Carbohydrate Research 403 (2015) 60–68

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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres

Minireview

Re-visiting the structure of heparin

Benito Casu ⇑, Annamaria Naggi, Giangiacomo Torri
G. Ronzoni Institute for Chemical and Biochemical Research, via G. Colombo, 81 20133 Milan, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The sulfated polysaccharide heparin has been used as a life-saving anticoagulant in clinics well before its
Received 13 June 2014 detailed structure was known. This mini-review is a survey of the evolution in the discovery of the pri-
Accepted 22 June 2014 mary and secondary structure of heparin. Highlights in this history include elucidation and synthesis of
Available online 3 July 2014
the specific sequence that binds to antithrombin, the development of low-molecular-weight heparins
currently used as antithrombotic drugs, and the most promising start of chemo-enzymatic synthesis.
Keywords: Special emphasis is given to peculiar conformational properties contributing to interaction with proteins
Heparin structure
that modulate different biological properties.
Active site for antithrombin
Low-molecular-weight heparins
Ó 2014 Elsevier Ltd. All rights reserved.
Heparin oligosaccharides
Protein binding
Conformations

1. Introduction 2. From ‘mysterious heparin’ to Wolfrom’s structure

Heparin is a well-known sulfated polysaccharide widely used as
an anticoagulant and antithrombotic drug and with perspective
uses in other therapeutic fields. This paper is an overview of
advances in the knowledge of structure and structure-activity rela-
tionships of heparin as evolved over the past few decades, with
glimpses on its early history. To meet with the space constraint
of mini-reviews, the authors referred to previous extended reviews
and books,1–7 to some landmark papers and to most recent articles
of groups active in the field for recognizing contributions that
could not be specifically mentioned in the present context. Such
an exercise implied some arbitrary choices: this overview follows
the strategy of reporting ‘case studies’ involving the research
groups of the authors. It was also meant as a tribute to Carbohy-
drate Research for its steadily contributing, since its early issues,
to the progresses in heparin research.
Abbreviations: GAG, glycosaminoglycan; GlcA, D-glucuronic acid; GlcA2SO3,
D-glucuronic acid 2-sulfate; GlcN, D-glucosamine; GlcNSO36SO3, D-glucosamine
N,6-sulfated; IdoA, L-iduronic acid; IdoA2SO3, L-iduronic acid 2-sulfate; TSD,
trisulfated disaccharide; AT, antithrombin; HA, high affinity for AT; LA, low-affinity
for AT; ATBR, antithrombin-binding region; LMWH, low-molecular-weight heparin;
GlcNSO33,6SO3, 3,6-O-sulfated D-glucosamine; ManNSO36SO3, D-mannosamine
6-sulfate; 1,6-anhydro-GlcNSO3, 1,6-anhydro-D-glucosamine N-sulfate; 1,6-anhydro-
ManNSO3, 1,6-anhydro-D-mannosamine N-sulfate; ULMWH, ultra-low-molecular-
weight heparin; FGFs, fibroblast growth factors; FGF1, fibroblast growth factor-1;
FGF2, fibroblast growth factor-2; FGFR, fibroblast growth factor receptor; 3OST-1 and
3OST-3, 3-O-sulfotranferases 1 and 3; K5, E. coli K5 polysaccharide; HQSCcos,
statistical correlation two-dimensional NMR spectroscopy.
⇑ Corresponding author.
E-mail address: casu@ronzoni.it (B. Casu).
Heparin and its anticoagulant activity were ‘discovered’ in Tor-
onto about 100 years ago by McLean and Howell, who were actu-
ally looking for a pro-coagulant substance extracted from tissues
and thought to be a phospholipid. It had taken some time to the
scientific community to realize that heparin is in fact a sulfated
polysaccharide belonging to the class of glycosaminoglycans
(GAG), constituted by alternating disaccharide sequences of an
uronic acid and a hexosamine. It is noteworthy that, while still
being a ‘mysterious’ entity, that is, before the exact nature of the
component carbohydrate residues and location of sulfate groups
along the polysaccharide chains were established, heparin started
to be successfully used as an anticoagulant in clinics, arising enthu-
siasm especially as a life-saving drug that permitted open-heart
surgery.8
Reviews on the history of heparin have been published by Rodé-
n9 and Barrowcliffe.10 Hereinafter some milestones of the early his-
tory are mentioned. In 1935, Jorpes established that the uronic acid
and aminosugar ratio of heparin was 1:1 as in chondroitin sulfates.
Since the sulfur content of heparin corresponded to about 2.5 sul-
fate groups per disaccharide unit and on the assumption of a reg-
ular structure of its polysaccharide backbone, heparin was
erroneously thought to be an oversulfated chondroitin sulfate. 
Some years later Wolfrom, who thought that D-glucuronic acid
(GlcA) was the only uronic acid component of heparin, proposed
 
Curiously, a major crisis in the use of heparin in clinics was caused in the late
2007 and early 2008 by contamination with an unnatural oversulfated chondroitin
sulfate (see Section 11).

