1
HEPARINS AND HEPARINOIDS: OCCURRENCE, STRUCTURE AND MECHANISM OF
ANTITHROMBOTIC AND HEMORRHAGIC ACTIVITIES
Helena B. Nader*, Carla C. Lopes, Hugo A.O. Rocha, Elizeu A. Santos and Carl P. Dietrich
Current Pharmaceutical Design vol 10 number 9, 951-966 (2004)
Abstract: The correlation between structure, anticloting, antithrombotic and
hemorrhagic activities of heparin, heparan sulfate, low molecular weight
heparins and heparin-like compounds from various sources that are in use in
clinical practice or under development is briefly reviewed. Heparin-like
molecules composed exclusively of iduronic acid 2-O-sulfate residues have
weak anticloting activities, whereas molecules that contain both iduronic
acid 2-O sulfate, iduronic acid and small amounts of glucuronic acid, such as
heparin, or mixed amounts of glucuronic and iduronic acids (mollusk
heparins) possess high anticloting and anti-Xa activities. These results also
suggest that a proper combination of these elements might produce a strong
antithrombotic agent.
Heparin isolated from shrimp mimics the
pharmacological activities of low molecular weight heparins. A heparan
sulfate derived from bovine pancreas and a sulfated fucan from brown algae
have a potent antithrombotic activity in arterial and venous thrombosis
model "in vivo" with a negligible activity upon the serine-proteases of the coagulation cascade "in vitro".
These and other results led to the hypothesis that antithrombotic activity of heparin and other
antithrombotic agents is due at least in part by their action on endothelial cells stimulating the synthesis
of an antithrombotic heparan sulfate. All the antithrombotic agents derived from heparin and other
heparinoids have hemorrhagic activity. Exceptions to this are a heparan sulfate from bovine pancreas and
a sulfated fucan derived from brown algae, which have no hemorrhagic activity but have high
antithrombotic activities "in vivo". Once the structure of these compounds are totally defined it will be
possible to design an ideal antithrombotic.
INTRODUCTION
The leading causes of death in the United
States are diseases that involve heart and blood
vessels, and as a consequence thrombosis [1].
The incidence of death for this disease in 1991
was almost two times higher than the next in line,
namely, cancer (Fig. 1). Possibly, with the
introduction of antithrombotic agents, particularly
heparin and its derivatives, death by heart
diseases have decreased substantially (about
30%) in 2000 when compared to malignant
cancer, which has increased in the last ten years.
Nevertheless, heart diseases are still the main
cause of death [1]. This explains the efforts to
discover and develop specific and more potent
antithrombotic agents.
The anticloting and antithrombotic activity of
heparin includes the blood itself, composed of
plasma proteins and lipids, and cells. The red
cells seem not to be a target for antithrombotic
agents, but on the other hand, white cells and
platelets are deeply involved in thrombus
formation.
The protease network in coagulation,
fibrinolysis and kallikrein-kinin system is
shown in Fig. 2. This cascade of events consists
of a series of activation of serine proteases and
modulation by specific inhibitors, called
serpins. The ultimate goal of the coagulation
system is the formation of clot that consists on
the limited proteolysis of a soluble protein from
plasma (fibrinogen) into an insoluble protein
(fibrin).
2
CANCER
1991-500,000
1999-553,000
2000-555,000
DISEASES OF THE
HEART
1991-975,000
1999-725,000
2000-700,000
CHRONIC
OBSTRUCTIVE
PULMONARY
DISORDER
1991-90,000
1999-124,000
2000-124,000
LEADING
CAUSES
OF MORTALITY
IN USA
OTHER CAUSES
400,000
TRAUMA/ACCIDENT
PULMONARY
90,000
DISEASE (FLU)
1991-80,000
1999-64,000
2000-62,000
Fig. (1). Causes of Death in USA.
Heparin acts as anticoagulant compound
because it forms a ternary complex with
antithrombin III and the different serine proteases
of the coagulation cascade. The inhibition of
thrombin by antithrombin is accelerated by more
than 1,000 times in the presence of heparin.
Heparin is also capable of potentiating the effect
of another serpin that is called heparin cofactor II
that is specific for thrombin. It also releases and
increases the synthesis of TFPI (tissue factor
pathway inhibitor) by endothelial cells.
Heparin was the first compound used as
anticoagulant and antithrombotic agent. Heparin
was isolated in 1916 by McLean in Canada from
a preparation of dog liver [2]. The commercial
heparin preparations, introduced in the clinical
use 60 years ago, are from hog and bovine
intestinal mucosa, as well as bovine lung.
Chemical and enzymatic analyses and NMR
spectroscopy have revealed the main structural
features of heparin. In our laboratory we have
shown that bovine lung heparin is mainly (8090%) composed of hexasaccharide repeats with
two disaccharides of αD-glucosamine, N,6disulfate-αL-iduronic acid,2-O-sulfate, and one
disaccharide of αD-glucosamine 2-6-disulfateβD-glucuronic or αL-iduronic acid as shown in
Fig. 3 [3]. Large variations occur among
heparins isolated from invertebrates and of
heparins isolated from different tissues and
species of vertebrates (See below).
OCCURRENCE
OF
HEPARIN
IN
VERTEBRATES AND INVERTEBRATES
Whereas heparan sulfates are ubiquitous
components of all tissue-organized animal life
forms [4-6] heparin, has shown a very peculiar
distribution in mammalian and other vertebrate
tissues as well as invertebrates. Since the earlier
studies, lung, intestine, and liver were the
tissues rich in heparin from a variety of
mammals. [5-8]. Table 1 shows a systematic
study involving nine mammalian species. These
analyses have shown that except for rabbit
tissues, heparin was present in lung, skin, ileum,
lymph nodes, thymus, and appendix of all
species. The absolute content of heparin varied
in the different tissues.
The lack of heparin in
3
INTRINSIC
PATHWAY
HMWK
XII
XIIa
EXTRINSIC
PATHWAY
HMWK
XI
III
XIa
(Tissue factor)
Phospholipid
Ca ++
VII
IX
Phospholipid
Ca++
VIIa
Antithombin III
IXa
VIIIa
Phospholipid/ Ca++
Protein Ca
X
+ Protein S
X
Xa
Protein C
Va
TFPI
Phospholipid/ Ca++
+ Thrombomodulin
Prothrombin
II
Thrombin
IIa
Heparin Cofactor II
tPA
Fibrinogen
Fibrin
Plasmin
XIIIa
Polymerization
(Clot)
Plasminogen
Depolymerization
(Fibrinolysis)
Fig. (2). Coagulation cascade.
rabbits was correlated with the absence of mast
cells in the species [8]. The amounts of the
heparins isolated from mammalian and other
vertebrate's tissues are also shown in Table 1. A
large variation on the concentration of heparin
among species is evident. Thus, bovine and dog
tissues contain the highest amounts of heparin. In
non-mammal vertebrates the amounts of heparin
is considerably less. An interesting characteristic
is that heparin is mainly distributed in tissues in
contact with the environment or in organs that
function as internal barriers against infection
and foreign bodies.
The anticoagulant activity and molecular
weight of some mammalian heparins is shown
in Table 2. It is interesting that the anticoagulant
activity varied from 60 up to 200 I.U./mg.
Likewise the molecular weight of the heparins
depending on the tissue of origin also shows a
large variation (11 kDa up to >150 KDa).
4
CH 2OSO3H
O
O
COOH
O
COOH
o
OH
OH
OSO3H
CH2OSO3H
O
O
O
OH
NSO3H
o
OH
COOH
O
O
OH
O
OH
OH
NSO3H
OSO3H
CH2OSO3H
O
COOH
O----R 1
NSO3H
n
Fig. (3). Basic hexasaccharide unit of heparin.
Table 1. Distribution of heparin in mammalian and other vertebrates
TISSUE
(µg/g dry
tissue)
Lung
Liver
Ileum
Kidney
Aorta
Brain
Muscle
Spleen
Skin
Lymph n.
