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

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 (80-90%) composed of hexasaccharide repeats with two disaccharides of αD-glucosamine, N,6-disulfate-αL-iduronic acid,2-O-sulfate, and one

disaccharide of αD-glucosamine 2-6-disulfate-βD-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

LEADING CAUSES

OF MORTALITY IN USA

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

PULMONARY DISEASE (FLU)

1991-80,000 1999-64,000 2000-62,000

CANCER 1991-500,000 1999-553,000 2000-555,000

TRAUMA/ACCIDENT 90,000

OTHER CAUSES 400,000

3

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).

XIIIa

Phospholipid Ca++

INTRINSIC PATHWAY

XII XIIa

XIXIa

IXIXa

Xa

VII VIIa

X X

TFPI

EXTRINSIC PATHWAY

+ Thrombomodulin

Prothrombin Thrombin

Heparin Cofactor II

Fibrinogen Fibrin

Polymerization(Clot)

VIIIa

Va

III (Tissue factor)

Antithombin III

Protein Ca + Protein S

tPA

PlasminogenPlasmin

Phospholipid/ Ca++

II IIa

Phospholipid/ Ca++

HMWK

HMWK

Phospholipid Ca++

Depolymerization (Fibrinolysis)

Protein C

4

Fig. (3). Basic hexasaccharide unit of heparin. Table 1. Distribution of heparin in mammalian and other vertebrates

VERTEBRATE SPECIES

TISSUE (µg/g dry

tissue) Rb GP Rat Dog Cat Pig Bov H Ck Sk Lizd Frg Fish Shrk

Lung <1 70 67 217 63 211 300 8 0.5 0.3 0 0.64 0.03 Liver <1 <1 <1 141 1 <1 50 <1 0 0 0.02 0.77 1.32 0 Ileum <1 27 1 400 87 113 1015 32 0.5 0.9 0.23 0 0 0

Kidney <1 4 <1 2 6 <1 26 <1 0.1 0 0 0.46 0.29 Aorta <1 <1 9 102 <1 2 150 <1 Brain <1 <1 <1 <1 <1 <1 <1 <1 0 0 0 0 0 0

Muscle <1 <1 36 9 <1 5 2 <1 0 0 0 11.4 0 0 Spleen <1 <1 <1 11 <1 <1 19 <1 0 11.9 Skin <1 <1 175 15 63 2 108 39 0.4 0 0 0 0 0

Lymph n. <1 11 5 160 74 242 180 41 Thymus <1 112 20 20 10 286 35

Appendix <1 17 38 20 47 Branchia 0.03 0

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.

O----R1OH

COOH

OHOHCOOH

OH

OH

O

OSO3H

OHCOOH

O

OH

CH2OSO3H

NSO3H

O

OSO3H

O

NSO3H

CH2OSO3HO

O

CH2OSO3H

NSO3H

O

O

O

O

n

o oCOOH

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. Several papers reported that mast cells could be derived from cells of thymus and lymph nodes and from the hematopoietic spleen tissue [18-21], others inferred that mast cells could develop locally from other cell types of the connective tissue [22]. If masts cells were derived from thymus then heparin should be absent from athymic (nude) mice if the correlation heparin/mast cells is valid. Analyses on the concentration of heparin in different tissues of athymic and normal mice has shown that heparin is present in the skin, lung, thymus and muscle of both lines [23]. These results would indicate that heparin in the athymic mice was not present in mast cells, or else, these mice, although athymic show the presence of mast cells. Other studies on the mast cell content of athymic mice have shown

that this strain show normal or even higher number of mast cells [25, 26] which then favors the second hypothesis.

It is now established that mast cells originate from haematopoyetic stem cells [27, 28], through the studies of mice of the genotypes W/Wv and S/S1d that are deficient in mast cells. Heparin is present in appreciable amounts in the skin of the breeders and of the normal progeny. On the other hand, no heparin was detected in the skin of the W/Wv genotype, which are deficient in mast cells [23]. No significant differences in the relative amounts of the other sulfated glycosaminoglycans, namely heparan sulfate, dermatan sulfate and

Table 3. Molecular weight and anticoagulant activity of heparins from various tissues and species

6

Mammal TISSUE AVERAGE M.W.

KDa

Anticogulant Activity (USP)

Dog Lung 10.7 171 Ileum 34 174 Liver 34 168 Spleen 48 165 Lymph node 51 136

Bovine Lung 13 100 Ileum 20 189 Liver 16 170 Spleen 15 Lymph node 13 151 Aorta 25 Skin 21 143 Kidney 12 140

Pig Lung 37 169 Ileum 23 175 Lymph node 20 174

Rat Lung 54 Skin >150 Muscle >150

Cat Lung 68 130 Ileum 21 178 Skin 54 132 Lymph node 74 120 Appendix 74 127

Hamster Lung 30 126 Man Lung 45 159

Ileum 42 120 Skin 60 Lymph node 37 121 Thymus 45 110 Appendix 47 126

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

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, 41-43]. 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.