http://dx.doi.org/10.1016/j.carres.2014.06.023
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
 B. Casu et al. / Carbohydrate Research 403 (2015) 60–68
the heparin structure to be represented by repeating units of alter-
nating 1,4-linked GlcA2SO3 and GlcNSO36SO3 residues, where the
configuration of glycosidic bonds was proposed to be a for both GlcA
and GlcN on the basis of optical rotation data.11 The configuration of
GlcA in heparin was subsequently demonstrated to be b.12
3. First re-visitation: L-iduronic acid enters into the picture
Wolfrom’s structure for heparin remained undisputed for sev-
eral years. Although in the meantime L-iduronic acid (IdoA) had
been detected in some heparins, it was generally thought that
these findings were reflecting residual contents of IdoA-containing
GAGs such as dermatan sulfate, which is difficult to totally remove
from heparin preparations. Undoubtedly, acceptance of Wolfrom’s
structure has been also a tribute to his outstanding reputation as a
carbohydrate chemist. However, such a consent was mainly due to
serious problems facing everybody who attempted to tackle the
heparin structure using methods available at the time for obtaining
di- and oligosaccharide fragments to reconstruct the structure of
the polysaccharide. In the late 1960s and early 1970s the applica-
tion of milder methods of acid hydrolysis and the advent of non-
destructive analytical techniques such as NMR spectroscopy rap-
idly changed the picture. In 1962, Cifonelli and Dorfman found
IdoA to be a major uronic acid component of heparin,13 and in
1968 preliminary NMR studies by Perlin indicated that the struc-
ture of heparin was not as simple as depicted by Wolfrom’s for-
mula, and that IdoA residues substantially contributed to the
structure of the polysaccharide.14 Analysis of higher resolution
(220 MHz) spectra15 and chemical studies16 together with inde-
pendent structural studies by Lindahl on nitrous-acid generated
fragments17 finally provided clear evidence that IdoA (mostly as
IdoA2SO3) was the prominent uronic acid of heparin. In the mean-
time, Wolfrom had admitted that his earlier results had led to an
incorrect conclusion.18 As reviewed in Refs. 1,10, the hydrolytic pro-
cedures previously applied were leading to the loss of IdoA-contain-
ing fragments; as a consequence, Wolfrom’s analysis was based on
the minor GlcA-containing ones. In a study exploiting for the first
time NMR spectroscopic analysis of enzymatic digests (provided by
Dietrich), Perlin also established the a-L-configuration of the
IdoA2SO3 residues of heparin.19 The optical rotation of heparin was
rationalized by studies with model synthetic iduronates.20 A com-
plete analysis of 1H and 13C NMR spectra of heparin, highlighting
its major components, was published in 1979.21 These findings led
to establish the structure of the major disaccharide repeating units
of heparin as shown in (Fig. 1A). The trisulfated disaccharide (TSD)
repeating sequences –IdoA2SO3–GlcNSO3,6SO3—were found to be
especially prominent (up to 90%) in preparations from beef lung,
an organ that for some time was the principal source of heparin,
and somewhat less represented (about 75%) in heparins from porcine
mucosa, which largely replaced beef lung as an animal tissue for
preparation of clinical heparins. The complement to 100% of uronic
acids was GlcA, usually associated also with N-acetylated (instead
of N-sulfated) glucosamine residues. Biosynthetic studies, especially
by the Uppsala group (reviewed in Ref. 22) definitively proved that
GlcA- and GlcNAc-containing sequences were actual constituents of
the heparin chains, their presence being the result of incomplete
modification of the biosynthetic precursor chains constituted by
repeating –GlcA–GlcNAc–sequences. These studies, together with
structural analysis of extensively purified heparins (see Section 11)
definitively established the concept that heparin was structurally
microheterogeneous. Heparins from different tissues and animal spe-
cies were found to have different contents of IdoA2SO3 and IdoA, and
to be heterogeneous also in terms of size, being composed of chains
of different length, their mean MW ranging from 10 to 20 KDa
depending also on the method of preparation.23,24
61
4. Discovery and synthesis of the binding site for antithrombin
Mechanism of anticoagulation
Soon after Rosenberg’s milestone finding that the anticoagulant
activity of heparin was mainly based on its ability to bind anti-
thrombin (AT), thus accelerating by several orders of magnitude
the AT-mediated inhibition of coagulation factors, in 1976 Rosen-
berg’s, Lindahl’s and Andersson’s groups independently discovered
that heparin was heterogeneous in terms of its interaction with AT.
In fact, they separated chains with high affinity (HA) from those
with low affinity (LA) for AT, and found that the anticoagulant
activity was largely associated with the HA chains, these latter con-
stituting only about one third of those of the unfractionated poly-
saccharide (UFH). That also was a landmark finding and a
surprising one, since the compositional differences between HA
and LA chains were subtle and not easily detectable. Rosenberg
observed that nitrous acid-generated fragments obtained from
HA heparin contained significantly more unsulfated IdoA than
the LA species. Similarly the Ronzoni and Choay groups noticed
marked differences in the NMR spectra of HA and LA oligosaccha-
rides. However, the structure of the antithrombin-binding region
(ATBR) remained elusive for some more time, until Lindahl’s group
established that the ATBR was a pentasaccharide (structure shown
in Fig. 1B). This pentasaccharide contains the typical 3-O-sulfated
GlcN, optionally also 6-O-sulfated, GlcNSO33,6SO3 residue.25 Note-
worthy, the unsulfated IdoA residue preceding this pentasaccha-
ride sequence, systematically found in HA species from porcine
mucosal heparin, did not significantly contribute to the affinity
for AT. Identification of sulfate groups essential for high-affinity
binding to AT (as depicted in Fig. 1B) was completed by the com-
bined application of classical methods for sulfated oligosaccharide
analysis; this latter work, which appeared in n. 100 of Carbohydrate
Research,26 remains an important reference in the field. All sulfate
groups essential and/or important for high-affinity binding to AT
(as depicted in Fig. 1B) were identified.27 The 3-O-sulfate group
taken as a marker of the ATBR was also characterized by a specific
signal in the 13C NMR spectra of HA heparin oligosaccharides
obtained with different methods.28 In variants of ATBR found in
other heparin sources, especially from bovine lung, the 3-O-sul-
fated GlcN residue is prevalently N-sulfated and the IdoA residue
preceding the active pentasaccharide is IdoA2SO3.29
Even before the structure of the ATBR was fully elucidated, the
Choay group started an ambitious program of chemical synthesis
of the active pentasaccharide, reaching its goal in 1983.30 (For
detailed coverage of discovery and synthesis of ATBR see
Refs. 31–33) In a collaboration between Sanofi and Organon, the
pentasaccharide was later developed as an antithrombotic drug
(fondaparinux, ArixtraÒ).33
Regarding the structure of heparin with respect to thrombin
inhibition it was shown, using chemical synthesis, that a hexadeca-
saccharide displaying an ATBR at the reducing end is required.33
The basis for the specificity of AT-mediated inhibition of thrombin
was later established by crystallographic studies using a synthetic
hexadecasaccharide.34
5. Minor sequences. Updated view of the heparin structure
Minor sequences, contributing to the structural microhetero-
geneity of heparin, include internal GlcA2SO3 residues, first discov-
ered by Conrad in liver HS (see Ref. 3) and identified in heparin
lyase I-generated fragments of porcine mucosal heparin.35,36 N-
unsubstituted GlcN residues are among the very minor sequences
of heparin (see Section 11). Some of the heparin chains terminate,
at their reducing end, with a ‘linkage region’ (LR) reminiscent of
the sequence –GlcA–Gal–Gal–Xyl–Ser linking the carbohydrate
62 B. Casu et al. / Carbohydrate Research 403 (2015) 60–68

(A)

(B)

Figure 1. (A) Major, trisulfated disaccharide (TSD) repeating units of heparin. (B) Hexasaccharidic heparin sequence containing the pentasaccharidic antithrombin-binding
region (ATBR). Typical residues of the ATBR are the 3-O-sulfated glucosamine GlcNSO33,6SO3 preceded by a nonsulfated GlcA. Sulfate groups essential for high-affinity
binding to AT (Refs. 27,32) are highlighted with ovals. In the highly sulfated heparins, such as bovine lung heparin, the 3-O-sulfated GlcN residue is prevalently N-sulfated,
and the IdoA residue preceding the active pentasaccharide is IdoA2SO3.29 In the synthetic pentasaccharide fondaparinux the 6-O-sulfated glucosamino residue is N-sulfated.33

chains to the original GAG proteoglycan; most often, the LR is par-
tially eroded during purification processes of heparin (Refs. 37, and
Refs. therein).
The established heterogeneity of heparin chains in terms of size,
sequence of the backbone residues and sulfation patterns, also
depending on animal source and ways of purification (Ref. 38)
makes it difficult to depict the heparin structure even in statistical
terms. A complete picture of heparin should consist of a list of its
numerous component chains, with indication of their relative con-
tents. Methods have been developed for sequencing heparin and
heparan sulfate oligosaccharides39,40 and for mapping heparin
chains by superimposing the structures of fragments obtained by
graded enzymatic cleavage41 and great progresses have been made
toward profiling all component species (for a review, see Ref. 42).
However, the isolation, sequencing and quantification of all the
heparin chains remain a formidable task. A way of depicting an
average heparin chain (used by Linhardt’s group)43 is adopted here
(Fig. 2A) for representing an updated picture of an ATBR-containing
heparin chain. The structure of the undegraded LR is reported in
Figure 2B.
6. Low-molecular-weight-heparins
The biological action of the active pentasaccharide stems on the
concept that it represents the essential heparin sequence that
strongly binds to AT and dramatically increases the rate of AT-
mediated inhibition of factor Xa while only marginally inhibiting
thrombin. In fact, besides containing the sequence of the ATBR,
in order to inhibit also thrombin (factor IIa) heparin chains should
be at least 18 residues long (for comprehensive reviews, see Refs.
32,33,44). The molecular basis for the interactions relevant to the
anticoagulant and antithrombotic activity of heparin species is
illustrated by the 3D structures (compared in Ref. 44) of AT, hepa-
rin-activated AT and complexes of AT with thrombin and other
coagulation factors as determined by X-ray crystallography
(mainly by Huntington’s group) or by NMR methods (by Mulloy’s
group). More detailed information on the mechanism of activation
of AT is given in Section 9.2.
The first report on the size-dependence of the anti-thrombin
and anti-factor Xa activities of heparin45 triggered the develop-
ment of low-molecular-weight heparins (LMWHs) as antithrom-
botic agents with a lower anticoagulant activity. Although the
original expectation that LMWHs would involve lower risks of
bleeding than UFH turned out to be incorrect, these mini-heparins
have met with increasing success, mainly because of their better
pharmacokinetic properties and easy control of therapeutic doses.
Originally obtained by size-fractionation of heparin, the most com-
mon LMWHs were obtained by partial depolymerization of UFH.
For a recent review, see Ref. 46. For discussions on relationship
between anticoagulant and antithrombotic activity of heparin see
Ref. 44; for development of LMWHs and their clinical use, see
Ref. 10.
LMWHs obtained with different depolymerization procedures,
and also those obtained with the same procedures but under
different reaction or purification conditions, have different compo-
sitional characteristics, as clearly shown, that is, by their oligosac-
charide mapping47 and NMR spectra48,49 and are considered
different therapeutic entities.50 In fact, different LMWHs not only
may have different end groups generated by the specific depoly-
merization procedures, but may differ from each other also in com-
positional terms, including their content of intact ATBR sequences.
As a typical example illustrated in Fig. 3, chains of LMWHs pre-
pared by cleavage of heparin with heparin lyase I terminate at their
nonreducing end with unsaturated uronic acid residues as in the
LMWHs prepared by chemical b-elimination. On the other hand,
the two LMWHs greatly differ at their ‘reducing’ terminals, where
the enzymatically-generated chains prevalently terminate with a
GlcNSO36SO3 residue, while a substantial proportion of the end
residues of the chemically-obtained ones are either ManNSO36SO3,
1,6-anhydro-GlcNSO3 or 1,6-anhydro-ManNSO3 (reviewed in Ref.
46). As an additional example of structural modification induced
in LMWHs by the specific method used for their preparation, enox-
aparin contains significantly more GlcA2SO3 residues than heparin
and the other LMWHs.49,51
A number of reducing end residues of the enzymatically-
derived LMWHs are constituted by the 3-O-sulfated residues
GlcNSO33,6SO3 typical of the ATBR. In fact, heparinase 1 cleaves
the ATBR sequence,52,53 resulting in a lower anti-Xa activity with
respect to chains of similar size containing intact ATBRs, such as
those obtainable under controlled chemical b-elimination.51 The
 B. Casu et al. / Carbohydrate Research 403 (2015) 60–68 63