Thymus
Appendix
Branchia
VERTEBRATE SPECIES
Rb
<1
GP
70
Rat
67
Dog
217
Cat
63
Pig
211
Bov
300
H
8
Ck
0.5
Sk
0.3
Lizd
0
Frg
0.64
Fish
0.03
Shrk
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
<1
27
4
<1
<1
<1
<1
<1
11
112
<1
1
<1
9
<1
36
<1
175
5
20
141
400
2
102
<1
9
11
15
160
20
17
1
87
6
<1
<1
<1
<1
63
74
<1
113
<1
2
<1
5
<1
2
242
10
50
1015
26
150
<1
2
19
108
180
286
20
<1
32
<1
<1
<1
<1
<1
39
41
35
47
0
0.5
0.1
0
0.9
0
0.02
0.23
0
0.77
0
0.46
1.32
0
0
0
0.29
0
0
0
0.4
0
0
0
0
0
11.4
0
0
0
0
0
0
0
0
11.9
0
0.03
0
38
Rb, rabbit, GP, guinea pig; Bov, bovine; H, human; Ck, chicken; Sk, snake; Lizd, lizard; Frg, frog; Shrk,
shark
As shown in Table 2 there was no correlation
between molecular weight and anticoagulant
activity of the heparins. All these results imply
that heparins have a large structural variation
depending of their origin. In invertebrates heparin
is found in few species, namely, mollusks
(eulamelibranchia), crustacean, annelid, tunicate
and possibly urochordate [4, 9, 10-12] (Table 3)
Again the anticoagulant activity and molecular
weight varied according to the species analyzed.
HEPARIN AND MAST CELLS
Since the discovery of mast cells by Paul
Ehrlich [13] and that the metachromatic activity
of these cells with basic dyes was related to
heparin [14,15], the question whether this
compound was only confined to the mast cells or
was also present in other cell structures has been
a matter of controversy [16]. The peculiar
distribution of heparin between fetal and adult
tissues raised again the question of whether
these heparins were related to the presence of
mast cells. Studies on the concentration of
heparin and content of mast cells in different
fetal and adult bovine tissues [17] have shown
that a good correlation between the mast cell
number and heparin concentration could be
obtained in all tissues analyzed. A few
differences could be observed among the mast
cell from the different tissues. The mast cells in
adult ileum were usually larger and contained
more granules when compared with the mast
cells from fetuses. In fetal spleen, the mast cells
were distributed homogeneously in the whole
organ whereas in adult spleen the mast cells
were only found in the capsule.
5
Table 2. Distribution of heparin in invertebrates
CLASS
Especies
Average
M.W.
(KDa)
Anticoagulant
Activity
(USP)
MOLLUSCS
Ciprinia islandica
95
Mactrus Pussula
100
Mercenaria mercenaria
18
348*
Anomalocardia brasiliana
32
320
Donax striatus
20
220
Tivela mactroides
25
180
CRUSTACEA
Ucides chordatus
60
Dedrocephalus brasiliensis
10
52
Penaeus brasiliensis
9
60
ANNELIDA
Aphrodite longicornis
Hermodice carunculata
ECHINODERMA
Mellita quinquisperforata
12
50
CNIDARIA
Physalia sp.
Mnemiopsis sp
Sipuncula nudus
*Antifactor IIa.
that this strain show normal or even higher
Several papers reported that mast cells could be
number of mast cells [25, 26] which then favors
derived from cells of thymus and lymph nodes
the second hypothesis.
and from the hematopoietic spleen tissue [18-21],
It is now established that mast cells
others inferred that mast cells could develop
originate from haematopoyetic stem cells [27,
locally from other cell types of the connective
28], through the studies of mice of the
tissue [22]. If masts cells were derived from
thymus then heparin should be absent from
genotypes W/Wv and S/S1d that are deficient in
athymic (nude) mice if the correlation
mast cells. Heparin is present in appreciable
heparin/mast cells is valid. Analyses on the
amounts in the skin of the breeders and of the
concentration of heparin in different tissues of
normal progeny. On the other hand, no heparin
athymic and normal mice has shown that heparin
was detected in the skin of the W/Wv genotype,
is present in the skin, lung, thymus and muscle of
which are deficient in mast cells [23]. No
both lines [23]. These results would indicate that
significant differences in the relative amounts of
heparin in the athymic mice was not present in
the other sulfated glycosaminoglycans, namely
mast cells, or else, these mice, although athymic
heparan sulfate, dermatan sulfate and
show the presence of mast cells. Other studies on
the mast cell content of athymic mice have shown
Table 3. Molecular weight and anticoagulant activity of heparins
from various tissues and species
6
Mammal
TISSUE
Dog
Lung
Ileum
Liver
Spleen
Lymph node
Lung
Ileum
Liver
Spleen
Lymph node
Aorta
Skin
Kidney
Lung
Ileum
Lymph node
Lung
Skin
Muscle
Lung
Ileum
Skin
Lymph node
Appendix
Lung
Lung
Ileum
Skin
Lymph node
Thymus
Appendix
Bovine
Pig
Rat
Cat
Hamster
Man
AVERAG
E M.W.
KDa
10.7
34
34
48
51
13
20
16
15
13
25
21
12
37
23
20
54
>150
>150
68
21
54
74
74
30
45
42
37
45
47
chondroitin sulfate were observed among the
breeders and four different littermates analyzed
[23]. This suggests that heparin is not replaced by
other sulfated glycosaminoglycan in the animals
that lack heparin. These results clearly indicate
that heparin is related to the presence of mast
cells.
The presence of heparin in mast cells of the
peritoneal cavity of rats [29-31] as well as in
mastocytomas [32, 33] has been extensively
documented. These cells and tumors have been
used for the studies of heparin biosynthesis by
different authors [34, 35]. Analyses of heparin
Anticogulant
Activity
(USP)
171
174
168
165
136
100
189
170
151
143
140
169
175
174
130
178
132
120
127
126
159
120
60
121
110
126
and other sulfated glycosaminoglycans in
several types of mastocytomas (cells in culture,
solid and ascytes tumors) have shown that
besides heparin, dermatan sulfate, chondroitin
sulfate and heparan sulfate were also present in
high amounts. In some instances, heparin could
not be detected in the tumors [36] or in mast
cells present in rat mucosa [37]. From these
combined data, we can conclude that heparin is
only present in mast cells but mast cells might
not contain heparin.
Heparin
and
other
sulfated
glycosaminoglycans as well as histamine were
7
found in various organs of Anomalocardia
brasiliana, a mollusk from the South Atlantic,
and quantified. The heparin was present in
granules in the cytoplasm [37-40]. A good
correlation between heparin and histamine
content was found in the labial palp, intestine,
ctenide, mantle, and foot tissues. The tissue
location of metachromatic cells, putatively
containing heparin, was identified histologically
by alcian blue, toluidine blue, Masson trichrome,
hematoxylin eosin and heparinase degradation
(Fig. 4A). Except for the foot, cells containing
metachromatic granules were found in the
epithelium surfaces of all the organs analyzed. In
ctenidium the basophilic cells are primarily
located around the acquiferum tubes and in minor
amounts in the epithelium of the filaments. The
basophilic cells are concentrated in the surface of
the internal side and the median folder of the
mantle (P). In these areas, these cells constitute
the only type of cells of the epithelium
independently of the stain used. The epithelium
facing the shell is devoid of metachromatic cells.
The metachromatic cells are extremely abundant
in the labial palp and they are uniformly
distributed in the ciliated epithelium of the
selection face of the crests. The basophilic cells
are intercalated in the intestinal mucosal
epithelium (Int.). These cells are rich of
metachromatic granules as shown in higher
magnification and toluidine blue staining.
An "in situ" identification of heparin using
nitrous acid and heparinase degradation has
established unequivocally the presence of this
compound in the metachromatic cells (Fig. 4B).
The location of mast cells at the epithelium
surface of mollusk tissues exposed to the
environment are very similar to the distribution of
mammalian and other vertebrate mast cells and
gives support to the suggestion for a role of
mast cells and its heparin, in the defense of the
organisms against virus, bacteria and foreign
bodies. Similar results were observed in
Tunicate [10].
