Table Of ContentNONANTICOAGULANT
ACTIONS OF
GLY COSAMINOGLY CANS
NONANTICOAGULANT
ACTIONS OF
GL YCOSAMINOGL YCANS
Edited by
Job Harenberg
University ofH eidelberg
Mannheim, Germany
and
Benito Casu
G. Ronzoni Institute
Milan, Italy
Plenum Press. New York and London
Library of Congress Cataloging-in-Publication Data
Nonanticoagulant actions of glycosaminoglycans I edited by Job
Harenberg and Benito Casu.
p. ern.
·Proceedings of the Fourth Symposium on Glycosaminoglycans,
Nonanticoagulant Actions of Glycosaminoglycans--results and
perspectives of the German--Italian collaboration. held October
6-9,1994. in LCiV6iiO. Italy"-··T.p. \It:::rso.
Includes bibliographical references and index.
1. Glycosaminoglycans--Congresses. 2. Heparin--Derivatives
-Congresses. 3. Thromboembolism--Chemotherapy--Congresses.
I. Harenberg. Job. II. Casu. Benito. III. Symposium on
Glycosaminoglycans, Nonanticoagulant Actions of Glycosaminoglycans
(4th 1994 Loveno. Italy)
[DNLM, 1. Glycosaminoglycans--pharmacology--congresses.
2. Glycosaminoglycans--therapeuticuse--congresses.
3. Anticoagulants--pharmacology--congresses. au 83 N812 1996J
RM340.N66 1996
615·.718--dc20
DNLM/DLC
for Library of Congress 96-10228
CIP
Proceedings of the Fourth Symposium on Glycosaminoglycans: Nonanticoagulant Actions of
Glycosaminoglycans - Results and Perspectives of the German - Italian Collaboration,
held October 6 - 8, 1994, in Loveno, Italy
ISBN-l3: 978-1-46\3-8021-4 e-ISBN-\3: 978-1-46\3-0371-8
001: 10.1007/978-1-46\3-0371-8
© 1996 Plenum Press, New York
Softcover reprint of the hardcover 1st edition 1996
A Division of Plenum Publishing Corporation
233 Spring Street, New York, N. Y. 10013
All rights reserved
1098765432 I
No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any
means, electronic, mechanical, photocopying, microfilming, recording, or otherwise, without written
permission from the Publisher
FOREWORD
Prophylaxis and treatment of thromboembolism have made one of the major impacts
in medicine. Heparin has widely been used as the most effective drug during the last 50 years.
However, its potential side effects have led to the search for equally effective but safer
alternatives. At present, the low-molecular-weight heparins are the most promising steps in
this direction.
Considerable interest has been generated at the same time in exploring other gly
cosaminoglycans of the nonheparin type for therapeutic use. The existence of these com
pounds has been known for a long time and substantial information has been gathered on
glycosaminoglycans such as heparinsulfate, dermatansulfate, and chondroitinsulfate. Many
of these substances are derived from animal or plant sources, and some of them have now
been synthesized.
The aim of the Fourth Symposium on Glycosaminoglycan Research at Villa Vigoni
in Loveno at Lake Comolltaly was to summarize the considerable new information in this
field. The articles of the present volume are mainly based on a German-Italian collaboration
supported by the Vigoni Program. The selected articles describe many different, nonheparin
glycosaminoglycans, some of them already in clinical trials as antithrombotic agents. In
particular, the interaction of glycosaminoglycans with some cellular elements of blood,
especially leukocytes and platelets, are discussed. New methods for their identification and
assays are described and considerable emphasis is placed on the pharmacokinetic aspects of
these new compounds. Particularly, some nonanticoagulant activities of the glycosamino
glycans are discussed in detail. There is little doubt concerning the use of glycosaminogly
cans as the treatment of choice for thromboembolic disorders in the future. And new
indications are possible as well.
We would like to express our gratitude to Professor Harenberg and Professor Casu
for organizing this outstanding symposium and for selecting and assembling these articles.
Klaus van Ackern and Dieter L. Heene
v
CONTENTS
1. Inequivalence of Glycosaminoglycans Using High-Performance Size Exclusion
Chromatography, Polyacrylamide Gel Electrophoresis and High-
Performance Capillary Electrophoresis ............................ .