(A)

(B)

Figure 2. (A) Simplified formula of a chain of porcine intestinal mucosal heparin containing an ATBR sequence, thus with high affinity for antithrombin. I = IdoA; G = GlcA in
the ATBR; G0 = GlcA toward the reducing end (RE) of the chain. The structure is from Ref. 43. (B) Intact linkage region (LR); see Ref. 38 and Refs. therein. GLR = GlcA in the
linkage region, Gal1 and Gal 2 are two D-galactose residues.

(A)

(B)

(C)

Figure 3. Simplified structures of the LMWHs tinzaparin (A), enoxaparin (B) and dalteparin (C), showing prevalent terminal residues at the reducing and reducing ends.
Internal sequences are indicated as in non-depolymerized heparin (From Ref. 91).

compositional and structural characteristics of the recently intro-
duced ‘generic’ (bioequivalent) LMWHs are required to closely
match those of the originators.54
7. Ultra-low-molecular-weight heparins
The success of the first ‘active’ synthetic pentasaccharide fonda-
parinux33 aroused interest in exploring the possible pharmaceuti-
cal exploitation of the lower-MW fractions of LMWH. Studies
have been made, mainly using controlled depolymerization of hep-
arin with recombinant heparin-lyases, to obtain LMW and ultra-
low-molecular-weight heparins (ULMWHs) richer in ATBR than
current LMWHs.55 ULMWHs essentially consisting of the pentasac-
charidic ATBR were successfully obtained by a chemo-enzymatic
approach.56 Also successful was the attempt to prepare ULMWHs
by adjusting the reaction conditions for the b-elimination reaction
64 B. Casu et al. / Carbohydrate Research 403 (2015) 60–68
used in the preparation of enoxaparin, to obtain a product consis-
tently richer in ATBR and with higher affinity for AT than enoxap-
arin,57 from which an octadecasaccharide containing three ATBRs
in a single chain was isolated.58
In parallel with similar efforts with heparan sulfates, a number
of heparin oligosaccharides have been isolated over the years,
mainly as fragments of heparin lyases- or nitrous acid-depolymer-
ized UHFs and LMWHs. A list of about one hundred heparin oligo-
saccharides (from tetrasaccharide to tetradecasaccharide)
identified up to the year 2001 (mainly by Linhardt–Toida’s, Lin-
dahl’s, Sugahara’s and Gallagher–Turnbull’s groups) is reported in
Ref. 4. More recently, the development of methods for heparin/
HS sequencing39–41 has permitted to add many more oligosaccha-
rides to that list. Linhardt’s group isolated and fully characterized
in heparin lyase I digests of porcine mucosal heparin up to 22 oli-
gosaccharides with size ranging from disaccharide to hexadecasac-
charide; notably, quite a few of these oligosaccharides terminate
with a reducing GlcNSO33,6SO3 residue.53 A number of oligosac-
charides containing the intact ATBR sequence were isolated from
the LMWH enoxaparin.51 (See also Section 9).
Some attempts to preserve and increase the content of ATBR-
containing chains as compared with that of current LMWHs have
met with success. These achievements were made both by using
heparin-lyases III that do not cleave the active pentasaccharide
sequence43,55 or by finding alkaline conditions for the b-elimina-
tion reaction that largely preserve the ATBR sequence.57
8. More than anticoagulation
8.1. Targeting heparin-binding proteins other than
antithrombin
Besides to AT, heparin binds to a large number of proteins and
modulates their biological functions.3,59–62 In fact, ‘heparin-bind-
ing proteins’ are so defined because binding to heparin is currently
used for their separation from other proteins, even if their actual
physiological binding polysaccharide is heparan sulfate (HS),
which is constituted by very much the same sequences of heparin
but in very different proportions and arrangements.62 The biosyn-
thesis of HS is similar to that of heparin,22 but its post-polymer
modification proceeds less homogeneously and is interrupted at
earlier stages than for heparin, leaving blocks of nonsulfated
sequences –GlcA–GlcNAc–(NA regions, occasionally 6-O-sulfated)
along with N,O-sulfated sequences (NS blocks) including also the
trisulfated disaccharides (TSD) units found in heparin. Several NA
and NS blocks are variously interrupted by mixed sequences.62
Although the heparin-like sequences are often involved in HS-pro-
tein interactions, the intrinsic structural diversity and organ and
tissue specificities of HSs suggest that also specific NA and/or
mixed sequences may contribute to binding and biological proper-
ties of this intriguing polysaccharide.63 However, unravelling spec-
ificities is complicated by current finding that some variability in
the sulfation pattern of a given heparin/HS sequence is compatible
with efficient binding to the same protein.60
Perception of the biological importance of HS and biochemical
studies on this GAG have been initially slow, both because of its
complexity and the low amounts obtainable from animal cells
and tissues. For this reason, heparin and heparin fragments have
been often used as a surrogate of HS sequences for obtaining pre-
liminary structure–activity information.62 These studies led to
realize that there was ‘more to heparin than anticoagulation’,64,65
and there were novel drug development opportunities for
heparin.66,67
Since most of the emerging therapeutic exploitations of heparin
do not require the ATBR (in fact, anticoagulant/antithrombotic
properties are often undesirable because of possible bleeding
effects), heparins, required for modulating most of the non-AT-
mediated activities should be non-anticoagulant. The anticoagu-
lant activity of heparin species can be substantially decreased
either by removing their chains with high affinity for AT, or by
modifying some of the ATBR groups or units that are essential for
high-affinity binding to AT. The first approach seems still not eco-
nomically feasible. As evident from inspection of the structure of
the pentasaccharide sequence of the ATBS (Fig. 1B), extensive mod-
ification of the sulfation pattern of heparin (as easily performed by
solvolytic desulfation or with controlled alkaline reaction)68–70
would remove some of the essential sulfate groups and decrease
the anticoagulant properties. Also other partial or extensive modi-
fications of heparin68–70 may involve loss of anticoagulant proper-
ties, but the biological properties of most of these derivatives still
do not seem to be systematically investigated. Removal of the
ATBR marker, that is, the 3-O-sulfate of the central GlcNSO33,6SO3,
A⁄) residue of the pentasaccharide would result in an especially
drastic decrease of the anticoagulant properties.32 A way of ‘inacti-
vating’ the ATBR consists in glycol-splitting, by periodate oxida-
tion, all nonsulfated uronic acid residues of heparin, including
the GlcA residue in the ATBR proved to be essential for high-affin-
ity binging to AT.71 Among the observed non-AT-related effects of