Some conclusions can be made from these
studies: 1) heparin seems to be present
exclusively in mast cells or mast-like cells in the
case vertebrates and mollusks; 2) Its primary
biological activity is not related with
antithrombotic activity since mollusks which do
not possess a coagulation system contains
heparin and rabbits that contain this system is
devoid of heparin; 3) many indirect evidences
suggest that heparin and its mast cell is involved
in defense mechanisms independently of the
immune system [38].
STRUCTURE OF HEPARINS
As could be inferred from the studies
described above regarding the differences in
molecular weight and anticoagulant activity, the
heparins are a class of molecules with a great
structural variability. As a classical example, the
commercial heparins from bovine lung and
bovine mucosa differ in the amounts and
content of their constituent disaccharides [3, 4143]. A systematic study on the structure of some
mammalian and invertebrate heparins has
shown that all the heparins contain two different
basic regions that vary according to the tissue
and species of origin (Fig. 5). These regions
vary among each other by the degree of
susceptibility to Flavobacterium heparinum
heparinase and heparitinase II [3, 43, 45]. Other
major variation is the glucuronic/iduronic acid
content.
Mollusk
8
Fig. (4). Histological and histochemical analyzes of mollusk tissues. A: Ct., Ctenide: the arrow
indicates the basophilic cells; F, ctenide filaments; AT, acquiferum tube. Mt., mantle: the arrow indicates
the medium fold of the mantle; P, periostrach; M, muscular fibers; E 1, epithelium of the internal cavity
of the mantle; E 2, epithelium facing the shell. L.P., labial palp. Int., intestine: IL, intestinal lumen; E,
epithelium; S.F., selection face; D.S., dorsal face; Ct. E., ctenide extremity. Ft, foot: E, epithelium; V,
vilosities; the arrow shows the pedal gland.
9
Fig. (5). Structural variability of heparin from different tissues and species.
CH2OSO3H
O
OH
OH
CH2OSO3H
O
O
COOH
O
O
OH
OH
OSO3H
NSO3H
O
OH
OH
OH
NSO3H
CH2OSO3H
O
O
OH
OH
CH2OSO3H
O
O
COOH
O
O
OH
OH
OH
NSO3H
O
NSO3H
0
n1
OSO3H
OH
OH
NSO3H
O
O
OH
O
OH
OH
OSO3H
O
O
COOH
OH
CH2OSO3H
0
O
OH
OH
NSO3H
OH
NSO3H
O
OH
OH
CH2OSO3H
O
O
0
OSO3H
OH
CH2OSO3H
O
O
+
0
OH
OH
OH
OH
NSO3H
Heparitinase II
COOH
OH
NSO3H
O
OH
NSO3H
Heparitinase II
COOH
OH
OH
NSO3H
Heparinase
CH2OSO3H
O
COOH
+
O
OSO3H
O
CH2OH
O
COOH
O
CH2OSO3H
O
COOH
O
CH 2OSO3H
O
O
COOH
OH
Heparinase
COOH
+
O
OSO3H
NSO3H
Heparinase
CH 2OSO3H
O
CH2OSO3H
O
O
COOH
OH
OH
+
OH
NSO3H
CH 2OH
O
COOH
O
0
OH
O
OH
0
OSO3H
OH
O
+
OH
NSO3H
CH2OSO3H
O
COOH
0
OH
OH
OH
heparin contains 64% of glucuronic acid and 36%
of iduronic acid whereas lung heparin contains
mostly iduronic acid (>90%) as shown in Fig. 6
[39]. Chondroitin sulfate and dermatan sulfate
used as controls of the experiment shows
glucuronic acid and iduronic acid respectively as
major constituents.
Fig.
(6). Uronic acid content of heparins.The heparins
were subjected to acid hydrolysis and
subsequently to electrophoresis at pH 2.8.
Heparan sulfate, dermatan sulfate and chondroitin
sulfate were used as controls.
A recent study of the anomeric regions of the
C NMR spectra of the shrimp heparin, bovine
lung and bovine mucosa heparin also confirm
these observations (Fig. 7). Thus, the three
13
OH
NSO3H
OH
OH
Heparitinase II
CH 2OSO3H
O
COOH
Bovine lung
Bovine Intestine
Anomalocardia brasiliana
Donnax striatus
MOLLUSCS
Tivela mactroides
n2
Heparitinase II
˜
˜
˜
˜
˜
n1
6
6
8
4
3
OH
NSO3H
n2
1
˜4
˜8
˜5
˜
˜5
heparins contain identical signals of high
intensity attributed to the anomeric carbons of
the N,6-sulfated hexosamine (98.2 ppm) and 2O sulfated iduronic acid residues (46). Besides
these, the shrimp heparin also contains
prominent signals at 98.4, 98.5 and 99.1 ppm
related to the anomeric carbons of hexosamine
and at 103.3 and 103.9 attributable to non
sulfated α-L-iduronic and β-D-glucuronic acid
residues, respectively. All these signals are also
present in bovine mucosa heparin but with
lower intensity and insinuated in bovine lung
heparin but with still lower intensity when
compared to the mucosa and shrimp heparins.
Thus, this show that shrimp heparin also
contains higher amounts of glucuronic acid
residues in the structure when compared with
mammalian heparins [46].
Comparing these results to those of the
proposed structure of the heparins from various
sources we could conclude that the main
differences between the heparins are the relative
proportions of glucuronic/iduronic acid and 2O-sulfated iduronic acid residues as shown in
Figs. 5 and 6. Other residues are also present in
minor proportions in heparin such as acetyl
groups and 3-O-sulfated residues of the
hexosamine moieties. This last residue that
occurs in one third
of the heparin molecules is responsible for
the binding of heparin to antithrombin [47-50].
10
BOVINE LUNG
BOVINE MUCOSA
SHRIMP
ppm
Fig. (7). 13C NMR of the heparin from shrimp Penaeus brasiliensis compared to heparin from
bovine lung and mucosa heparins.
CORRELATION BETWEEN STRUCTURE
AND ANTITHROMBOTIC ACTIVITIES
11
the coagulation cascade. A complication factor
for this hypothesis is the finding that each
heparin is indeed a population of molecules with
different molecular weights. This is exemplified
by the electrophocusing technique where
heparin is fractionated in at least 21 components
(Fig. 8A).
If we observe the coagulation cascade and
its multiple enzymatic systems and co-factors
(Fig. 2) and the structural diversity of the
heparins one could suggest that ideally a
specific heparin structure would act on a
specific step of
A
B
A
B
M.W.
KDa
3.0
3.2
3.4
4.3
4.6
4.8
6.0
7.3
9 .5
1 0.5
11.5
1 4.0
1 6.0
1 8.0
1 9.5
2 1.0
2 3.5
2 5.0
2 9.5
3 0.0
3 7.5
1
2 3
4 5
1
2 3
4 5
I.U ./mg
U SP
M.W.
KDa
"In vivo"
antithrombotic
activity (U /mg)
10-20
3.0
3.2
95
20-60
3.4
4.3
4.6
4.8
6.0
7.3
130
70-150
140
200-300
130
9.5
10.5
11.5
14.0
16.0
18.0
19.5
21.0
23.5
25.0
29.5
30.0
37.5
Origin
Ori gin
LMW-HEPARIN
HEPARIN
Fig. (8). Anticoagulant , antithrombotic activities of heparin fractions obtained by electrophocusing. A,
Electrophocusing of Heparin. B, Low Molecular Weight Heparin prepared by the Fenton reaction. USP,
United States Pharmacopea anticoagulant assay; LMW, low molecular weight; 1 to 5, Heparins from
different pharmaceutical companies.
The components have different anticloting
activities but nevertheless about the same
antithrombotic activity "in vivo" [51-53]. These
results were the basis for producing LMWheparins by depolymerization of the parent
molecule by the Fenton reaction as shown in
Fig. 8B [53]. In spite of low anticoagulant
activity the LMW-heparins they exhibited the
same "in vivo" antithrombotic activity as well as
anti-Xa activity (Table 4).
Table 4. "In vitro" anticoagulant activities and "in vivo" antithrombotic activities of
heparins, LMW-heparins and bovine pancreas heparan sulfate.