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Fig. (5). Structural variability of heparin from different tissues and species.

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 13C NMR spectra of the shrimp heparin, bovine lung and bovine mucosa heparin also confirm these observations (Fig. 7). Thus, the three

heparins contain identical signals of high intensity attributed to the anomeric carbons of the N,6-sulfated hexosamine (98.2 ppm) and 2-O 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 2-O-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].

OHCOOH

OSO3H

OH

NSO3H

OHCOOH

OHOHCOOH

OH

OHOSO3H NSO3H NSO3H

OHCOOH

0

OHOH

COOH

OH

OHOSO3H NSO3H NSO3H

Heparinase

OHOH

NSO3H

+OH

O

OH

NSO3H

CH2OSO3H

OH

OH

CH2OSO3HO O

CH2OSO3HO O O

CH2OSO3HO

CH2OSO3HO

OSO3H

OH

COOH

OH

NSO3H

0OH

+

CH2OSO3HO O O O

CH2OSO3HO

CH2OSO3H

OO

O O OO

O OO

Heparinase Heparinase Heparitinase II Heparitinase II Heparitinase II

OHCOOH

OH

OSO3H NSO3H

CH2OSO3H

OH

COOH

OH

OH NSO3H

OH

COOH

OH

OH NSO3H

O O OCH2OH

O O O

O O OO

OO

CH2OSO3H

n1n2

Heparitinase II

OSO3H

OH

COOH

OH

NSO3H

0OH

+

CH2OSO3HO O

OH

COOH

OH

NSO3H

0OH

CH2OSO3HO O

OH

OSO3H

OH

COOH

OH

NSO3H

0OH

+

CH2OSO3HO O

OH

COOH

OH

NSO3H

0OH

+

CH2OSO3HO O

OH

COOH

OH

NSO3H

0OH

O O

OH OH

CH2OH

˜ ˜

n1 n2Bovine lung 6 1˜ ˜Bovine Intestine ˜ ˜6 4

Anomalocardia brasiliana ˜ ˜8 8

Tivela mactroidesDonnax striatus ˜ ˜4 5

3 5MOLLUSCS

10

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

ppm

BOVINE LUNG

BOVINE MUCOSA

SHRIMP

11

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

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).

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 LMW-heparins 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.

"In vitro" (U/mg) Sample MW KDa USP APTT Anti-Xa

"In vivo" Antitrombotic

3.0 3.2 3.4 4.3 4.6

4.8 6.0 7.3

9.5 10.5 11.5 14.0 16.018.0 19.5 21.0 23.5 25.0 29.5 30.0 37.5

10-20

20-60

70-150

200-300

M.W. KDa I.U./mg

USP

Origin

"In vivo" antithrombotic activity (U/mg)

95

130

140

130

1 2 3 4 5

A B

1 2 3 4 5

A

3.0 3.2 3.4 4.3 4.6

4.8 6.0 7.3

9.5 10.5 11.5 14.0 16.018.0 19.5 21.0 23.5 25.0 29.5 30.0 37.5

M.W. KDa

Origin

LMW-HEPARINHEPARIN

B

12

Chrom. Y.W. activity (U/mg) Heparin Intestine 15,1 140 135 106 69 176

Lung 9.8 130 74 90 60 96 pancreas 9.8 140 154 129 68 84

LMW-heparin oxidation 4.5 48 37 87 162 119

Molec. sieving 4.6 49 31 100 187 161 heparitinase II 5.8 45 22 78 265 146

Heparan sulfate bovine pancreas

25.5 <5 <5 <5 <5 126

Heparan sulfate bovine lung

15 <5 <5 <5 <5 20

APTT, activated partial thromboplastin time; Chr, chromogenic assay; Y.W., Yin and Wessler assay

These pioneer results led the pharmaceutical industries in the 80´s to search for different methods of heparin depolymerization besides the Fenton reaction (Ardeparin and Parnaparin) such as nitrous acid degradation (Nadroparin and Dalteparin), esterification and beta-elimination (Enoxaparin) and heparinase degradation (Tinzaparin). The method of preparation produces structural differences between the commercial LMW-heparins [54] and, consequently, the FDA considers them different drugs. These LMW-heparins have largely replaced the conventional heparin as judged by the sales in 2001 of the two types of heparin, around 2.0 billion and 300 million dollars, respectively.