heparins,67 only two extensively investigated, that is, interaction
with fibroblast growth factors and heparanase, are shortly
reviewed here. (For other non-AT-dependent structure–activity
studies using libraries of heparin derivatives and fragments stud-
ied, u.a., as inhibitors of chemokines and b-secretase, see Refs.
72,73, respectively.)
8.2. Interactions with growth factors
Fibroblast growth factors (FGFs) utilize cell surface HS as a
co-receptor in the assembly of signaling complexes with FGF-
receptors on the plasma membrane. The best known members
of their family, which control angiogenesis and wound healing,
are FGF1 and FGF2. The molecular mechanism for their activa-
tion involves complexation of the FGF with a HS chain, which
favors dimerization of the cytokine and also acts as template
for formation of a ternary complex involving FGF receptors.
(Extensively reviewed in Ref. 74). First indication on structural
requirements for heparin sequences that bind to FGF1 and
FGF2, especially on the requirement of 6-O-sulfate groups in
the case of FGF1 were obtained using selectively desulfated
heparins as models.75
More detailed knowledge on the heparin/HS FGF-binding
sequences, such as identification of the minimal ones required
for binding, i.g., the –IdoA2SO3–GlcNSO3,6SO3–IdoA2SO3– motif
for FGF1 and –IdoA2SO3–GlcNSO3–IdoA2SO3– for FGF2, was subse-
quently obtained by Lindahl’s and other groups using libraries of
oligosaccharides (reviewed in Ref. 4). Also synthetic oligosaccha-
rides (such a tetrasaccharide76 and a hexasaccharide77) were used
for studying the sequence and size requirements for heparin bind-
ing to FGF2. Structural requirements for formation of ternary hep-
arin/FGF/FGFR-receptors are still not well defined. For FGF1, a
heparin octasaccharide is the shortest oligomer capable of dimeriz-
ing the growth factor and assembling the active ternary com-
plex.78,79 Diversification of the structural determinants of FGFs–
heparin interactions and implications for binding specificity were
also established.80
Among the non-anticoagulant heparins, some glycol-split deriv-
atives (prepared from partially 2-O-desulfated heparins to obtain
up to a total of about 50% glycol-split uronic acids) showed to be
efficient antagonists of FGF2 and potent angiogenesis
inhibitors.81,82
 B. Casu et al. / Carbohydrate Research 403 (2015) 60–68
8.3. Non-anticoagulant heparins as heparanase inhibitors
The multiple functions of heparanase in cancer,83 inflammation
and other diseases84 are now widely recognized. Most of these
functions are associated with the enzymatic activity of heparanase,
which, acting as a b-D-glucuronidase, cleaves the HS chains at the
level of GlcA residues. Since the first reports on structural require-
ments of heparin/HS to act as substrate for the enzyme,85 non-anti-
coagulant heparins were investigated as inhibitors of the
enzymatic activity of heparanase, and some of them (especially
glycol-split heparins) were found to be promising as antimetastatic
and anticancer agents.86 These results are discussed in wider con-
texts in Refs. 87,88. Structural requirements of heparin chains to
act as heparanase substrates or inhibitors were also studied using
chemo-enzymatically synthesized heparin oligosaccharides.89 In
the framework of further studies on heparin-derived heparanase
inhibitors, NMR and LC/MS methods have been developed for
structural characterization of glycol-split heparins90 and glycol-split
LMWHs.91 Microarray assays,92 and saturation-transfer difference
(STD) NMR measurements93 were used for biological evaluation
(including inhibition of heparanase) of synthetic heparin mimetic
hexasaccharides.93
9. Conformational aspects
9.1. Conformation of unbound heparin sequences
In mid 1980s, the molecular conformation of GAG component
residues was a matter of debate, especially as regards the IdoA-
containing sequences, such as those of heparin/HS and dermatan
sulfate, with apparently conflicting results between data obtained
from kinetics of periodate oxidation and from emerging NMR indi-
cations from synthetic methyl iduronates20 and urinary dermatan
sulfate.94,95 NMR analysis of the synthetic pentasaccharide, corre-
sponding to the ATBR of heparin, provided evidence for the confor-
mational peculiarity of its sulfated iduronate residue.30 A full
analysis of the NMR spectra of heparin and synthetic heparin
mono- and oligosaccharides combined with molecular mechanics
calculations clearly showed that IdoA2SO3 and IdoA residues are
in a dynamic conformational equilibrium among three iso-ener-
getic forms (1C4, 2S0 and 4C1),96,97 as predicted by a theoretical
study on iduronate monosaccharides.98 The conformer populations
of the three forms could widely differ depending on sulfation on
the same residue or in that of neighbor residues, as well as on
extrinsic factors such as the presence of calcium ions.97 Notably,
whereas IdoA2SO3 residues in internal ‘regular’ TDS sequences
show a relative population of conformers 1C4:2S0 of about 60:40,
such a ratio is practically reversed (to 40:60) in favor of the 2S0
form when the adjacent GlcNSO36SO3 residue is 3-O-sulfated, as
in the ATBR.96,97,99 Since the relative orientations of the substituent
groups significantly differ in the two conformations, involving, i.e.,
different spacing between sulfate and/or carboxylate groups, such
a behavior was soon perceived to have an important role on bind-
ing and associated biological properties of iduronic acid-containing
GAGs.100 Studies on synthetic pentasaccharides with iduronate
residues ‘locked’ in the 2S0 conformation reinforced the concept
that this local internal flexibility (also referred to as ‘plasticity’)
favors the adjustment of IdoA/IdoA2SO3-containing sequences to
relevant binding sites of proteins.101 As exemplified for the penta-
saccharide fondaparinux, 15N NMR chemical shifts of the sulfamido
groups can provide additional conformational information.102
NMR, molecular dynamics and modeling studies by Mulloy’s group
generated the presently accepted 3D structure for the ‘regular’
regions of heparin chains with iduronate residues in different local
conformations.103,104 NMR and molecular dynamics studies on a
heparin-derived hexasaccharide further confirmed conformational
65
equilibria of internal sulfated iduronate residues.105 As illustrated
in Ref. 106, Mulloy’s structures of heparin are represented by heli-
ces with alternate triplets of sulfate groups on both sides of the
chains irrespective of the conformation of the iduronate residues.
This shape is intrinsically the result of the a-L-a-D- alternate glyco-
sidic linkages of iduronate and glucosamine residues in the repeat-
ing TSD units. Analysis of available crystal coordinates of heparin
oligosaccharide-protein complexes has shown that the linear prop-