Reinhard MaIsch, Job Harenberg, Lukas Piazolo, and Dieter L. Heene
2. New NMR Spectroscopic Approaches for Structural Analysis of
Glycosaminoglycans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 15
Giangiacomo Torri
3. Application of Mass Spectrometry to the Analysis of Natural and Synthetic
Sulfated Oligosaccharides ...................................... " 27
Luigi Silvestro, Simona Rizea Savu, P. A. van Veelen, and P. L. Jacobs
4. Monoclonal Antibody Directed against Heparin and Heparin-Fractions. . . . . . .. 47
Gunter Huhle, Job Harenberg, Reinhard MaIsch, and Dieter L. Heene
5. Simulation of Glycosaminoglycan Structures by Chemical Modifications of
E. coli Polysaccharides K5 and K4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 59
Annamaria Naggi
6. Pharmacology of Synthetic and Biotechnology-Derived Homologues and
Analogues of Heparin ........................................... 65
Walter Jeske, Jawed Fareed, Debra Hoppensteadt, and Benito Casu
7. Protein Binding of Sulfated Glycosaminoglycans: Searching for Specificity .. " 89
Benito Casu
8. Non-Anticoagulant Actions of Glycosaminoglycans: Protein Binding Studies ... 101
Alfonso Iorio, Adriano Alatri, and Giancarlo Agnelli
9. Binding of Glycosaminoglycans to Leukocytes Using Fluorescent Labeled
GAG-Derivatives ............................................... 113
Job Harenberg, Reinhard MaIsch, Lukas Piazolo, Gunter Huhle, and
Dieter L. Heene
10. Intact Biological Activity and Binding to Granulocytes of
LMM-Heparin-Tyramine-Fitc ..................................... 127
Lukas Piazolo, Job Harenberg, Reinhard MaIsch, and Dieter L. Heene
vii
viii Contents
11. A Detailed Evaluation of the Structural and Biological Effects of Alkaline
O-Desulfation Reactions of Heparin ................................ 139
Kevin R. Holme, Weisheng Liang, Zicheng Yang, France Lapierre,
Patrick N. Shaklee, and Lun Lam
12. Glycosaminoglycans and Related Structures as Potential Inhibitors for
Erythrocyte Infection by Plasmodiumfalciparum Malaria .............. 163
Roger A. Laine
13. The Interaction of Basic Fibroblast Growth Factor (bFGF) with Heparan
Sulfate Proteoglycans: Biochemical Bases and Biological Implications ... 171
Marco Rusnati, D. Coltrini, Pasqua Oreste, Giorgio Zoppetti, and
Marco Presta
14. Binding of 125I_bFGF to Rat Aortic Smooth Muscle Cells: Effect of Natural and
Chemically Modified Heparins and Heparan Sulfates .................. 189
Laura Giorgini, Annamaria Naggi, and Giancarlo Ghiselli
IS. Modulatory Role of Heparin and Heparan Sulfates on Angiogenesis .......... 201
Giovanni Camussi, E. Battaglia, Enrico Lupia, and G. Montrucchio
16. Involvement of Thrombin on GAGs Release in Different Cellular Systems ..... 209
Domenico Rotilio, Antonio Tamburro, B. Mariani, O. Lancia, and
Francesco Peracchia
17. TFPI Release by GAGs and Its Role in Their Mechanism of Action ........... 227
Piotr Radziwon, 1. Schenk, B. Boczkowska-Radziwon, Jawed Fareed, and
Hans K. Breddin
18. Biological Activities and Effects on the Platelet Aggregation of a Structurally
Defined Curdlan Sulfate ......................................... 235
Susanne Alban, Walter Jeske, Debra Hoppensteadt, Jawed Fareed, and
Gerhard Franz
19. Influence of Glycosaminoglycans on Natural Killer Cell Activity ............. 243
Sabine Johann and Reinhold Forster
20. Non-Anticoagulant Actions of Glycosaminoglycans (GAGs): The
Therapeutical Approach to Alzheimer's Disease ...................... 249
Umberto Comelli
21. Therapy with Glycosaminoglycans in Nephrology ......................... 281
Giovanni Gambaro, Miriam Barbanti, Egidio Marchi, and Bruno Baggio
Index ................................................................. 287
1
INEQUIVA LENCE OF
GLYCOSAMINOGL YCANS USING
HIGH-PERFORMANCE SIZE EXCLUSION
CHROMATOGRAPHY, POLYACRYLAMIDE
GEL ELECTROPHORESIS AND
HIGH-PERFORMANCE CAPILLARY
ELECTROPHORESIS
Reinhard MaIsch, Job Harenberg, Lukas Piazolo, and Dieter L. Heene
1st Dept. of Medicine, Faculty of Clinical Medicine Mannheim
University of Heidelberg
Theodor Kutzer Ufer, 68167 Mannheim, Germany
1. INTRODUCTION
Biologically active sulfated polysaccharides like heparin and derma tan sulfate are
commonly known GAGs (l)* Although its primary application as anticoagulant heparin can
be considered as polyelectrolytic drug displaying a variety of biological activities. Heparins
can be easily neutralized in vitro and in vivo by an equigravimetric amount of protamine or
polybrene.
