

1.25 1.22

2.34

0.89 0.91

0.14 0.15

30:6X 48 : 52

54

87.9 12.1

0.93 0.95

1.52

0.71 0.82

0.26 0.21

65 : 35 78 : 22

Not active 25

16.1 23.3

1.20 1.17

2.11 2.0

0.84 0.79

0.19 0.19

49:51 69:31

I2 90

90.0 10.0

0.89 0.86

1.56 1.49

0.75 0.81

0.21 0.20

64:36 12 : 38

Not active 32

79.1 20.9

1.06 1.12

2.24 1.82

0.80 0.85

0.16 0.14

45 : 55 68 : 32

Total-SO, (mole/mole

h

AFFINITY

Anticoagulant N-SO,

N-acetyl

GlcUA

COLUMN

Total LPL’ acuvitv

: IdUA activity

LPI.: HI.

(R of heparin standard)

hexosamine)

f-Sulodexrde 1.5 M NaCl fraction’

Low affinity ’ High affinity 2.0 M NaCl fraction Low affinity High affinity Sulfated

1.98

8

25 30

12:X8 31:69

2x

2x

lh:84 13.x:

GAG

Preparation A 1.5 M NaCl fraction Low affinity High affinity 2.0 M NaCl fraction Low affinity High affinity Preparation B 1.5 M NaCl fraction Low affinity High affinity 2.0 M NaCl fraction Low affinity High affinity

1.68

I0 120

’

’

31

IX:82

26

17:X3

62 52

43: 17

32 26

13 .67 15:x5

55 30

45 : 55 15:x5

51149

It GAG recoveries in antithrombin III (AT) affinity chromatography were 95- 100%. h Values were expressed per mole of hexosamine since hexosamine content in the fractions was conststent. 26.2-29.4s of lyophilired weight. ’ Values shown are post-GAG plasma activities; post-heparin plasma activity, 8.3 PM of fatty acid released/h/ml plasma. with LPL:HL= 74~26. ’ Fractions refer to Dowex-1 Cl- column GAG fractions eluted at 1.5 M or 2.0 M NaCl concentration. ’ Low-affinity fractions were those GAG which did not bind to antithrombin and eluted from the column in the wash buffer: high-affinity fractions were those which reacted with antithrombin III and eluted from the column by 2.5 M NaCI. ’ Activity < 5% of heparin standard (160 units/mg). Ahhreuiarions: HL = hepatic lipase; LPL = lipoprotein lipase; other abbreviations are same as in Table 3.

147

lipase activity than post-GAG plasma.

lipoprotein

lipase

activity

in

Discussion The chemical analyses of f-Sulodexide indicate that it is a mixture of chondroitin sulfates and heparin with the latter as the predominant component. The sulfated GAG preparations contained largely chondroitin sulfates and heparin with lesser amounts of heparan sulfate. Since chondroitin sulfates do not contribute much to anticoagulant activity and post-GAG plasma lipoprotein lipase activity we did not attempt to quantitate them individually, but attempted to isolate GAG with biologic activity from these materials. Fractionation of f-Sulodexide and the sulfated GAG preparation on Dowex-1 Cl- column resulted only in partial success. Although most of the GAG material eluted from the column in 1.5 M and 2.0 M NaCl concentrations there was considerable overlapping of GAG in the fractions. Hydrolysis of GAG fractions by chondroitinase ABC completely removed chondroitin sulfates from the preparations. It is evident from the analyses that the chondroitin sulfate-free fractions are mostly heparin or heparin-like GAG but with lower total sulfate content than heparin and with lower molecular weight. The anticoagulant activity of heparin is often attributed to its N-sulfate ester groups and also to iduronosulfate esters [30]. Lindahl [31] suggested that high-affinity binding of heparin to antithrombin III requires a specific sequence of sugar units in the GAG. Molecular size of heparin also seems to be important in the anticoagulant activity of heparin [32]. In this study we did not see any correlation between N-SO, ester groups and antithrombin III affinity of GAG but high-affinity fractions had consistently lower total sulfate content than low-affinity fractions from the same preparation. The high-affinity fractions had lower contents of iduronic acid than the low-affinity fractions. The observations suggest that total sulfate, N-SO, and iduronic acid contents are not solely responsible for antithrombin III affinity of these GAG but other chemical characteristics might contribute to this activity. Recently Rosenberg et al. [33] observed that a

highly active preparation of heparin had greater amounts of glucuronic acid and lower amounts of total sulfate and N-sulfate ester groups than in a relatively inactive preparation. A tetrasaccharide sequence with nonsulfated iduronic acid at the nonreducing end was showed more activity for antithrombin III binding than a tetrasaccharide with a sulfated iduronic acid at the nonreducing end. Hook et al. [34] observed that the smallest heparin fragment that contained intact antithrombin binding sequence had the size of an octasaccharide. The entire octasaccharide is perhaps not needed for antithrombin III binding. Although the GAG fractions of this study are larger than the size of an octasaccharide, the number of active octasaccharide sequences needed for anticoagulant activity in the preparations might be fewer than in heparin thus resulting in the lower anticoagulant activity. Post-GAG plasma lipoprotein lipase activities of the GAG fractions suggest that this activity is independent of antithrombin III affinity of GAG. The ability of lipoprotein lipase release into the circulation is not unique for heparin, but several polyanions also release lipase [35]. Since the GAG fractions had similar net anionic charges they exhibited similar post-GAG plasma lipase activity irrespective of monosaccharide sequences required for antithrombin III affinity. Thus it is possible to obtain heparin-like preparations with little anticoagulant activity but with lipoprotein lipase releasing ability by selective modification of heparin or heparin-like GAG. f-Sulodexide seems to be one such compound. The high-affinity fractions from sulfated GAG preparations had anticoagulant activity similar to heparin but had only 30-60% lipoprotein lipase releasing ability of heparin and this could be due to their lower total sulfate content than heparin. GAG with low anticoagulant activity but with high lipoprotein lipase ability are potentially important as a hypolipidemic agent in the prevention and treatment of atherosclerosis. Acknowledgements We thank Professor P. Bianchini and Drs. G. Mascellani and E. Marchi for a critical review of the manuscript and for helpful suggestions.

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