agation of heparin chains is interrupted at the level of the binding
sites to proteins by ‘kinks’ generated by changes in local conforma-
tion as would be observed on going from a 1C4 to 2S0 form.107 A crit-
ical overview of the influence of substitution pattern and cation
binding on conformation and activity of heparin derivatives is
reported in Ref. 108.
9.2. Conformation of protein-bound sequences
Complexation with proteins is among the extrinsic factors that
can drive the conformations toward a unique form. Conceivably,
only one of the favored conformations of iduronate residues is rep-
resented in co-crystal structures. Thus, in co-crystals of a complex
with AT and a sulfated pentasaccharide, the IdoA2SO3 residue was
found to be 100% in the 2S0 form.109 Such a AT-complexation-
induced drive toward the 2S0 conformation was found also in solu-
tion.110 Milestone papers unraveled the influence of structure,
chain length and local conformations in the regulation of thrombin
activity by AT and heparin,111 and provided evidence for an
induced-fit model of allosteric activation of AT112 and for the
involvement of the unique trisaccharide toward the nonreducing
end of the ATBR in mediating the early steps of AT activation.113
A notable example of protein-binding-induced selection of a local
conformation was the finding that in co-crystals of FGF-2 and a
heparin hexasaccharide containing two IdoA2SO3 residues, one of
these residues is in the 1C4 and the other in the 2S0 conforma-
tion.114 (See Ref. 4). However, 1C4 is the only conformation in solu-
tion of IdoA2SO3 residues of synthetic heparin/HS tetrasaccharides
complexed with both FGF1115 and FGF2.76 A recent, still unex-
plained but certainly meaningful example of different conforma-
tions selected in the solid state by apparently similar sulfated
iduronate residues of heptasaccharidic and tetrasacccharidic sub-
strates of two different sulfotransferases (3OST-1 and 3OST-3) is
illustrated in Figure 4.116
A series of papers deals with the structural and biochemical
effects of chain extension of the ATBR. The first of these contribu-
tions analyzed the crystallographic coordinates of the AT complex
with a synthetic heptasaccharide corresponding to the pentasac-
charidic active sequence extended by a disaccharidic unit toward
the reducing end. Its somewhat higher affinity for AT is explainable
by additional contacts with AT exerted by the disaccharide elonga-
tion.117 Similarly, in NMR/molecular mechanics studies in solution
on octa- and higher heparin oligosaccharides isolated from the
LMWH enoxaparin (reviewed in Ref. 51), elongation (with disac-
charidic units unmodified by the depolymerization process) of
the ATBR toward both the reducing and the non-reducing end
allowed better contacts with AT and higher affinity for the protein.
Noteworthy, an octasaccharide featuring an unusual GlcA resi-
due just preceding the ATBR showed to have a tenfold higher affin-
ity and more favorable contacts with the AT basic residues than the
homologous octasaccharide with IdoA in the same position as nor-
mally found in HA chains isolated from porcine mucosal hepa-
rin.118 Also, as expected from X-ray data on a synthetic variant of
the ATBR pentasaccharide,109 an octasaccharide containing a 3-O-
sulfate group also in the other GlcNSO36SO3 residue of the ATBR
had higher affinity and better contacts than its homologous ‘nor-
mal’ octasaccharide.119 Although some literature data (reviewed
in Ref. 46) indicated that some chains of LMWHs could contain
66 B. Casu et al. / Carbohydrate Research 403 (2015) 60–68
(A)
(B)
Figure 4. Conformation of heparin/HS oligosaccharides in co-crystals with 3-O-sulfotransferases.116 (A) Chemoenzymatically synthesised heptasaccharide substrate
crystallized with 3OST-1; (B) tetrasaccharide generated by cleavage of A with heparin lyase-I, crystallized with 3OST-3. Note that the conformation of the central IdoA2SO3
residue of A is 1C4, while in B is 2S0.
more than one ATBR,49 rather unexpectedly a dodecasaccharide
containing two pentasaccharidic ATBR separated by a nonsulfated
IdoA residue was isolated from enoxaparin; interestingly, one of
the two pentasaccharide sequences binds to AT more strongly than
the other and the two types of complexes are in a dynamic equilib-
rium120 On the other hand, in octasaccharides terminating with the
1,6-anhydro residues typical of enoxaparin, the terminal units
were found not contribute to the AT binding, thus rationalizing
the lower affinity for AT (and lower anti-Xa activity) of these spe-
cies.121 As regards the conformational aspects, all the above-men-
tioned ATBR-containing oligosaccharides were found to bind to AT
with the same geometry as the (core) pentasaccharide; in all of
them, the IdoA2SO3 residue is in the 2S0 form. However, in the
AT-binding oligosaccharides so far studied, the iduronate residues,
irrespective of being sulfated or nonsulfated, select in the bound
state either the 2S0 or the 1C4 conformation depending on their
position along the oligosaccharide chain even in those cases in
which they preferred in the unbound state the other conforma-
tion.51 While confirming that the conformational plasticity plays
a decisive role in modulating the protein binding of heparin
sequences, these results imply that the conformation of iduronate
residues that predominate in the absence of binding proteins is not
necessarily the same adopted in the bound state. Conceivably, the
most favored conformation of heparin/HS sequences in the bound
state is determined by a balance of several interactions, to which
the plastic iduronate residue contributes in different ways depend-
ing on the structure and conformation of the active site of the
protein.
Structural data and concepts on heparin complexes with pro-
teins other than AT are presented and discussed in recent mini-
reviews.122–125
10. Toward a biotechnological heparin
Since early 1990, concerns on possible shortage of heparin of
animal origin stimulated studies aimed at generating heparins by
exploiting available biosynthetic enzymes in combination with
regioselective chemical modification reactions. Discovery of the
microbial polysaccharide K5, which is exclusively constituted by
(GlcA–GlcNAc)n sequences as the heparin biosynthetic precursor
N-acetyl heparosan, triggered an European project aimed at explor-
ing whether such an approach could afford heparin/HS-like spe-
cies. Our group participated in the project by investigating the
possibility of sulfation at specific positions both of unmodified
K5 backbone and derivatives obtained by enzymatic partial C-5-
epimerization of GlcA residues. After chemical N-deacetylation
and N-sulfation, a selective O-sulfation at position 6 of the GlcNAc
residues was achieved. Under different conditions, a selective 3-O-
sulfation of GlcA, and complete O-sulfation of the polysaccharide