The biosynthesis of heparin and dermatan sulfate takes place in several mammalian
and non mammalian tissues and results in preoteoglycans with different sulfation patterns
(2).
Low molecular mass heparins (LMMH) differ from heparin in their efficacy and side
effects (3). Recently dermatan sulfates have been also proven to be anti thrombotic agents.
The fragments of glycosaminoglycans described herein are produced by different depolym
erization processes like nitrous acid cleavage and B-elimination .
• Abbreviations used: GAGs, glycosaminoglycans; CZE, capillary zone electrophoresis; HPCE, high
performance capillary electrophoresis; LMMH, low molecular mass heparin; p, polydispersity; M, average
molecular mass; Mm, mass average molecular mass; IdoA, i-iduronic acid; GlcNS03, glucosamine N-sulfate; '"
UA, 4-deoxy-a-L-threo-hex-4-enopyranosoyl uronic acid; G1cNAc, 2-deoxy-2-acetamidoglycopyranose; SDS,.
sodium dodecyl sulfate; SAX, strong anion exchange; Am, anhydromannose; AU, absorption unit; Da,
dalton.
Nonanticoagulant Actions of Glycosarninoglycans, Edited by J. Harenberg and B. Casu
Plenum Press, New York, 1996 1
2 R. MaIsch et al.
The structure of the main disaccharide of heparin and chondroitin sulfates has been
established by nmr analysis (4). Different terminal residues have been also studied by nmr
techniques. The compositional analysis of heparin and dermatan sulfates was performed by
high performance anion exchange chromatography (HPAEC) (5) and high performance
capillary electrophoresis (6). The high performance size exclusion chromatography
(HPSEC) (7) and polyacrylamide gel electrophoresis (PAGE) (8, 9) have been used to
determine the average molecular mass and the polydispersity of the glycosaminoglycans.
The migration in PAGE and the retention time in HPSEC of heparin oligosaccharides depend
on their molecular mass and their charge density.
Selective enzymatic cleavage of heparin by heparinase I, II and III and of chondroitin
sulfate by chondroitin sulfate ABC was performed. The resulting disaccharides with the
general structure ~ UA 2X (1 ~ 4)-D-G1cNY6X for heparin and heparan sulfate and ~ UA
2X (1 ~ 3)-D-GalNY6X for chondroitin sulfate absorb specifically at 232 nm. Strong anion
exchange chromatography methods were described for their detection and purification (5).
High performance capillary electrophoresis (HPCE) methods were developed for the com
position analysis of chondroitin sulfates, heparin and heparan sulfate using enzymatic
cleavage (5, 10). As heparin and chondroitin sulfates can be detected specifically by these
methods they are examined for the detection of cross contamination of glycosaminoglycans.
Here we describe the microheterogenity and purity of various LMM- glycosamino
glycan preparations using different chromatographic and electrophoretic methods. In future
it is important to study the different biological effects of glycosaminoglycans independent
from the variation of the analytical methods for their determination. This should be accom
plished by the comparison of different methods.
2. AIMS
The aims of the study were the analysis oflow molecular GAGs by high performance
size exclusion chromatography (HPSEC), the development of the molecular mass determi
nation in small PAGE gels, the comparison of PAGE with HPSEC and the analysis of GAGs
by capillary electrophoresis. Different electrophoretic and chromatographic methods were
used because the variation ofthe different analytical methods was determined. The variation
of the methods should be regarded differently of their biological effects.
