sign in sign up
<<
Home , Heparin

1

HEPARINS AND : OCCURRENCE, STRUCTURE AND MECHANISM OF 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 , , low molecular weight 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 2-O-sulfate residues have weak anticloting activities, whereas molecules that contain both iduronic acid 2-O sulfate, iduronic acid and small amounts of , 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 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 model "in vivo" with a negligible activity upon the serine-proteases of the 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 plasma proteins and lipids, and cells. The red The leading causes of death in the United cells seem not to be a target for antithrombotic States are that involve heart and blood agents, but on the other hand, white cells and vessels, and as a consequence thrombosis [1]. are deeply involved in The incidence of death for this in 1991 formation. was almost two times higher than the next in line, The protease network in coagulation, namely, (Fig. 1). Possibly, with the and kallikrein-kinin system is introduction of antithrombotic agents, particularly shown in Fig. 2. This cascade of events consists heparin and its derivatives, death by heart of a series of activation of serine proteases and diseases have decreased substantially (about modulation by specific inhibitors, called 30%) in 2000 when compared to malignant . The ultimate goal of the coagulation cancer, which has increased in the last ten years. system is the formation of clot that consists on Nevertheless, heart diseases are still the main the limited proteolysis of a soluble protein from cause of death [1]. This explains the efforts to plasma (fibrinogen) into an insoluble protein discover and develop specific and more potent (fibrin). antithrombotic agents. The anticloting and antithrombotic activity of heparin includes the blood itself, composed of 2 DISEASES OF THE CANCER HEART 1991-500,000 1991-975,000 1999-553,000 1999-725,000 2000-555,000 2000-700,000 CHRONIC OBSTRUCTIVE LEADING CAUSES OTHER CAUSES PULMONARY 400,000 DISORDER OF MORTALITY 1991-90,000 IN USA 1999-124,000 2000-124,000

PULMONARY TRAUMA/ACCIDENT DISEASE (FLU) 90,000 1991-80,000 1999-64,000 2000-62,000 Fig. (1). Causes of Death in USA.

Heparin acts as compound of αD-glucosamine 2-6-disulfate- because it forms a ternary complex with βD-glucuronic or αL-iduronic acid as shown in III and the different serine proteases Fig. 3 [3]. Large variations occur among of the coagulation cascade. The inhibition of heparins isolated from invertebrates and of by antithrombin is accelerated by more heparins isolated from different tissues and than 1,000 times in the presence of heparin. species of vertebrates (See below). Heparin is also capable of potentiating the effect of another that is called heparin cofactor II that is specific for thrombin. It also releases and OCCURRENCE OF HEPARIN IN increases the synthesis of TFPI (tissue factor VERTEBRATES AND INVERTEBRATES pathway inhibitor) by endothelial cells. Heparin was the first compound used as Whereas heparan sulfates are ubiquitous anticoagulant and antithrombotic agent. Heparin components of all tissue-organized animal life was isolated in 1916 by McLean in Canada from forms [4-6] heparin, has shown a very peculiar a preparation of dog [2]. The commercial distribution in mammalian and other vertebrate heparin preparations, introduced in the clinical tissues as well as invertebrates. Since the earlier use 60 years ago, are from hog and bovine studies, lung, intestine, and liver were the intestinal mucosa, as well as bovine lung. tissues rich in heparin from a variety of Chemical and enzymatic analyses and NMR . [5-8]. Table 1 shows a systematic spectroscopy have revealed the main structural study involving nine mammalian species. These features of heparin. In our laboratory we have analyses have shown that except for rabbit shown that bovine lung heparin is mainly (80- tissues, heparin was present in lung, skin, ileum, 90%) composed of hexasaccharide repeats with lymph nodes, thymus, and appendix of all two of αD-glucosamine, N,6- species. The absolute content of heparin varied disulfate-αL-iduronic acid,2-O-sulfate, and one in the different tissues. The lack of heparin in 3

INTRINSIC PATHWAY

HMWK XII XIIa EXTRINSIC PATH WAY HMWK XI XIa III (Tissue factor)

Phospholipid Phospholipid Antithombin III Ca++ IX Ca++ VII IXa VIIa VIIIa Phospholipid/ Ca++ Protein Ca X + Protein S Xa X

Va TFPI Phospholipid/ Ca++

+ Thrombomodulin

Prothrombin Thrombin II IIa Heparin Cofactor II tPA Fibrinogen Fibrin

XIIIa Plasmin Plasminogen

Polymerization Depolymerization (Clot) (Fibrinolysis)