were obtained.126 Some of these K5 derivatives inhibited Factor
Xa, with potential antithrombotic properties.127 In a parallel study,
conditions for C-5 epimerization of GlcA residues were optimized
to achieve up to 50% conversion to IdoA.128 Chemical O-sulfation
of N-sulfated, C-5-epimerized HMW and LMW K5 polysaccharide
afforded several derivatives, which were significantly 3-O-sulfated
at the aminosugar, but almost invariably also at the GlcA residue.
Efforts to reduce the unnatural 3-O-sulfation of GlcA and increase
the content of N,3-O-sulfated GlcN by exploiting a strategy of
graded solvolyic desulfation of fully sulfated, partially 5-O-epimer-
ized K5, have generated products with biological properties similar
to those of heparin and LMWHs.129 Thus, although the structure of
the obtained ‘neoheparins’ significantly differed from that of hep-
arins of animal origin, the above studies showed that some of the
biological properties of these latter could be simulated and
modulated.
A more recent project of an U.S.A. consortium exploiting a full
series of recombinant biosynthetic enzymes had afforded homoge-
neous ULMW heparins and has great potential for generating hep-
arin/HS oligosaccharides with almost unlimited possibilities for
tailoring specific sequences.130
11. The ‘heparin crisis’ and its (beneficial) fall-outs
In late 2007 and early 2008 a series of adverse events in patients
receiving heparin injections occurred in U.S.A. and in some Euro-
pean countries, causing more than one hundred deaths. As a reac-
tion to these dramatic events, the U.S. Food and Drug
Administration (FDA) recalled suspected heparin vials and orga-
nized their analysis by a consortium of academic groups (including
our own), coordinated by Ram Sasisekharan. It was soon shown
that the adverse events were caused by an ‘unnatural’ contami-
nant, a fully O-sulfated chondroitin sulfate.131
 B. Casu et al. / Carbohydrate Research 403 (2015) 60–68
The realization that the current methods of pharmacopoeial
controls for heparin were inadequate to detect unexpected con-
taminants triggered an explosion of analytical studies and propos-
als for orthogonal approaches, most of which involving
combination of NMR and separation methods. A survey of these
novel analytical methods (examples of which are reported in Refs.
132–135) and on their expected contribution to an increased safety
in the use of pharmaceutical heparins is outside the scope of this
review. However, it is worth mentioning that some sophisticated
methods for obtaining and analyzing NMR data, such as the two-
dimensional correlation spectroscopy-filtering with iterative radia-
tion sampling136 and statistical correlation two-dimensional
(HSQCcos) spectroscopy,137 contributed not only to detect
unknown contaminants, but also to identify previously undetected,
very minor components of heparin. Among these components,
internal N-unsubstituted GlcN,6SO3 residues and 3-O-sulfated
GlcNSO3,6SO3 residues at the nonreducing terminal of heparin
chains were recently identified in unfractionated heparin.137 New
criteria for characterization of currently marketed heparin prod-
ucts were reported by the Federal Food and Drug Administration
(FDA) agency.138
12. Open issues and perspectives
In spite of its being almost 100 years old, heparin has success-
fully overcome critical times such as the just mentioned crisis
and has not lost its reputation as a life-saving drug.139 Although,
for one side, heparin is facing competition from novel synthetic
antithrombotics, from the other side most of the prospective devel-
opments for heparin-related oligo- and polysaccharides as HS
mimics140 are widely open. In spite of the steady advances in
methods of structural characterization of heparin and understand-
ing of its interactions with proteins mostly associated with increas-
ing interest in structure and functions of HS and HS
proteoglycans,141 there are still several open issues in the field that
could easily fill the professional life of other generations of
researchers. For example, understanding of biological function
and potential pharmaceutical exploitation of 3-O-sulfated GlcN
residues in different sequences of heparin and HS is still at its
beginning.142 In fact, in spite of the many pathophysiological func-
tions continuously discovered for HSs, only a few drugs exploiting
these functions are being developed.140
Acknowledgements
The authors gratefully thank Ulf Lindahl and Maurice Petitou for
critical reading of the manuscript and Antonella Bisio, Giuseppe
Cassinelli and Marco Guerrini for useful discussions. Two of them
(B.C. and G.T.) wish to express their gratitude to Arthur Perlin for
introducing them into the heparin field. Part of the work described
in this mini-review has been financially supported by several agen-
cies. Among current Grants, those from National Institutes of
Health, United States (Number R01-CA138535) and Finlombardia
SpA, Italy (Fondo promozione accordi istituzionali) are acknowl-
edged. The authors wish to thank also the Villa Vigoni Foundation
for hosting, since more than twenty years, yearly Glycosaminogly-
can Symposia, with special emphasis on heparin.
References
1. Casu, B. In Adv. Carbohydr. Chem. Biochem.; Stuart Tipson, R., Derek Horton,
Eds.; , 1985; Vol. 43, pp 51–134.
}rk, I., Lindahl, U., Eds.; Plenum
2. Adv. Carbohydr. Chem. Biochem.; Lane, D. A., Bjo
Press: New York, 1992.
3. Conrad, E. Heparin Binding Proteins; Academic Press: San Diego, CA, 1998.
4. Casu, B.; Lindahl, U. Adv. Carbohydr. Chem. Biochem. 2001, 57, 159–206.
67
5. Linhardt, R. J. J. Med. Chem. 2003, 46, 2551–2564.
6. Chemistry and Biology of Heparin and Heparan Sulfate; Garg, H. G., Linhardt, J.,
Hales, C. A., Eds.; Elsevier: Amsterdam, 2005.
7. Heparin—A Century of Progress; Lever, R., Mulloy, B., Page, C. P., Eds.; Springer:
Berlin, 2012.
8. Bigelow, W. G. Misterious Heparin; McGraw-Hill Ryerson: Toronto, 1990.
9. Rodén, L.; Ananth, S.; Campbell, P.; Crenton, T.; Ekborg, G.; Manzella, S.;
Pillion, D.; Meezan, E. In Ref. 2, 1992; pp 1–20
10. Barrowcliffe, T. W. Ref. 7, 2012; pp 3–22.
11. Wolfrom, M. L.; Rice, F. A. J. Am. Chem. Soc. 1946, 68, 532.
12. Helting, T.; Lindahl, U. J. Biol. Chem. 1971, 246, 5442–5447.
13. Cifonelli, J. A.; Dorfman, A. Biochem. Biophys. Res. Commun. 1962, 7, 41–46.
14. Perlin, A. S.; Mazurek, M.; Jaques, L. B.; Kavanagh, L. W. Carbohydr. Res. 1968,
7, 369–379.
15. Perlin, A. S.; Casu, B.; Sanderson, G. R.; Johnson, L. F. Can. J. Chem. 1970, 48,
2260–2268.
16. Perlin, A. S.; Sanderson, G. S. Carbohydr. Res. 1970, 12, 183–192.
17. Lindahl, U.; Axelsson, O. J. Biol. Chem. 1971, 246, 74–82.
18. Wolfrom, M. L.; Honda, S.; Wang, P. Y. Carbohydr. Res. 1969, 10, 185–259.
19. Perlin, A. S.; Mackie, D. M.; Dietrich, C. P. Carbohydr. Res. 1971, 18, 145–194.
20. Perlin, A. S.; Casu, B.; Tse, J.; Sanderson, G. R. Carbohydr. Res. 1972, 21, 123–