Sample
MW
KDa
USP
"In vitro" (U/mg)
APTT
Anti-Xa
"In vivo"
Antitrombotic
12
Heparin
Intestine
Lung
pancreas
LMW-heparin
oxidation
Molec. sieving
heparitinase II
Heparan sulfate
bovine pancreas
Heparan sulfate
bovine lung
Chrom.
Y.W.
activity (U/mg)
15,1
9.8
9.8
140
130
140
135
74
154
106
90
129
69
60
68
176
96
84
4.5
4.6
5.8
25.5
48
49
45
<5
37
31
22
<5
87
100
78
<5
162
187
265
<5
119
161
146
126
15
<5
<5
<5
<5
20
APTT, activated partial thromboplastin time; Chr, chromogenic assay; Y.W., Yin and
Wessler assay
standard heparin and LMW-heparin bind to
These pioneer results led the pharmaceutical
antithrombin, which has a binding site for
industries in the 80´s to search for different
thrombin and another for factor Xa. While
methods of heparin depolymerization besides the
standard heparin can bind to both sites, LMWFenton reaction (Ardeparin and Parnaparin) such
heparin can interact only with the site for factor
as nitrous acid degradation (Nadroparin and
Xa, which explains its low anti-IIa activity and
Dalteparin), esterification and beta-elimination
high anti-Xa activity "in vitro."
(Enoxaparin) and heparinase degradation
Other structural characteristics of the
(Tinzaparin). The method of preparation produces
heparins contribute to the anticloting and
structural differences between the commercial
antithrombotic activity. Thus, a heparin isolated
LMW-heparins [54] and, consequently, the FDA
from the shrimp Penaeus brasiliensis with a
considers them different drugs. These LMWmolecular weight of 10 KDa has a low
heparins have largely replaced the conventional
anticloting activity and a potent anti-Xa activity
heparin as judged by the sales in 2001 of the two
"in vitro" (Table 5) and antithrombotic activity
types of heparin, around 2.0 billion and 300
"in vivo" similarly to the LMW-heparins [46].
million dollars, respectively.
As shown before its structure differs
Due to the parallelism of high "in vivo"
substantially from heparin and LMW-heparins
antithrombotic activity and high anti-Xa activity
regarding the type of uronic acid and degree of
it is believed that the pharmacological activity of
sulfation.
The
mollusk
Anomalocardia
the LMW-heparins as antithrombotics are due to
brasiliana, which also contains large amounts
the inhibition of factor Xa. Indeed, some of the
of glucuronic residues, has a very high
commercial LMW-heparins are sold by the
anticloting activity by the USP assay (320
potency of their anti-Xa units. The
IU/mg) and an anti-Xa activity similar to normal
biodisponibility is also measured by this activity.
heparins [37,55,56].
Regarding the structural differences for activity
of the two cofactors, namely aXA and aIIa, both
Table 5. Effect of LMW-heparin and shrimp heparin in the
induction of thrombosis by laser shots.
Agent
Dosage
mg/kg
Injection
route
Minutes after
injection
5
15
13
Saline
LMW-heparin
LMW-heparin
Shrimp heparin
LMW-heparin
Shrimp heparin
Shrimp heparin
1.0
2.5
1.0
0.5
0.25
0.5
SC
SC
SC
SC
IV
IV
IV
Number of laser
shots
2/3
3
7
5
5
3
5/7
6/7
10
7
6/5
4/5
6/6
6/5
SC, subcutaneous; IV, intravenous
Structural studies of the region of heparin
responsible for the binding site of antithrombin
led to the synthesis of a pentasaccharide
GlcNR(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)IdoA(2-OSO3)-GlcNSO3(6-OSO3) (where R
represents either a sulfate or an acetyl group and OSO3 represents an O-sulfate/ester sulfate group,
with locations of O-sulfate groups indicated in
parentheses) with high affinity for antithrombin
[50,57]. This first chemically defined compound
with anti-Xa activity is now in clinical trials [58].
Several attempts to modify preexisting
glycosaminoglycans for increase of anticloting
activity are being performed. As an example, Osulfation
of
sulfaminoheparosan,
a
glycosaminoglucuronan with the structure→4)-βD-GlcA(1→4)- β−D-GlcNSO(3)(-)-(1→,
obtained by N-deacetylation and N-sulfation of
the capsular polysaccharide from E. coli K5 has
an increased anti-Xa activity. Some of the
products contained the trisulfated aminosugar
GlcNSO(3)(-)3,6SO(3)(-), which is a marker
component of the pentasaccharide sequence
through which heparin binds to antithrombin
[59,60]. Depending on the reaction conditions,
the products showed different proportions of
components with high affinity for antithrombin.
A high-affinity subtraction, with 36 KDa, was
shown to cause conformational changes in the
molecule very similar to those induced by highaffinity heparin. The anti-Xa activity was 170
units/mg, similar to that of the third international
heparin standard and markedly higher than
activities of previously described heparin
analogues. Another preparation, of 13 KDa,
exhibited an anti-Xa activity of 70 units/mg.
These findings suggest that the modified bacterial
polysaccharide interacts with antithrombin and
promotes its anticoagulant action in a manner
similar to that of heparin. Similar to the mollusk
heparin these molecules contain only glucuronic
acid in their structures suggesting that iduronic
acid may play a minor role for the anti-Xa
activity. Supporting this suggestion, another
heparin-like compound, a polysaccharide
prepared from the giant African snail Achatina
fulica, which has a repeating disaccharide
structure of →4)-2-deoxy-2-acetamido-α-Dglucopyranose (1→ pentasaccharide→4)-2sulfo-α-L-idopyranosyl- uronic acid (1→ was
chemically modified and tested for its
pharmacological activity [61].
After Ndeacetylation, acharan sulfate was N-sulfonated
using either chlorosulfonic acid-pyridine or
sulfur trioxide-trimethylamine complex. The
sulfate level in these products ranged from 22 to
24%(w/w), significantly less than that of
heparin (36%, w/w) whereas the molecular
weight of both N-sulfoacharan sulfates were
comparable with that of heparin. "In vitro"
anticoagulant activity showed that Nsulfoacharan
sulfate
derivatives
were
moderately active for the inhibition of thrombin
and neither product showed any measurable
anti-factor Xa activity. The differences in the
activities of N-sulfoacharan sulfates produced
by these two methods are probably ascribable to
a small level of concomitant O-sulfonation
obtained when using chlorosulfonic acidpyridine.
Analyzing all this data, we can conclude
that glycosaminoglycans with high iduronic
residues are not crucial for the anticoagulant
activity of the compounds. Molecules composed
exclusively of iduronic acid 2-O-sulfate have
weak activities, whereas molecules that contain
both iduronic acid 2-O sulfate, iduronic acid and
small amounts of glucuronic acid, such as
14
heparin, or mixed amounts of glucuronic and
iduronic acids (mollusk heparin) possess high
anticloting and anti-Xa activity. These results
also suggest that a proper combination of these
elements might furnish the ideal antithrombotic
agent.
Opposite to the antithrombotics described
above, a heparan sulfate (molecules that contain a
small region similar to heparin) derived from
pancreas with negligible anti-IIa and anti-Xa
activity (< 5 I.U./mg) is a potent antithrombotic
"in vivo" (Table 4) measured by a variety of
methods including vena cavae ligature [62,63].
Furthermore, it was shown that this heparan
sulfate has also a potent inhibitory effect of
arterial thrombosis. The bovine lung heparan
sulfate has a low "in vivo" antithrombotic activity
in both venous (Table 4) and arterial vessels [64].
This variation of activity of heparan sulfates is
due to the findings that the structure of these
compounds varies according to tissue and species
of origin [64,65]. Another example of
antithrombotic compounds "in vivo" without "in
vitro" activities is a sulfated fucan isolated from
the brown algae Spatoglossum schroderi [Hugo
A. O. Rocha, personal communication]. These
last results cast some doubts that the "in vivo"
antithrombotic activity of heparin, LMWheparins and other heparinoids is mainly related
with the inhibition of Factor -Xa.
EFFECT OF HEPARIN AND OTHER
ANTITHROMBOTIC COMPOUNDS ON
VASCULAR ENDOTHELIAL CELLS
We have so far discussed the possible site of
action of these drugs in the protease network of
coagulation. As previously mentioned, the vessel
wall is another site of action for antithrombotic
compounds.