3. MATERIALS AND METHODS
The following unfractionated and low molecular mass GAGs were used:
Unfractionated and low molecular mass sodium-heparin and Innohep® was obtained
by Braun Melsungen AG; Germany. Dermatan sulafte and low molecular mass dermatan
sulafte were obtained from Alfa Wassermann, Bologna, Italy. Chondroitin sulfate A was from
Sigma, Deisenhofen, Germany. Clexane® was from Rhone Poulenc Rorer, Kaln, Germany.
Fragmin®was obtained from Kabi Pfrimmer, Erlangen, Germany. Fraxiparin®was provided
from Sanofi-Wintrop, Muinch, Germany. LMMH-Merckle was from Merckle, Ulm, Ger
many, Mono-Embolex® was generously provided by Sandoz AG, Numberg, Germany.
Reviparin® was provided from Nordmark AG, Uetersen, Germany. Boric acid no B 7901,
tris no T 6191, were of research grade and obtained from Sigma GmbH, Deisenhofen,
Germany. Glycerol No 4094 was from Merck AG, Darmstadt, Germany and 3-dimethy
lamino-propionitril (DNPN) from Fluka Feinchemikalien GmbH, Neu Ulm, Germany.
Acrylamide No 10675, N, N-metylenebisacrylamide No 29195, ethlyene diamine tetraacetic
Inequivalence of Glycosaminoglycans 3
acid No 11278 and glycine No 23390 of research grade were obtained from Serva Heidelberg
Germany.
Oligosaccharides prepared by synthesis ranging from di- to dodecasaccharide were
generously provided by Dr. M. Petitou and Dr. L. Lormeau from the Institute Choay, Paris,
France. Heparin fractions were obtained by size exclusion chromatography (HPSEC).
Heparin , chondroitin sulfate and chondroitin sulfate disaccharides were obtained from
Sigma Chemical St. Louis USA. Heparinase I was generously supplied from Baxter Diag
nostics Inc, Deerfield, USA, and Chondroitinase ABC was purified from Proteus vulgaris
was from Seikagaku Cooperation, Tokyo, Japan. All the other reagents used were of
analytical grade.
3.1. High Performance Size Exclusion Chromatography (HPSEC)
A system consisting of a multi solvent delivery system (model 600 from Millipore
Waters GmbH, Eschborn, Germany) with a Waters 600 E system controller, an injector
(model U6K from Millipore Waters), 20 ~l sample loop, (LKB model 2154-100, LKB,
Bromma, Sweden), a column (Ultropac TSK G 2000 SW 7.5 x 600 mm i.d, partiele size 10
~m. LKB No.2135-260, a precolumn (Ultrapac TSK SWP, 75 7.5 i.d., 10 ~m, LKB No
2135-075) was connected between the injector and the pump, a photodiode array detector
(model Waters 991) with a computer (NEe Power Mate SX Plus) and the 990/991 fore
groundlbackground software (Millipore Waters) were used.
3.1.1. Chromatographic Conditions. The eluent was aqueous 0.1 M sodium chloride
(filtered and degassed before use) at a rate of I mllmin. The detector was set at a wavelength
range from 190 to 300 nm. Vo of the column was determined with dextran sulfate (M =
300.000 Da) and Vt with sodium azide. 20 ~l of the heparin stock solutions were injected at
a concentration of 10 mg/ml. The column was calibrated with standards in the international
collaborative study for the standardization of molecular mass of heparins. The method was
established by the subcommittee of the XIII. ISTH Congress in Amsterdam (11).
3.1.2. Calculations. Chromatographic data obtained from the elution curve (absor
bance expressed as peak height in millimeters every 0.5 ml) and elution volume (in
milliliters) were used for calculating the average molecular mass according to Yau et al (12).