132.
21. Gatti, G.; Casu, B.; Hamer, A. S.; Perlin, A. S. Macromolecules 1979, 12, 1001–
1007.
22. Carlsson, P.; Kjellén, L. Ref. 7, 2012; pp 23–41.
23. Mulloy, B. Ref. 7, 2012; pp 77–98.
24. Bertini, S.; Bisio, A.; Torri, G.; Bensi, D.; Terbojevich, M. Biomacromolecules
2005, 6, 168–173.
25. Lindahl, U.; Bäckstro } m, G.; Thunberg, L.; Leder, I. G. Proc. Natl. Acad. Sci. U.S.A.
1980, 77, 6551–6555.
26. Thunberg, L.; Bäckstro }m, G.; Lindahl, U. Carbohydr. Res. 1982, 100, 393–410.
27. Lindahl, U.; Thunberg, L.; Bäckstro } m, G.; Riesenfeld, J.; Nordling, K.; Bjo
}rk, I. J.
Biol. Chem. 1984, 259, 12368–12376.
28. Casu, B.; Oreste, P.; Torri, G.; Zoppetti, G.; Lormeau, J. C.; Petitou, M.; Sinaÿ, P.
Biochem. J. 1981, 197, 599–609.
29. Loganathan, D.; Wang, H. M.; Mallis, L. M.; Linhardt, R. J. Biochemistry 1990,
29, 4362–4368.
30. Choay, J.; Petitou, M.; Lormeau, J.-C.; Sinaÿ, P.; Casu, B.; Gatti, G. Biochem.
Biophys. Res. Commun. 1983, 116, 492–499.
31. Petitou, M.; Casu, B.; Lindahl, U. Biochimie 2003, 85, 83–89.
32. Van Boeckel, C. A. A.; Petitou, M. Angew. Chem., Int. Ed. 1993, 32, 1671–1688.
33. Petitou, M.; van Boeckel, C. A. Angew. Chem., Int. Ed. 2004, 43, 3118–3133.
34. Dementiev, A.; Petitou, M.; Herbert, J. M.; Gettings, P. G. Nat. Struct. Biol. 2004,
11, 863–867.
35. Yamada, S.; Murakami, T.; Tsuda, H.; Yoshida, K.; Sugahara, K. J. Biol. Chem.
1995, 270, 8696–8705.
36. Chai, W.; Hounsell, E. F.; Bauer, C. J.; Lawson, A. M. Carbohydr. Res. 1995, 269,
139–156.
37. Iacomini, M.; Casu, B.; Guerrini, M.; Naggi, A.; Pirola, A.; Torri, G. Anal.
Biochem. 1999, 274, 50–58.
38. Casu, B.; Torri, G.; Vercellotti, J. R. Pharmacol. Res. Comm. 1979, 11, 297–309.
39. Venkataraman, G.; Shriver, Z.; Raman, R.; Sasisekharan, R. Science 1999, 86,
537–542.
40. Turnbull, J. E.; Hopwood, J. J.; Gallagher, J. T. Proc. Natl. Acad. Sci. U.S.A. 1999,
96, 2698–2703.
41. Linhardt, R. J.; Rice, K. G.; Kim, Y. S.; Loshe, D. L.; Wang, H. M.; Loganathan, E.
D. Biochem. J. 1988, 254, 781–787.
42. Shriver, Z.; Capila, I.; Venkataraman, R. Ref. 7, 2000, pp 159–175.
43. Xiao, Z.; Tappen, B. R.; Ly, M.; Zhao, W.; Canova, L. P.; Guan, H.; Linhardt, R. J. J.
Med. Chem. 2011, 54, 603–610.
44. Gray E.; Hogwood, J.; Mulloy, B. Ref. 7, 2012; pp 43–61.
45. Andersson, L.-O.; Barrowcliffe, T. V.; Holmer, E.; Johnson, E. A.; Sims, G. E.
Thromb. Res. 1976, 6, 575–583.
46. Guerrini, M.; Bisio, A. Ref. 7, 2012; pp 127–157.
47. Linhardt, R. J.; Loganathan, D.; Al-Hakim, A.; Wang, H.-M.; Walenga, J. M.;
Hoppensteadt, D.; Fareed, J. J. Med. Chem. 1990, 33, 1639–1645.
48. Guerrini, M.; Guglieri, S.; Naggi, A.; Sasisekharan, R.; Torri, G. Semin. Thromb.
Hemost. 2007, 33, 478–487.
49. Bisio, A.; Vecchietti, D.; Citterio, L.; Guerrini, M.; Raman, R.; Bertini, S.; Eisele,
G.; Naggi, A.; Sasisekharan, R.; Torri, G. Thromb. Haemost. 2009, 102, 865–873.
50. Fareed, J.; Haas, S.; Sahahara, E. Semin. Thromb. Hemost. 1999, 3, 1–14.
51. Guerrini, M.; Mourier, P.; Torri, G.; Viskov, C. Glycoconjugate J. 2014. in press.
52. Shriver, Z.; Sundaram, M.; Venkataraman, G.; Fareed, J.; Linhardt, J. R.;
Biemann, K.; Sasisekharan, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10365–
10370.
53. Xiao, Z.; Zhao, W.; Yang, Z.; Zhang, Z.; Guan, H.; Linhardt, R. J. Glycobiology
2011, 21, 13–22.
54. Harenberg, J.; Walenga, J.; Torri, G.; Dahl, O. E.; Drouet, L.; Fareed, J. J. Thromb.
Haemost. 2013, 334, 1421–1425.
55. Sundaram, M.; Shriver, Z.; Zhao, G.; Venkataraman, G.; Langer, R.;
Sasisekharan, R. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 651–656.
56. Xu, Y.; Masuko, S.; Takieddin, M.; Xu, H.; Liu, R.; Jing, J.; Mousa, S. A.; Linhardt,
R. J.; Liu, J. Science 2011, 334, 498–501.
57. Lima, M. A.; Viscov, C.; Herrman, F.; Gray, A. L.; de Faris, E. H.; Cavalheir, R. P.;
Sassaki, G. L.; Hoppensteadt, D.; Fareed, J.; Nader, H. B. Thromb. Haemost. 2013,
109, 471–478.
68 B. Casu et al. / Carbohydrate Research 403 (2015) 60–68
58. Mourier, P. A.; Guichard, O. Y.; Herman, F.; Viskov, C. Anal. Biochem. 2014, 15,
453–457.
59. Capila, I.; Linhardt, R. J. Angew. Chem., Int. Ed. 2002, 41, 391–412.
60. Kreuger, J.; Spillmann, D.; Li, J. P.; Lindahl, U. J. Cell Biol. 2006, 174, 323–327.
61. Lindahl, U. Thromb. Haemost. 2007, 96, 109–115.
62. Gallagher, J.T. Ref. 7, 2012; pp 347–360.
63. Xu, D.; Esko, J. D. Annu. Rev. Biochem. 2014. in press.
64. Lindahl, U.; Lidholt, K.; Spillmann, D.; Kjellén, L. Thromb. Res. 1994, 75, 1–32.
65. Lindahl, U. Glycoconju. J. 2000, 17, 597–605.
66. Lever, L.; Page, C. P. Nat. Rev. Drug Disc. 2002, 57, 159–206.
67. Lever, R.; Page, C.P.; Ref. 7, pp 281–305.
68. Yates, E. A.; Santini, F.; Guerrini, M.; Naggi, A.; Torri, G.; Casu, B. Carbohydr.
Res. 1996, 294, 15–27.
69. Fernandéz, C.; Hattan, C. M.; Kerns, R. J. Carbohydr. Res. 2006, 341, 1253–1265.
70. Yates, E. A.; Guimond, S. E.; Turnbull, J. E. J. Med. Chem. 2004, 47, 277–280.
71. Naggi, A. Ref. 6, 2005, pp 461–481.
72. Shute, J. Ref. 7, 2012; pp 307–324.
73. Schwo }rer, R.; Zubkova, O. V.; Turnbull, J. E.; Tyler, P. C. Chemistry 2013, 19,
6817–6823.
74. Mohammadi, M.; Olsen, S. K.; Ibrahimi, O. A. Cytokine Growth Factor Rev. 2005,
16, 107–137.
75. Maccarana, M.; Casu, B.; Lindahl, U. J. Biol. Chem. 1993, 268, 23898–23905.
76. Guglieri, S.; Hricovini, M.; Raman, R.; Polito, L.; Torri, B.; Casu, B.;
Sasisekharan, R.; Guerrini, M. Biochemistry 2008, 47, 13862–13869.
77. Sapa, N.; Cabannes, E.; Petitou, M.; Imberty, A. Glycobiology 2011, 21, 1181–
1193.
78. Jastrebova, N.; Varwildemeersch, M.; Lindahl, U.; Spillmann, D. J. Biol. Chem.
2010, 285, 26842–26851.
79. Brown, A.; Robinson, C. J.; Gallagher, J. T.; Blundell, T. L. Biophys. J. 2013, 104,
1720–1730.
80. Xu, R.; Ori, A.; Rudd, T. R.; Uniewicz, K. A.; Ahmed, Y. A.; Guimond, S. E.;
Skidmore, M. A.; Siligardi, G.; Yates, E. A.; Fernig, D. G. J. Biol. Chem. 2012, 287,
40061–40073.
81. Casu, B.; Guerrini, M.; Naggi, A.; Perez, M.; Torri, G.; Ribatti, D.; Carminati, P.;
Giannini, G.; Penco, S.; Pisano, C.; Belleri, M.; Rusnati, M.; Presta, M.
Biochemistry 2002, 41, 10519–10528.
82. Casu, B.; Guerrini, M.; Guglieri, A.; Naggi, A.; Perez, M.; Torri, G.; Cassinelli, G.;
Ribatti, D.; Carminati, P.; Giannini, G.; Penco, S.; Pisano, C.; Belleri, M.; Rusnati,
M.; Presta, M. J. Med. Chem. 2004, 47, 838–848.
83. Ramani, V. C.; Puroshothaman, A.; Stewart, M. D.; Thompson, C. A.; Vlodavsky,
I.; Au, J. L.; Sanderson, R. D. FEBS J. 2013, 280, 2294–2306.
84. Vlodavsky, I.; Iozzo, R. V.; Sanderson, R. D. Matrix Biol. 2013, 32, 220–222.
85. Sandbäck-Pikas, D.; Li, J. P.; Vlodavsky, I.; Lindahl, U. J. Biol. Chem. 1998, 273,
18870–18877.
86. Naggi, A.; Casu, B.; Perez, M.; Torri, G.; Cassinelli, G.; Penco, S.; Pisano, C.;
Giannini, G.; Ishai-Michaeli, R.; Vlodavsky, I. J. Biol. Chem. 2005, 280, 12103–
12113.
87. Vlodavsky, I.; Ilan, N.; Naggi, A.; Casu, B. Curr. Pharm. Des. 2007, 13, 2057–