Fig. (2). Coagulation cascade. rabbits was correlated with the absence of mast contact with the environment or in organs that cells in the species [8]. The amounts of the function as internal barriers against infection heparins isolated from mammalian and other and foreign bodies. vertebrate's tissues are also shown in Table 1. A The anticoagulant activity and molecular large variation on the concentration of heparin weight of some mammalian heparins is shown among species is evident. Thus, bovine and dog in Table 2. It is interesting that the anticoagulant tissues contain the highest amounts of heparin. In activity varied from 60 up to 200 I.U./mg. non- vertebrates the amounts of heparin Likewise the molecular weight of the heparins is considerably less. An interesting characteristic depending on the tissue of origin also shows a is that heparin is mainly distributed in tissues in large variation (11 kDa up to >150 KDa). 4

CH2OSO3H CH2OSO3H COOH CH2OSO3H O O O O O O COOH COOH COOH o o O O OH OH O OH OH O OH OH O----R1 OH OSO3H NSO3H OSO3H NSO3H NSO3H n

Fig. (3). Basic hexasaccharide unit of heparin.

Table 1. Distribution of heparin in mammalian and other vertebrates

TISSUE VERTEBRATE SPECIES (µg/g dry tissue) Rb GP Rat Dog Cat 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, ; Ck, chicken; Sk, snake; Lizd, lizard; Frg, frog; Shrk, shark

As shown in Table 2 there was no correlation a matter of controversy [16]. The peculiar between molecular weight and anticoagulant distribution of heparin between fetal and adult activity of the heparins. All these results imply tissues raised again the question of whether that heparins have a large structural variation these heparins were related to the presence of depending of their origin. In invertebrates heparin mast cells. Studies on the concentration of is found in few species, namely, mollusks heparin and content of mast cells in different (eulamelibranchia), crustacean, annelid, tunicate fetal and adult bovine tissues [17] have shown and possibly urochordate [4, 9, 10-12] (Table 3) that a good correlation between the Again the anticoagulant activity and molecular number and heparin concentration could be weight varied according to the species analyzed. obtained in all tissues analyzed. A few differences could be observed among the mast HEPARIN AND MAST CELLS cell from the different tissues. The mast cells in adult ileum were usually larger and contained Since the discovery of mast cells by Paul more granules when compared with the mast Ehrlich [13] and that the metachromatic activity cells from fetuses. In fetal spleen, the mast cells of these cells with basic dyes was related to were distributed homogeneously in the whole heparin [14,15], the question whether this organ whereas in adult spleen the mast cells compound was only confined to the mast cells or were only found in the capsule. was also present in other cell structures has been 5

Table 2. Distribution of heparin in invertebrates

CLASS Average Anticoagulant Especies M.W. Activity (KDa) (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 that this strain show normal or even higher derived from cells of thymus and lymph nodes number of mast cells [25, 26] which then favors and from the hematopoietic spleen tissue [18-21], the second hypothesis. others inferred that mast cells could develop It is now established that mast cells locally from other cell types of the connective originate from haematopoyetic stem cells [27, tissue [22]. If masts cells were derived from 28], through the studies of mice of the 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 , namely mast cells, or else, these mice, although athymic heparan 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 AVERAG Anticogulant E M.W. Activity KDa (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