3.2. Preparation of Gradient Polyacrylamide Gels
,
A linear gradient polyacrylamide resolving gel (8 x 7 cm, 1,5 mm consisting of 20
to 30 total percent acrylamide (T) with a superimposed 2-5 percent (w/v) cross linker N,
N-bisacrylamide (C) gradient was prepared. A gradient of 0-10 percent glycerol was also
present for extra gradient stabilization. Samples wells of 5 mm width were formed in a 4
percent T and 0,4 percent C (w/v) stacking gel. A modified buffer system ofLaemmli was
=
used and comprised 0,4 M tris.hel, 0,4 M boric acid and 0,01 M sodium-EDTApH 8,3 in
the resolving gel, and 0,2 M tris.hcl, 0,2 M boric acid and 0,005 M sodium-EDTApH = 8,3
=
in the stacking gel, and 0,2 M glycine and 25 mM tris.hel, pH 8,3 as the electrode buffer.
3.2.1. Electrophoresis ofGlycosaminoglycans. Oligosaccharide samples (10 - 20
~g) containing 10 % glycerol (v/v) were loaded into the wells in a volume of 20 to 30 ~l.
Electrophoresis was performed at 140 V while the samples concentrated and migrated
through the stacking gel, and then decreased to 70 V after the sample had entered the
resolving gel. The gels were cooled by a circulating tap water. Electrophoresis was termi
nated after about 3 -4 hours.
4 R. MaIsch et al.
3.2.3. Fixation and Staining. At the end of the electrophoresis run, each gel was
removed and immediately immersed in 1 % (w/v) aqueous alcian blue containing 1 percent
acetic acid or a 1 % acridine orange solution prepared in 20 % ethanol for 24 hours with
gentle agitation. The gel was de stained by frequent changes of des tilled water and 5 % acetic
acid within 24 hours
3.3. Separation of Glycosaminoglycans by HPCE
The experiments were performed on a PACE 2050 from Beckmann Instruments;
Fullerton, CA, USA equipped with a variable wavelength ultraviolet detector. System
operation and data management were controlled using Gold-software from Beckmann
Instruments running on a IBM personal computer. In both modes the samples were analyzed
using a 50 cm x 50 J..lm capillary cartridge (no 727604) from Beckmann Instruments,
Fullerton, CA, USA. The concentration of the stock solutions was for heparin-disaccharides
1 mg/ml and for heparin-oligosaccharides and heparin preparations 10 mg/m!.
3.3.1. Normal Polarity Method. Electrophoresis was performed using a sodium
tetraborate 10 mM and 50 mM boric acid pH = 8.5. The compounds were detected at 200
and 230 nm respectively. The other conditions were: data rate: 5 Hz, rise time: 1s , range
(AU): 0.05, polarity: direct, run time: 30 min, voltage: 18 kV, temperature: 25° C, injection:
high pressure 10 or 15 sec.
3.3.2. Reversed Polarity Method. Electrophoresis was run using a 20 mM sodium
phosphate buffer adjusted with hydrochloric acid to pH = 3.5. The other conditions were:
data rate: 5 Hz, rise time: 1s , range (AU): 0.2, polarity: indirect, wavelengths: 230 nm, time:
60 min, voltage: 12 kV, temperature: 25° C, injection: high pressure 15 sec.
4. RESULTS
Unfractionated and low molecular mass glycosaminoglycans were analyzed using
HPSEC, PAGE and HPCE. Their differences were characterized by their elution profiles,
spectral absorbance, molecular mass, polydispersity and migration time.
4.1 High Performance Size Exclusion Chromatography
Four unfractionated GAGs and seven different LMM-GAGs were analyzed with a
TSK G2000 SW column by high performance size exclusion chromatography (HPSEC). It
can be seen that the GAGs are eluted from the column in different elution profiles and
retention times (Fig. 1).
Unfractionated glycosaminoglycans are eluted before low molecular mass com
pounds. The unfractionated and LMM-glycosaminoglycans (GAGs) were analyzed 5 times
to determine the within-assay coefficient of variation the standard deviation of the average
molecular mass and polydispersity P of each GAG with the HPSEC method.
4.1.1. Determination of the Average Molecular Mass by HPSEC. The average mo
lecular mass, Mm of the LMM-GAGs ranged from 4629 to 9084 and from 11272 to 20428
Dalton for unfractionated GAGs. The mean and the standard deviation of the average
molecular mass Mm and the polydispersity are given in Table 1.
The standard variation of Mm of the GAGs ranged from 0.65 to 9.26 %. The
polydispersity P (Mm/Mn) showed a variation from 0.92 to 6.96 %. The average mean