In the eighties Colburn and
Buonassisi [66] have shown that an endothelial
cell line in culture shows blood compatibility,
that is, their surface does not promote clotting.
Thus, when the surface of endothelial cells
changes, there is a chance of thrombus formation.
One of the compounds present at the cell surface
and extracellular matrix of endothelial cells is a
heparan sulfate proteoglycan. This heparan
sulfate, which has been totally sequenced [67],
has shown some heparin sequences and possesses
antithrombotic activity in different models [66].
This activity was absent in the heparan sulfate
from the adjacent smooth muscle cells of
arterial vessel [66].
When heparin is given to a patient, the
endothelium is possibly one of the sites of
action for the compound. Endothelial cells from
rabbit aorta [68] and human umbilical cord
exposed to heparin increase the synthesis of the
antithrombotic heparan sulfate present at the
cell surface, as well as the one released to the
medium [69]. As shown in Fig. 9, this effect is
also elicited by LMW-heparins [70,71]. Heparin
was fragmented with heparinase from
Flavobacterium heparinum [45]. The different
fragments were also tested as elicitors of the
synthesis of endothelial heparan sulfate [70]. It
was shown that the minimum structural
requirement to produce the enhancement in the
synthesis of the antithrombotic heparan sulfate
is a pentasulfated tetrasaccharide (Fig. 9). Ndesulfation of heparin completely abolishes the
stimulatory activity. Other sulfated compounds,
such as lactobionic acids, that consist of sulfated
lactose linked by a sequence from 3 to 12
carbons, a cyclic octaphenol-octasulfonic acid
(compound Y), dextran sulfate, oversulfated
chondroitin sulfates, a fucan from brown
seaweed [70-72] and other compounds that
possess antithrombotic activity also increase the
synthesis of the endothelial antithrombotic
heparan sulfate (Fig. 9).
The increased synthesis of heparan sulfate
chains is observed when the cells are exposed to
heparin and other structurally unrelated
antithrombotic agents. This lead to the
hypothesis that the antithrombotic activity of
these compounds "in vivo" could be related, at
least in part, to the increased production of this
peculiar heparan sulfate by endothelial cells. In
favor of this hypothesis are the findings that the
heparin-tetrasaccharide and compound Y which
are antithrombotic agents "in vivo" exhibit a
negligible activity "in vitro" upon the serineproteases of the coagulation cascade as
previously observed for bovine pancreas
heparan sulfate.
A protein of 47 KDa that binds heparin and
other antithrombotic agents with high affinity
has been isolated from the proteins of the
endothelial cell surface (79). Fractionation of
15
the surface proteins by heparin-affinity
chromatography reveals the enrichment of two
major protein bands (47 and 28 KDa) that are
present in very small amounts in the crude cell
surface extracts (Fig 10A). The binding of the
proteins with heparin and GL522 in the presence
of 0.5M NaCl reveals that besides the 47 and 28
KDa several proteins bind with [125I]-heparin and
[14C]-GL522 whereas only a 47KDa protein bind
with heparin in the presence of 1M NaCl (Fig.
10B). Likewise, the 47 KDa is the only protein
that binds [14C]-GL522 in the presence of 1 M
NaCl (Fig. 10C).
MECHANISM OF THE HEMORRHAGIC
ACTIVITY OF HEPARIN AND LMWHEPARINS
The main drawback in heparin and
heparinoids antithrombotic therapy is their
hemorrhagic activity. Thus, several reports have
shown that heparin, LMW-heparins and other
sulfated antithrombotics, except for bovine
pancreas heparan sulfate and a sulfated fucan
isolated from brown alga, produce bleeding in
some patients. The extent of bleeding of LMW-
heparins in animal experiments as well as in
humans is less pronounced or equal to heparin
[73-77]. These differences could be related to
the structural differences of the LMW-heparins
used [54, 78]. The neutralization of the bleeding
produced by LMW-heparins is still under study.
In two bleeding models in rats, protamine failed
to reverse the bleeding activity of these
heparins.
Cruz and coworkers in the earlier sixties
have shown that the antihemostatic effect of
heparin was independent of blood cells like
platelets and was related to special structures of
the damaged tissue [80]. Thus, when heparin
was applied topically to skin wounds it
produced enhanced bleeding from small vessels
and capillaries. [81, 82]. This antihemostatic
activity persisted even after extensive washing
of the preparation with saline solutions,
suggesting that heparin molecule binds to a
receptor of the wound resulting in the
uncontrollable hemorrhage. Among the several
proteins tested to counteract the inhibition of
hemostasis produced by heparin, namely,
plasma and serum proteins, tropomyosin and
actin, only non denaturated myosin was able to
LOW MOLECULAR WEIGHT
HEPARINS (100 µg/ml)
16
LMW-HEPASE
pK 10169
Op 386
CY 222
CY 216
HEPARIN
CONTROL
500
CS
HS
1000
1500
2000
GLYCOSAMINOGLYCANS
(cpm/µg cell protein)
2500
HEPARIN
N-DESULFATED HEPARIN
SULEPAROID
LW 10082
LW 10282
DEXTRAN SULFATE
GAGPS
MPS
LACTOBIONIC ACID
COMPOUND Y
CONTROL
0
HEPARIN
TETR ASACCHARIDES (100 µg/ml)
ANTITHROMBOTIC AGENTS (100 µg/ml)
0
1000
2000
3000
GLYCOSAMINOGLYCANS
(cp m/ µg cell p rot ein)
4000
NONE
HEPARIN
TETRA- 6S
TETRA- 5S
TETRA- 4S
0
1000
2000
3000
4000
GLYCOSAMINOGLYCANS
(cpm/ µg cell protein)
Fig. (9). Stimulation of heparan sulfate synthesis in endothelial cells by different antithrombotic
agents. GAGPS, MPS, mixture of glycosaminoglycans from Organon; Op 386, CY 222, CY 216, LW
10082, LW 10282, LMW-heparins from different pharmaceutical industries; Suleparoid, oversulfated
chondroitin sulfate; Compound Y, cyclic octaphenolocta- sulfonic acid; Tetra, 4S, 5S, 6S, tetra-, penta- ,
hexa- sulfated tetrasaccharides obtained from heparin by heparinase degradation
17
reverse the inhibitory activity. Likewise, among
the nucleotide phosphates tested, such as UTP,
ITP, GTP, CTP, AMP, only ATP and ADP at
low concentrations (10-5 M) were able to
dislodge the residual heparin bound to the
receptor counteracting its inhibitory activity [81].
extensive washing with saline. This effect,
nevertheless, could be reversed by ATP and/or
myosin [81, 85]. Table 6 shows that the
bleeding produced by the LMW-heparins and
well as heparin are totally reversed by 10-4 M of
ATP. A significant reduction of bleeding
caused by heparin was also observed in patients
subjected to cardiopulmonary bypass surgery,
topically applying ATP in the thoracic cavity
(Fig. 11). Topical application of protamine in 8
patients failed to produce the reversion of
bleeding due to the findings that the molecular
weight of the circulating heparin was in the
order of 6KDa, that corresponds to a LMWheparin [86]. The putative binding site of
heparin and their fragments is a purinergic
receptor of the smooth muscle cells [87]. The
antithrombotics would cause vasodilatation by
competing with ATP or ADP for the receptor.
Table 6. reversion of the hemorrhagic
activity of heparinand LMW heparins by
ATP
Fig. (10). Isolation of a 47KDa protein with high
affinity for heparin at the surface of endothelial
cells. GL-522, octaphenoloctasulfonic acid,
compound Y.
"In vitro" experiments have shown that
heparin inhibits competitively the hydrolysis of
ATP by myosin ATPase [81, 83, 84]. These
combined results suggested that heparin was
binding to a myosin-like molecule of the smooth
muscular cells inhibiting the contractility of the
vessels and thus producing the increase of
bleeding. The minimum structural requirement of
the heparin molecule capable of producing
bleeding was a heparin-derived disaccharide with
a sulfate at the C-6 position of the hexosamine
residue [85].