sulfate were observed among the and other sulfated glycosaminoglycans in breeders and four different littermates analyzed several types of (cells in culture, [23]. This suggests that heparin is not replaced by solid and ascytes tumors) have shown that other sulfated in the animals besides heparin, dermatan sulfate, chondroitin that lack heparin. These results clearly indicate sulfate and heparan sulfate were also present in that heparin is related to the presence of mast high amounts. In some instances, heparin could cells. not be detected in the tumors [36] or in mast The presence of heparin in mast cells of the cells present in rat mucosa [37]. From these peritoneal cavity of rats [29-31] as well as in combined data, we can conclude that heparin is mastocytomas [32, 33] has been extensively only present in mast cells but mast cells might documented. These cells and tumors have been not contain heparin. used for the studies of heparin biosynthesis by Heparin and other sulfated different authors [34, 35]. Analyses of heparin glycosaminoglycans as well as histamine were 7 found in various organs of Anomalocardia gives support to the suggestion for a role of brasiliana, a mollusk from the South Atlantic, mast cells and its heparin, in the defense of the and quantified. The heparin was present in organisms against virus, bacteria and foreign granules in the cytoplasm [37-40]. A good bodies. Similar results were observed in correlation between heparin and histamine Tunicate [10]. content was found in the labial palp, intestine, Some conclusions can be made from these ctenide, mantle, and foot tissues. The tissue studies: 1) heparin seems to be present location of metachromatic cells, putatively exclusively in mast cells or mast-like cells in the containing heparin, was identified histologically case vertebrates and mollusks; 2) Its primary by alcian blue, toluidine blue, Masson trichrome, biological activity is not related with hematoxylin eosin and heparinase degradation antithrombotic activity since mollusks which do (Fig. 4A). Except for the foot, cells containing not possess a coagulation system contains metachromatic granules were found in the heparin and rabbits that contain this system is epithelium surfaces of all the organs analyzed. In devoid of heparin; 3) many indirect evidences ctenidium the basophilic cells are primarily suggest that heparin and its mast cell is involved located around the acquiferum tubes and in minor in defense mechanisms independently of the amounts in the epithelium of the filaments. The immune system [38]. basophilic cells are concentrated in the surface of the internal side and the median folder of the STRUCTURE OF HEPARINS mantle (P). In these areas, these cells constitute the only type of cells of the epithelium As could be inferred from the studies independently of the stain used. The epithelium described above regarding the differences in facing the shell is devoid of metachromatic cells. molecular weight and anticoagulant activity, the The metachromatic cells are extremely abundant heparins are a class of molecules with a great in the labial palp and they are uniformly structural variability. As a classical example, the distributed in the ciliated epithelium of the commercial heparins from bovine lung and selection face of the crests. The basophilic cells bovine mucosa differ in the amounts and are intercalated in the intestinal mucosal content of their constituent disaccharides [3, 41- epithelium (Int.). These cells are rich of 43]. A systematic study on the structure of some metachromatic granules as shown in higher mammalian and invertebrate heparins has magnification and toluidine blue staining. shown that all the heparins contain two different An "in situ" identification of heparin using basic regions that vary according to the tissue and heparinase degradation has and species of origin (Fig. 5). These regions established unequivocally the presence of this vary among each other by the degree of compound in the metachromatic cells (Fig. 4B). susceptibility to Flavobacterium heparinum The location of mast cells at the epithelium heparinase and heparitinase II [3, 43, 45]. Other surface of mollusk tissues exposed to the major variation is the glucuronic/iduronic acid environment are very similar to the distribution of content. Mollusk mammalian and other vertebrate mast cells and 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. CH OSO H COOH CH OH CH2OSO3H CH2OSO3H CH2OSO3H CH2OSO3H CH2OSO3H COOH 2 3 2 O O O O O O O O OOO OO COOH COOH COOH COOH O O O O OH O OH OH O OH O OH O OH OH O OH O OH O OH OH O OH OH OH OH NSO H NSO3H OSO3H NSO3H OSO3H NSO3HNSOOH 3H OSO3H NSO3H OH NSO3H 3 n n1 2 Heparinase Heparinase Heparinase Heparitinase II Heparitinase II Heparitinase II

CH2OSO3H COOH CH OSO H COOH CH OSO H CH OSO H 2 3 2 3 2 3 CH OSO H CH OH O OO O O O 0 COOH CH2OSO3H COOH 2 3 COOH 2 COOH OO OO OO + + O OH OH 0 OH OH O OH O OH OH + + OH OH OH OH OH 0 OH OH 0 OH OH 0 OH OH OH OH NSO H OSO H NSO H OSO H NSO HNSOOH H 3 3 3 3 3 3 OH OH OSO3H NSO3H NSO3H NSO3H Heparitinase II n1 n2 COOH CH2OSO3H COOH CH2OSO3H OO OO Bovine lung 6 1 + Bovine Intestine ˜ 64˜ OH 0 OH OH 0 OH ˜˜ OH OH Anomalocardia brasiliana 88 MOLLUSCS Donnax striatus ˜ 45 OSO3H NSO H OH NSO H ˜ 3 3 Tivela mactroides ˜ 3 ˜ ˜ ˜ 5