2073.
88. Casu, B.; Vlodavsky, I.; Sanderson, R. D. Pathophysiol. Haemost. Thromb. 2007,
36, 195–203.
89. Peterson, S.; Liu, J. J. Biol. Chem. 2012, 287, 34836–34843.
90. Alekseeva, A.; Casu, B.; Torri, G.; Pierro, S.; Naggi, A. Anal. Biochem. 2013, 434,
112–122.
91. Alekseeva, A.; Casu, B.; Cassinelli, G.; Guerrini, G.; Torri, G.; Naggi, A. Anal.
Bioanal. Chem. 2014, 406, 249–265.
92. Noti, C.; de Paz, J. L.; Polito, L.; Seeberger, P. H. Chem. Eur. J. 2006, 12, 8664–
8686.
93. Roy, S.; El Hadri, A.; Richard, S.; Denis, F.; Holte, K.; Duffner, J.; Yu, F.;
Galcheva-Gargiova, Z.; Capila, I.; Schultes, B.; Petitou, M.; Kaundinya, G. V. J.
Med. Chem. 2014, 115.
94. Gatti, G.; Casu, B.; Torri, G.; Vercellotti, J. R. Carbohydr. Res. 1979, 68, C3–C7.
95. Casu, B.; Choay, J.; Ferro, D. R.; Gatti, G.; Jacquinet, J.-C.; Petitou, M.; Provasoli,
A.; Ragazzi, M.; Sinaÿ, P.; Torri, G. Nature 1986, 322, 215–216.
96. Ferro, D. R.; Provasoli, A.; Ragazzi, M.; Torri, G.; Casu, B.; Gatti, G.; Jacquinet, J.-
C.; Sinaÿ, P.; Petitou, M.; Choay, J. J. Am. Chem. Soc. 1986, 1908, 6773–6778.
97. Ragazzi, M.; Ferro, D. R.; Perly, B.; Sinaÿ, P.; Petitou, M.; Choay, J. Carbohydr.
Res. 1990, 195, 169–185.
98. Ragazzi, M.; Ferro, D. A.; Provasoli, A. J. Comput. Chem. 1986, 7, 105–112.
99. Ferro, D. R.; Provasoli, A.; Ragazzi, M.; Casu, B.; Torri, G.; Bossenec, V.; Perly,
B.; Sinaÿ, P.; Petitou, M.; Choay, J. Carbohydr. Res. 1990, 195, 157–167.
100. Casu, B.; Petitou, M.; Provasoli, M.; Sinaÿ, P. Trends Biol. Sci. 1988, 13, 221–225.
101. Das, S. K.; Mallet, J.-M.; Esnault, J.; Driguez, P.-A.; Duchaussoy, P.; Sizun, P.;
Hérault, J.-P.; Herbert, J.-M.; Petitou, M.; Sinaÿ, P. Angew. Chem., Int. Ed. 2001,
40, 1670–1673.
102. Larngeslay, D. J.; Beecher, C. N.; Naggi, A.; Guerrini, M.; Torri, G.; Larive, C. K.
Anal. Chem. 2013, 85, 1247–1255.
103. Mulloy, B.; Forster, M. J.; Jones, C.; Davies, D. B. Biochem. J. 1993, 293, 849–858.
104. Mulloy, B.; Forster, M.; Jones, C.; Davies, D. B. Glycobiology 2000, 10, 1147–
1158.
105. Mikhailov, D.; Linhardt, R. J.; Mayo, K. H. Biochem. J. 1997, 328, 51–61.
106. Mulloy B.; Ref. 7, 2012; pp 77–98.
107. Raman, R.; Sasisekharan, V.; Sasisekharan, R. Chem. Biol. 2005, 12, 267–277.
108. Rudd, T. R.; Guimond, S. E.; Skidmore, M. A.; Duchesne, R.; Guerrini, M.; Torri,
G.; Cosentino, C.; Brown, A.; Clarke, D. T.; Turnbull, J. E.; Fernig, D. G.; Yates, E.
A. Glycobiology 2007, 17, 983–993.
109. Jin, L.; Abrahams, J. P.; Skinner, R.; Petitou, M.; Pike, R. N.; Carrell, R. W. Proc.
Natl. Sci. USA 1997, 14683–14688.
110. Hricovini, M.; Guerrini, M.; Bisio, A.; Torri, G.; Petitou, M.; Casu, B. Biochem. J.
2001, 359, 265–272.
111. Olson, S. T.; Bjo}rk, I. Semin. Thromb. Hemost. 1994, 20, 373–409.
112. Desai, U. R.; Petitou, M.; Bjo }rk, I.; Olson, S. T. Biochemistry 1998, 37, 13033–
13041.
113. Petitou, M.; Barzu, T.; Herault, J.-P.; Herbert, J.-M. Glycobiology 1997, 7, 323–
327.
114. Faham, S.; Hileman, R. E.; Fromm, J. R.; Linhardt, R. J.; Rees, D. C. Science 1996,
271, 116–120.
115. Guerrini, M.; Agulles, T.; Bisio, A.; Hricovini, M.; Lay, L.; Paoletti, L.; Sturiale,
L.; Torri, G.; Casu, B. Biochem. Biophys. Res. Commun. 2002, 292, 222–230.
116. Liu, J.; Moon, A. F.; Sheng, J.; Pedersen, L. C. Curr. Opin. Struct. Biol. 2012, 22,
570–577.
117. Belzar, K. J.; Dafforn, T. R.; Petitou, M.; Carrell, R. W.; Huntington, J. A. J. Biol.
Chem. 2000, 275, 8733–8741.
118. Guerrini, M.; Guglieri, S.; Casu, B.; Torri, G.; Mourier, P.; Boudier, C.; Viskov, C.
J. Biol. Chem. 2008, 283, 26662–26675.
119. Guerrini, M.; Elli, S.; Mourier, P.; Rudd, T. R.; Gaudesi, D.; Casu, B.; Boudier, C.;
Torri, G.; Viskov, C. Biochem. J. 2013, 449, 343–351.
120. Viskov, C.; Elli, S.; Urso, E.; Gaudesi, D.; Mourier, P.; Herman, F.; Boudier, C.;
Casu, B.; Torri, G.; Guerrini, M. J. Biol. Chem. 2013, 288, 25895–25907.
121. Guerrini, M.; Elli, S.; Gaudesi, D.; Torri, G.; Casu, B.; Mourier, P.; Herman, F.;
Boudier, C.; Lorenz, M.; Viskov, C. J. Med. Chem. 2010, 53, 8030–8040.
122. Imberty, A.; Lortat-Jacob, H.; Pérez, S. Carbohydr. Res. 2007, 342, 430–439.
123. Rudd, T. R.; Skidmore, M. A.; Guerrini, M.; Hricovini, M.; Powell, A. K.;
Siligardi, G.; Yates, E. A. Curr. Opin. Struct. Biol. 2010, 20, 567–574.
124. Casu, B.; Naggi, A.; Torri, G. Matrix Biol. 2010, 29, 442–452.
125. Gonzáles-Corrochano, J.; Rolando-Horcajo, M.; Javier Cañada, F.; Nieto, P.;
Martin-Lomas, M.; Giménez-Gallego, G.; Jiménez-Barbero, G. ChemBioChem
2013, 14, 1732–1744.
126. Casu, B.; Grazioli, M.; Guerrini, M.; Naggi, A.; Torri, G.; Oreste, P.; Tursi, F.;
Zoppetti, G.; Razi, N.; Feizi, E.; Lindahl, U. Carbohydr. Res. 1994, 263, 271–284.
} rk, I.; Naggi, A.; Casu, B.; Lindahl, U. Biochem. J. 1995, 309,
127. Razi, N.; Feizi, E.; Bjo
429–430.
128. Oreste, P.; Zoppetti, G. Ref. 7, 2012; pp 403–422.
129. Lindahl, U.; Li, J.-P.; Kusche-Gullberg, M.; Salmivirta, M.; Alaranta, S.;
Veromaa, T.; Emeis, J.; Roberts, I.; Trevor, C.; Oreste, P.; Naggi, A.; Torri, G.;
Casu, B. J. Med. Chem. 2005, 48, 349–352.
130. Xu, Y.; Cai, C.; Chandarajoti, K.; Hsieh, P. H.; Li, L.; Pham, T. Q.; Sparkenbaugh,
J.; Sheng, J.; Key, N. S.; Pawlinski, R.; Harris, E. N.; Linhardt, R. J.; Liu, J. Nat.
Chem. Biol. 2014, 10, 248–250.
131. Guerrini, M.; Beccati, D.; Shriver, Z.; Naggi, A.; Viswanathan, K.; Bisio, A.;
Capila, I.; Lansing, J. C.; Guglieri, S.; Fraser, B.; Al-Akim, A.; Gunay, N. S.; Zhang,
Z.; Robinson, L.; Buhse, L.; Nasr, M.; Woodcock, I.; Langer, R.; Venkataraman,
G.; Linhardt, R. J.; Casu, B.; Torri, G.; Sasisekharan, R. Nat. Biotechnol. 2008, 26,
669–675.
132. Guerrini, M.; Zhang, Z.; Shriver, Z.; Naggi, A.; Masuko, S.; Langer, R.; Casu, B.;
Linhardt, R. J.; Torri, G.; Sasisekharan, R. Proc. Natl. Acad. Sci. U.S.A. 2009,
10616956–10616961.
133. Korir, A.; Larive, C. K. Anal. Bioanal. Chem. 2009, 393, 155–169.
134. Lima, M. A.; Rudd, T. R.; de Farias, E. H.; Ebner, L. F.; Gesteira, T. F.; de Souza, L.
M.; Mendes, A.; Cordula, C. R.; Martins, J. R.; Hoppensteadt, D.; Fareed, J.;
Sassaki, G. L.; Yates, E. A.; Tersarol, I. L.; Nader, H. B. PlosOne 2011, 6, e15970.
135. Shriver, Z.; Capila, I, Venkataraman, G.; Sasisekharan, R. Ref. 7, 2012; pp 159–
176.
136. Rudd, T. R.; Macchi, E.; Gardini, C.; Muzi, L.; Guerrini, M.; Yates, E. A.; Torri, G.
Anal. Chem. 2012, 84, 6841–6847.
137. Rudd, T. R.; Macchi, E.; Muzi, L.; Ferro, M.; Gaudesi, D.; Torri, G.; Casu, B.;
Guerrini, M.; Yates, E. A. Anal. Chem. 2013, 85, 7487–7493.
138. Ye, H.; Toby, T. K.; Sommers, C. D.; Ghastriani, H.; They, M. L.; Ye, W.; Kolinski,
R. R.; Buhse, L. F.; Al-Akim, A.; Keire, D. A. J. Pharm. Biomed. Anal. 2013, 85, 99–
107.
139. Weitz, D. S.; Weitz, J. I. J. Thromb. Thrombolysis 2010, 29, 199–207.
140. Lindahl, U.; Kjellén, L. J. Int. Med. 2013, 273, 555–571.
141. Lindahl, U. Matrix Biol. 2014, 36, 3–7.
142. Thacker, B. E.; Xu, D.; Lawrence, R.; Esko, J. D. Matrix Biol. 2013, 35, 60–72.