REVERSION OF THE ANTIHEMOSTATIC
ACTIVITY BY ATP
Compound
Dosis
(µg/ml)
Heparin
Heparin
LMW-heparin1
LMW-heparin1
LMW-heparin2
LMW-heparin2
LMW-heparin3
LMW-heparin3
200
400
200
400
200
400
200
400
Bleeding
potency
Saline ATP x
10-4 M
5.3
0.7
10.3
1.0
2.5
0.3
4.2
0.4
2.0
0.2
3.8
0.3
2.3
0.1
4.1
0.5
1, enoxaparin; 2, dalteparin; 3, nadroparin.
IN
SEARCH
FOR
ANTITHROMBOTIC.
AN
IDEAL
Contrasting with cancer, a significant
reduction of death caused directly or indirectly
by thrombosis has been observed in the last ten
years [1]. This was probably due to the
As described above heparin binds to the
wounded tissue in such a way that its
antihemostatic effect persisted even after
Fig. (11). Effect of topical application of ATP or protamine upon the volume of blood oozed from
patients after cardiovascular surgery with extracorporeal circulation. Before closure, the thoracic cavity of
BLOOD VOLUME IN THE DRAINS (ml)
18
1000
p< 0.08
800
p< 0.005
p>0.1
600
400
200
0
CONTROLS
10 µM
50 µM
ATP
100 µM
0.1 mg/ml
PROTAMINE
the patients submitted to cardiovascular surgery, was washed with 500 ml of physiological solution
containing different amounts of ATP or protamine as indicated. The total blood volume oozed from the
patients were collected with two thoracic drains in flasks containing 200 ml of saline. The statistical
significance was measured by the Students t test.
introduction of LMW-heparins, which
popularized the use of antithrombotic therapy
with sulfated polysaccharides. Nevertheless,
these compounds have an unwanted effect,
which is the production of hemorrhage, which
could be serious in some patients. Thus, a
search for this class of compounds without
hemorrhagic activity is actively being
pursued. The heparan sulfate from bovine
pancreas and the sulfated fucan from brown
algae that are potent antithrombotic
compounds "in vivo" and devoid of
hemorrhagic activity suggest that a proper
structural modification of heparin, LMWheparins or other sulfated polysaccharides
without hemorrhagic activity is worth
pursuing. Alternatively, ATP or ATP
derivatives that bind to the smooth muscle
cells but are resistant to ATPases could be
used to displace heparin and its fragments
from the purinergic receptor. These
compounds would be used to decrease the
hemorrhage of patients caused by these
compounds.
BLOOD VOLUME IN THE DRAINS (ml)
19
1000
p< 0.08
800
p< 0.005
p>0.1
600
400
200
0
CONTROLS
10 µM
50 µM
100 µM
ATP
0.1 mg/ml
PROTAMINE
Fig. (11). Effect of topical application of ATP or protamine upon the volume of blood oozed from
patients after cardiovascular surgery with extracorporeal circulation. Before closure, the thoracic cavity of
the patients submitted to cardiovascular surgery, was washed with 500 ml of physiological solution
containing different amounts of ATP or protamine as indicated. The total blood volume oozed from the
patients were collected with two thoracic drains in flasks containing 200 ml of saline. The statistical
significance was measured by the Students t test.
introduction of LMW-heparins, which
popularized the use of antithrombotic
therapy with sulfated polysaccharides.
Nevertheless, these compounds have an
unwanted effect, which is the
production of hemorrhage, which could
be serious in some patients. Thus, a
search for this class of compounds
without hemorrhagic activity is
actively being pursued. The heparan
sulfate from bovine pancreas and the
sulfated fucan from brown algae that
are potent antithrombotic compounds
"in vivo" and devoid of hemorrhagic
activity suggest that a proper structural
modification of heparin, LMWheparins
or
other
sulfated
polysaccharides without hemorrhagic
activity
is
worth
pursuing.
Alternatively, ATP or ATP derivatives
that bind to the smooth muscle cells
but are resistant to ATPases could be
used to displace heparin and its
fragments from the purinergic
receptor. These compounds would be
used to decrease the hemorrhage of
patients caused by these compounds.
REFERENCES
[1]
[2]
[3]
National Vital Statistics Reports
2003; 51:4-9
McLean J. The discovery of heparin.
Circulation 1959; 19:75-8
Silva ME,
Dietrich CP. The
structure of heparin Characterization
of the products formed from heparin
be the action of a heparinase and a
heparitinase from Flavobacterium
20
heparinum. J Biol Chem 1975; 250:
6841-6
[4] Cassaro CMF,
Dietrich CP.
Distribution
of
sulfated
mucopolysaccharides in invertebrates.
J Biol Chem 1977; 252: 2254-61
[5] Toledo OMS, Dietrich CP. Tissue
specific distribution of sulfated
mucopolysaccharides in mammals.
Biochim Biophys Acta 1977; 498 :
114-22
[6] Barlow GH, Coen LJ, Mozen MM. A
biological
chemical
physical
comparison of heparin from different
mammalian
species.
Biochim
Biophys Acta 1964; 83: 272-7
[7] Hashimoto K, Matsuno M, Yosizawa
Z, Shibata T . A dextrorotatory
polysaccharide
of
very
high
anticoagulant activity newly isolated
from the whale's lung
intestine.
Tohoku J Exp Med 1963; 81: 93-5
[8] Nader HB, Takahashi HK, Straus AH,
Dietrich CP. Selective distribution of
heparin in mammals: conspicuous
presence of heparin in lymphoid
tissues. Biochim Biophys Acta 1980;
627: 40-8
[9] Medeiros GF, Mendes A,
Castro
RAB, Baú EC, Nader HB, Dietrich
CP. (2000) Distribution of sulfated
glycosaminoglycans in the animal
kingdom: widespread occurrence of
heparin-like
compounds
in
invertebrates. Biochim Biophys Acta
2000; 1475: 287-294
[10] Cavalcante
MCM,
Allodi
S,
Valente AP, Straus AH, Takahashi
HK , Mourao PA S, et.al. Occurrence
of heparin in the invertebrate Styela
plicata (Tunicata) is restricted to cell
layers facing the outside environment An ancient role in defense? J Biol
Chem 2000; 275: 36189-96
[11] Burson
SL,
Fahrenbach
MJ,
Frommhagen LH,
Riccardi BA,
Brown RA, Brockman JA, et al.
Isolation
purification of mactins
heparin-like
anticoagulants
from
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
mollusca. J Amer Chem Soc 1956;
78: 5874-8
Rahemtulla F,
Løvtrup S. The
comparative
biochemistry
of
invertebrate mucopolysaccharides-V
Mollusca. Comp Biochem Physiol
B 1976; 53: 15-8
Ehlich P. Beiträge zur Kenntnis der
granulierten Bindegewebszellen und
der eosinophilen Leukocythen. Arch
Anat Physiol 1879: 166-9
Holmgren H,
Willander O.
Beitrag zur Kenntnis der Chemie und
Funktion
der
Ehrlichschen
Mastzellen. Z Mikrosk Anat Forsch
1937; 42: 242-78
Jorpes E, Holmgren H, Willander
O.
Ueber das Vorkommen von
Heparin in den Gefässwänden und in
den Augen
Ein Beitrag zur
Physiologie
der
Ehrlichschen
Mastzellen. Z Mikrosk Anat Forsch
1937; 42: 279-301
Jaques
LB
Debnath
AK.
Simultaneous evaluation of tissue
heparin mast cells in small tissue
samples. Am J Physiol 1970; 219:
1155-60
Nader HB, Straus AH, Takahashi
HK ,
Dietrich CP. Selective
appearance of heparin in mammalian
tissues during development. Biochim
Biophys Acta 1982; 714: 292-297
Csaba G, Toro I , Modo K.