Due to the parallelism of high "in vivo" antithrombotic activity and high anti-Xa activity it is believed that the pharmacological activity of the LMW-heparins as antithrombotics are due to the inhibition of factor Xa. Indeed, some of the commercial LMW-heparins are sold by the potency of their anti-Xa units. The biodisponibility is also measured by this activity. Regarding the structural differences for activity of the two cofactors, namely aXA and aIIa, both

standard heparin and LMW-heparin bind to antithrombin, which has a binding site for thrombin and another for factor Xa. While standard heparin can bind to both sites, LMW-heparin can interact only with the site for factor Xa, which explains its low anti-IIa activity and high anti-Xa activity "in vitro."

Other structural characteristics of the heparins contribute to the anticloting and antithrombotic activity. Thus, a heparin isolated from the shrimp Penaeus brasiliensis with a molecular weight of 10 KDa has a low anticloting activity and a potent anti-Xa activity "in vitro" (Table 5) and antithrombotic activity "in vivo" similarly to the LMW-heparins [46]. As shown before its structure differs substantially from heparin and LMW-heparins regarding the type of uronic acid and degree of sulfation. The mollusk Anomalocardia brasiliana, which also contains large amounts of glucuronic residues, has a very high anticloting activity by the USP assay (320 IU/mg) and an anti-Xa activity similar to normal heparins [37,55,56].

Table 5. Effect of LMW-heparin and shrimp heparin in the induction of thrombosis by laser shots.

Minutes after injection

Agent Dosage mg/kg

Injection route

5 15

13

Number of laser shots

Saline - SC 2/3 3 LMW-heparin 1.0 SC 7 5 LMW-heparin 2.5 SC 5 3 Shrimp heparin 1.0 SC 5/7 6/7 LMW-heparin 0.5 IV 10 7 Shrimp heparin 0.25 IV 6/5 4/5 Shrimp heparin 0.5 IV 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, O-sulfation of sulfaminoheparosan, a glycosaminoglucuronan with the structure→4)-β-D-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 high-affinity 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-D-glucopyranose (1→ pentasaccharide→4)-2-sulfo-α-L-idopyranosyl- uronic acid (1→ was chemically modified and tested for its pharmacological activity [61]. After N-deacetylation, 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 N-sulfoacharan 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 acid-pyridine.

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, LMW-heparins 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). N-desulfation 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 serine-proteases 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 LMW-HEPARINS

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

16

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

40003000200010000

CONTROL

MPS GAGPS

DEXTRAN SULFATELW 10282LW 10082

SULEPAROIDN-DESULFATED HEPARIN

HEPARIN

GLYCOSAMINOGLYCANS (cpm/ µg cell prot ein)

AN

TIT

HR

OM

BO

TIC

AG

EN

TS

(100

µg/

ml)

25002000150010005000

CONTROL

HEPARIN

CY 216

CY 222

Op 386

pK 10169

LMW-HEPASE

GLYCOSAMINOGLYCANS (cpm/µg cell protein)

LOW

MO

LEC

ULA

R W

EIG

HT

HE

PAR

INS

(100

µg/

ml)

COMPOUND Y

HS CS

LACTOBIONIC ACID

TETRA- 4S

TETRA- 5S

TETRA- 6S

HEPARIN

NONE

40003000200010000

GLYCOSAMINOGLYCANS (cpm/ µg cell protein)

HEP

AR

IN

TETR

ASA

CCH

AR

IDE

S (1

00 µ

g/m

l)

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].

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

As described above heparin binds to the

wounded tissue in such a way that its antihemostatic effect persisted even after

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 LMW-heparin [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

Bleeding potency

Compound Dosis (µg/ml)

Saline ATP x 10-4 M

Heparin 200 5.3 0.7 Heparin 400 10.3 1.0

LMW-heparin1 200 2.5 0.3 LMW-heparin1 400 4.2 0.4 LMW-heparin2 200 2.0 0.2 LMW-heparin2 400 3.8 0.3 LMW-heparin3 200 2.3 0.1 LMW-heparin3 400 4.1 0.5

1, enoxaparin; 2, dalteparin; 3, nadroparin.

IN SEARCH FOR AN IDEAL ANTITHROMBOTIC.

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

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

18

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, LMW-heparins 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.

BLO

OD

VO

LUM

E IN

TH

E D

RA

INS

(ml)

CONTROLS 10 µM 50 µM 100 µM

0

200

400

600

800

1000

ATP

0.1 mg/ml

PROTAMINE

p< 0.005p< 0.08 p>0.1

19

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, LMW-heparins 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

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BLO

OD

VO

LUM

E IN

TH

E D

RA

INS

(ml)

CONTROLS 10 µM 50 µM 100 µM

0

200

400

600

800

1000

ATP

0.1 mg/ml

PROTAMINE

p< 0.005p< 0.08 p>0.1

20

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