(1962) The behaviour of the thymus
in conditions associated with tissue
proliferation. Acta Anatom 1962; 48
: 114-21
Ginsburg H, Lagunoff D. The "in
vitro" differentiation of mast cells. J
Cell Biol 1967; 48 :685-97
Ishizaka T, Okudaira H, Mauser
LE, Ishizaka K. Development of rat
mast cells "in vitro" I- Differentiation
of mast cells from thymus cells. J
Immunol 1976; 116: 747-754
Burnet
FM. The probable
relationship of some or all mast cells
to the T-cell system. Cell Immunol
1977; 30: 358-60
21
[22] Selye
H. The mast cells. 1965;
Butterworth Washington D C
[23] Straus AH, Nader HB,
Dietrich
CP. Absence of heparin or heparin like
compounds in mast cell-free tissues
animals. Biochim Biophys Acta 1982;
717: 478 -85
[24] Keller R, Hess, MW Riley, JF.
Mast cells in the skin of normal
hairless athymic mice. Experientia
1979; 32: 171-2
[25] Wlodarski K. Mast cells in the pinna
of
Balb/c
"nude"
(nu/nu)
heterozygotes (nu/+) mice. Experientia
1976; 32: 1591-2
[27] Kitamura Y, Go S, Hatanaka K.
Decrease of mast cells in W/Wv their
increase
by
bone
marrow
transplantation. Blood 1978; 52: 44752
[28] Kitamura Y
Go S. Decreased
production of mast cells in S1/S1d
anemic mice, Blood 1979; 53: 492-7
[29] Marshall JS, Kawabori S, Nielsen
L ,
Bienenstock
J.
(1994)
Morphological
functionalcharacteristics of peritoneal mast-cells
from young-rats. Cell Tissue Res
1994; 276: 565-570
[30] Pejler G, Berg L. Regulation of rat
mast-cell protease-1 activity - protease
inhibition is prevented by heparin
proteoglycan. Eur J Biochem 1995;
233: 192-9
[31] Amihai D, Trachtenberg S, Terkel
J , Hammel I. (2001) The structure of
mast cell secretory granules in the
blind mole rat (Spalax ehrenbergi). J
Struct Biol 2001; 136: 96-100
[32] Dunn
TB,
Potter
MJ. A
transplantable mast-cell neoplasm in
the mouse. J Natl Cancer Inst 1957;
18: 587-601
[33] Furth J, Hagen P,
Hirsch EI.
Transplantable mastocytoma in the
mouse containing histamine heparin
5-hydroxy- triptamine. Proc Soc
Exptl Biol Med 1957;95 :824-28
[34] Silbert JE. Incorporation of 35SO4
endogenous heparin by a microsomal
[35]
[36]
[37]
[37]
[38]
[39]
[40]
[41]
fraction from mast cell tumors. J
Biol Chem 1967; 242: 2301-5
Lindahl U. Enzymes involved in the
formation of the carbohydrate
structure of heparin. Meth Enzymol
1972; 28:676-84 Academic Press.
New York
Nader HB, Marx W, Spolter L.
Glycosaminoglycans of some mouse
mastocytomas. Biochim Biophys
Acta 1980; 631: 463-78
Dietrich CP, Paiva JF, Moraes
CT, Takahashi HK, Porcionatto
MA,
Nader
HB. Isolation
characterization of a heparin with
high anticoagulant activity from
Anomalocardia brasiliana. Biochim
Biophys Acta 1985; 843: 1-7
Enerbäck L, Kolset SO, Kusche
M, Hjerpe A, Lindahl U. (1985)
Glycosaminoglycans in ral mucosal
mast-cells. Biochem J 1985; 227:
661-8
Nader HB, Dietrich CP. Natural
occurrence possible biological role
of heparin in Heparin: Chemical
Biological Properties
Clinical
Applications (D A Lane U Lindahl
eds) Edward Arnold Publishers. 1989;
London pp81-96
Santos EA (1997) Heparina do
molusco Anomalocardia brasiliana:
A. Natureza dos resíduos de ácidos
urônicos. B. Distribuição em tecidos
e correlação com mastócitos. Tese de
Doutorado ; 1997. Escola Paulista de
Medicina São Paulo SP, Brazil
Santos EA , Menezes LR, Pereira
NML, Andrade GPV, Nader HB,
Dietrich CP. Mast cells are present
in epithelial layers of different tissues
of the mollusk Anomalocardia
brasiliana "In situ" characterization
of heparin a correlation of heparin
histamine concentration. Histochem
J; 2003 (in press)
Jaques
LB.
(1980) HeparinsAnionic
Polyelectrolyte
Drugs.
Pharmacol Rev 1980; 31: 99-166
22
[42] Casu B, Guerrini M, Naggi A,
Torri G, De-Ambrosi L, Boveri G,
et al. (1996) Characterization of
sulfation patterns of beef pig mucosal
heparins
by
nuclear
magnetic
resonance
spectroscopy.
Arzneimittelforschung 1996; 46: 472-7
[43] HB Nader, CP Dietrich. Extraction
characterization of heparins
In
Analytical techniques to evaluate the
structure
function of natural
polysaccharides glycosaminoglycans.
Ed Nicola Volpi. Research Sign Post
2002; 23-9
[44] Nader
HB,
Porcionatto
MA,
Moraes CT,
Dietrich CP. (1990)
Purification
substrate specificity of
heparinase heparitinase I heparitinase
II from Flavobacterium heparinum. J
Biol Chem 1990; 265: 6807-13
[45] Nader HB, Chavante SF, Santos
EA,
Oliveira FW,
Paiva JF,
Jerônimo SMB, et al. (1999) Heparan
sulfates heparins: similar compounds
performing the same functions in
vertebrates
invertebrates? Brazil J
Med Biol Res 1999; 32: 529-38
[46] Dietrich CP,
Paiva JF,
Castro
RAB,
Chavante SF,
Jeske W,
Fareed J, et al
Structural features
anticloting activities of a novel natural
low molecular weight heparin from the
shrimp Penaeus brasiliensis. Biochim
Biophys Acta 1999; 1428: 273-83
[47] Lindahl U, Backstrom G, Thunberg
L, Leder I G. Evidence for a 3-O
sulfated D-glucosamine residue in the
antithrombin-binding sequence of
heparin Proc Natl Acad Sci USA
1980; 77: 6551-5
[48] Olson ST, Bjork I, Sheffer R,
Craig PA, Shore JD, Choay J.
Role of the antithrombin-binding
pentasaccharide in heparin acceleration
of antithrombin-proteinase reactions resolution
of
the
antithrombin
conformational change contribution to
heparin rate enhancement. J
Biol
Chem 1992; 267: 12528-38
[49] Casu B,
Lindahl U. Structure
biological interactions of heparin
heparan sulfate.
Adv. Carbohyd
Chem Biochem 2001; 57: 159-206
[50] Choay J, Petitou M, Lormeau JC,
Sinay P, Casu B, Gatti G. Structureactivity relationship in heparin - a
synthetic pentasaccharide with highaffinity for anti-thrombin-iii eliciting
high anti-factor-xa activity. Biochem
Biophys Res Commun 1983; 116:
492-9
[51] Nader
HB,
McDuffie
NM,
Dietrich
CP.
(1974)
Heparin
fractionation by electrofocusing:
Presence of 21 components of
different
molecular
weights.
Biochem
Biophys Res Commun
1974; 57: 488-93
[52] McDuffie NM, Nader HB, Dietrich
CP. Electrophocusing of heparin
Fractionation of heparin into 21
components distinguishable from
other acidic mucopolysaccharides
Biopolymers 1975; 14: 1473-86
[53] Dietrich CP,
Nader HB, and
McDuffie
NM.
(1975)
Electrophocusing
of
heparin
Presence of 21 monomeric dimeric
molecular
species
in
heparin
preparations. An Acad Brasil Ci
1975; 47: 301-9
[54] Dietrich CP, Shinjo SK, Moraes FA ,
Castro RAB, Mendes A, Nader HB.
Structural features bleeding activity
of
commercial
LMW-heparins:
Neutralization by ATP protamine.
Sem Thromb Hemost 1999; 25: 4350
[55] Pejler G, Danielsson A , Bjork I,
Lindahl U, Dietrich CP, Nader HB
Structure
antithrombin-binding
properties of heparin isolated from
the clams Anomalocardia brasiliana
and Tivela mactroides. J Biol Chem
1987; 262: 1413-21
[56] Dietrich CP, Nader HB, Paiva JF,
Tersariol ILS. Santos EA. Holme
KR. et al. Heparin in molluscs:
Chemical
enzymatic degradation
23
[57]
[58]
[59]
[60]
[61]
[62]
[63]
[64]
13C 1H nmr spectroscopical evidences
for the maintainance of the structure
through evolution. Int J Biol Macromol
1989; 11: 361-366
Lindahl U, Thunberg L , Backstrom
G,
Riesenfeld J,
Nordling K,
Bjork
I. Extension
structural
variability of the antithrombin-binding
sequence in heparin. J Biol Chem
1984; 259: 12368-76
Walenga JM, Jeske WP, Samama
MM, Frapaise FX, Bick RL, Fareed
J. Fondaparinux: a synthetic heparin
pentasaccharide
as
a
new
antithrombotic agent.
Exp
Opin
Invest Drugs 2002; 11: 397-407
Casu B, Grazioli G, Razi N, Guerrini
M, Naggi A, Torri G, et al. Heparinlike compounds prepared by chemical
modification
of
capsular
polysaccharide from Escherichia-coli
K5. Carbohyd Res 1994; 263: 271-84
Naggi A, Torri G, Casu B, Oreste P,
Zoppetti G, Li JP, et al. Toward a
biotechnological
heparin
through
combined chemical
enzymatic
modification of the Escherichia coli K5
polysaccharide. Semin
Thromb
Hemost 2001; 27: 437-43
Wu SJ, Chun MW, Shin KH, Toida
T, Park Y, Linhardt RJ, et al.
Chemical sulfonation
anticoagulant
activity of acharan sulfate. Thromb
Res 1998; 92: 273-81
Bianchini P, Osima B, Parma B,
Nader HB, Dietrich CP. (1985) Lack
of correlation between "in vitro" "in
vivo" antithrombotic activity of heparin
fractions related compounds: Heparan
sulfate as an antithrombotic agent "in
vivo". Thromb Res 1985; 40: 597-607
Bianchini P, Osima B, Parma B,
Dietrich CP, Takahashi HK, Nader
HB Structural studies "in vivo" "in
vitro" pharmacological activities of
heparin fractions fragments prepared
by
chemical
enzymic
depolimerization. Thromb Res 1985;
40: 49-58
Dietrich CP, Nader HB and Straus
[65]
[66]
[67]
[68]
[69]
[70]
[71]
[72]
AH. Structural diferences of heparan
sulfates according to the tissue and
species of origin. Biochem Biophys
Res Commun 1983; 111:865 -71
Dietrich CP, Tersariol ILS, Toma L,
Moraes CT Porcionatto MA, Oliveira
FW and Nader HB. Sequencing of
heparan sulfate: Identification of
variable and constant oligosaccharide
regions in eight heparan sulfates from
different origins Cell Molecul Biol
1998; 44: 417-29
Colburn P, Buonassisi V. Anticlotting activity of endothelial cell
cultures
heparan
sulfate
proteoglycans. Biochem
Biophys
Res Commun 1982; 104: 220-227
Nader HB, Dietrich CP, Buonassisi
V, Colburn P. Heparin sequences in
the heparan sulfate chains of an
endothelial cell proteoglycan. Proc
Natl Acad Sci USA 1987; 84: 356569
Buonassisi V, Venter JC. Hormone
neurotransmitter receptors in an
established vascular endothelial cell
line.
Proc Natl Acad Sci USA
1976;73:1612-16
Nader HB, Buonassisi V, Colburn P,
Dietrich CP. Heparin stimulates the
synthesis
modifies the sulfation
pattern
of
heparan
sulfate
proteoglycan from endothelial cells. J
Cell Physiol 1989;140:305-310
Pinhal MAS, Santos IAN, Silva IF,
Dietrich CP, Nader HB. Stimulation
of the synthesis of an antithrombotic
heparan sulfate from endothelial cells
by heparin
its fragments.
Thrombosis Haemostasis 1995;74:
1169-1174
Pinhal MAS, Walenga JM, Jeske
W, Hoppensteadt D, Dietrich CP,
Fareed J, et al. Antithrombotic agents
stimulate the synthesis modify the
sulfation pattern of a heparan sulfate
proteoglycan from endothelial cells.
Thromb Res 1994;74:143-153
Nader HB, Pinhal MAS, Baú EC,
Castro
RAB,
Medeiros
GF,
24
[73]
[74]
[75]
[76]
[77]
[78]
[79]
Chavante SF, et al. Development of
new heparin-like compounds other
antithrombotic drugs and
their
interaction with vascular endothelial
cells. Brazil J Med Biol Res 2001;34:
699-709
Bagge L, Wahlberg T, Holmer E,
Tydén H, Nyström SO, Malm T.
Low-molecular-weight
heparin
(Fragmin)
versus
heparin
for
anticoagulation
during
cardiopulmonary bypass in open heart
surgery using a pig model. Blood
Coagul Fibrinol 1994;5:265-272
Colwell CW Jr.Recent advances in the
use of low molecular weight heparins
as prophylaxis for deep vein
thrombosis. Orthopedics 1994;17: 5-7
Haas S, Flosbach CW (1993)
Prevention
of
postoperative
thromboembolism with Enoxaparin in
general surgery: a German multicenter
trial. Sem Thromb Hemost 1993;19:
164-173
Hirsh J. Comparison of the relative
efficacy
safety of low molecular
weight heparin unfractionated heparin
for the treatment of venous thrombosis.
Haemostasis 1996; 26: 189-198
Martineau P, Tawil N. Low-molecularweight heparins in the treatment of
deep-vein
thrombosis.
Ann
Pharmacother 1998;32: 588-598
Fareed J, Jeske W, Hoppensteadt D,
Clarizio R, Walenga JM. Lowmolecular-weight
heparins:
pharmacologic profile
product
differentiation.
Amer J Cardiol
1998;82: 3L-10L
Pinhal, MAS, Trindade, E, Fareed, J,
Dietrich, CP and Nader, HB Heparin
and a cyclic octaphenol-octasulfonic
acid (GL-522-Y-1) bind with high
affinity to a 47KDa protein from
vascular endothelial cell surface and
stimulate the synthesis and structural
changes of an antithrombotic heparan
sulfate.Thromb Res 2001; 103: 35-45
[80] Cruz WO. The significance of a
smooth muscle component in
hemostasis. Proc Soc Exptl Biol Med
1965;119: 876-883
[81] Cruz
WO,
Dietrich
CP.
Antihemostatic effect of heparin
counteracted
by
adenosine
triphosphate. Proc Soc Exptl Biol
Med 1967;126: 420-426
[82] Nader HB Dietrich CP. Effect of
heparitin
sulfate
fractions
on
hemostasis Proc Soc Exptl Biol Med
1974;146: 504-508
[83] Nader HB, Tersariol ILS, Dietrich
CP. Antihemostatic activity of
heparin
disaccharides
oligosaccharides
obtained
by
chemical enzymatic fragmentation:
Reversal of the hemorrhagic activity
by ATP
myosin Thromb Res
1989;54: 207-214
[84] Tersariol ILS, Dietrich CP, Nader
HB. Uncoupling of actomyosin
ATPase by heparin
its fragments
Eur J Biochem 1997;245: 40-46
[85] Dietrich CP, Tersariol ILS, Silva RG,
Bianchini P, Nader HB. Dependence
of the C-6 sulfate of the glucosamine
moiety and 1-4 glycosidic linkage of
heparin disaccharides for production
of hemorrhage.
Reversal of the
antihemostatic activity of heparin
their fragments by ATP and myosin.
Sem Thromb Hemost 1991;17: 65-73
[86] Garcia-Jr HV, Buffolo E, Nader HB.
Dietrich CP (1994) Reduction of
blood loss produced by heparin after
topical application of adenosine
triphosphate in cardiopulmonary bypass operations. Ann Thorac Surg
1994;57: 956-959.
[87] Nader HB, Tersariol ILS and Dietrich
CP. Structural requirements of
heparin disaccharides responsible for
hemorrhage: Reversion of the
antihemostatic effect by ATP. FASEB
J 1989; 3: 2420-24
25