heparin contains 64% of glucuronic acid and 36% heparins contain identical signals of high of iduronic acid whereas lung heparin contains intensity attributed to the anomeric carbons of mostly iduronic acid (>90%) as shown in Fig. 6 the N,6-sulfated hexosamine (98.2 ppm) and 2- [39]. and dermatan sulfate O sulfated iduronic acid residues (46). Besides used as controls of the experiment shows these, the shrimp heparin also contains glucuronic acid and iduronic acid respectively as prominent signals at 98.4, 98.5 and 99.1 ppm major constituents. 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 Fig. proportions of glucuronic/iduronic acid and 2- (6). Uronic acid content of heparins.The heparins O-sulfated iduronic acid residues as shown in were subjected to acid hydrolysis and Figs. 5 and 6. Other residues are also present in subsequently to electrophoresis at pH 2.8. minor proportions in heparin such as acetyl Heparan sulfate, dermatan sulfate and chondroitin groups and 3-O-sulfated residues of the sulfate were used as controls. hexosamine moieties. This last residue that occurs in one third A recent study of the anomeric regions of the 13C NMR spectra of the shrimp heparin, bovine of the heparin molecules is responsible for lung and bovine mucosa heparin also confirm the binding of heparin to antithrombin [47-50]. these observations (Fig. 7). Thus, the three 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 If we observe the coagulation cascade and for this hypothesis is the finding that each its multiple enzymatic systems and co-factors heparin is indeed a population of molecules with (Fig. 2) and the structural diversity of the different molecular weights. This is exemplified heparins one could suggest that ideally a by the electrophocusing technique where specific heparin structure would act on a heparin is fractionated in at least 21 components specific step of (Fig. 8A).

A B

A M.W. B M.W. I.U./mg "In vivo" KDa KDa USP antithrombotic activity (U /mg) 3.0 3.0 3.2 3.2 10-20 3.4 95 3.4 4.3 4.3 4.6 4.6

4.8 4.8 6.0 6.0 20-60 130 7.3 7.3 9.5 9.5 10.5 10.5 11. 5 11.5 70-150 140 14.0 14.0 16.0 16.0 18.0 18.0 19.5 19.5 21.0 23.5 21.0 200-300 130 23.5 25.0 25.0 29.5 29.5 30.0 30.0 37.5 37.5

1 2 3 4 5 Origin 1 2 3 4 5 Origin 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 molecule by the Fenton reaction as shown in activities but nevertheless about the same Fig. 8B [53]. In spite of low anticoagulant antithrombotic activity "in vivo" [51-53]. These activity the LMW-heparins they exhibited the results were the basis for producing LMW- same "in vivo" antithrombotic activity as well as heparins by depolymerization of the parent 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 "In vitro" (U/mg) "In vivo" KDa USP APTT Anti-Xa Antitrombotic 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 25.5 <5 <5 <5 <5 126 bovine pancreas Heparan sulfate 15 <5 <5 <5 <5 20 bovine lung

APTT, activated partial thromboplastin time; Chr, chromogenic assay; Y.W., Yin and Wessler assay These pioneer results led the pharmaceutical standard heparin and LMW-heparin bind to industries in the 80´s to search for different antithrombin, which has a binding site for methods of heparin depolymerization besides the thrombin and another for factor Xa. While Fenton reaction (Ardeparin and Parnaparin) such standard heparin can bind to both sites, LMW- as nitrous acid degradation (Nadroparin and heparin can interact only with the site for factor Dalteparin), esterification and beta-elimination Xa, which explains its low anti-IIa activity and (Enoxaparin) and heparinase degradation high anti-Xa activity "in vitro." (Tinzaparin). The method of preparation produces Other structural characteristics of the structural differences between the commercial heparins contribute to the anticloting and LMW-heparins [54] and, consequently, the FDA antithrombotic activity. Thus, a heparin isolated considers them different . These LMW- from the shrimp Penaeus brasiliensis with a heparins have largely replaced the conventional molecular weight of 10 KDa has a low heparin as judged by the sales in 2001 of the two anticloting activity and a potent anti-Xa activity types of heparin, around 2.0 billion and 300 "in vitro" (Table 5) and antithrombotic activity million dollars, respectively. "in vivo" similarly to the LMW-heparins [46]. Due to the parallelism of high "in vivo" As shown before its structure differs antithrombotic activity and high anti-Xa activity substantially from heparin and LMW-heparins it is believed that the pharmacological activity of regarding the type of uronic acid and degree of the LMW-heparins as are due to . The mollusk Anomalocardia the inhibition of factor Xa. Indeed, some of the brasiliana, which also contains large amounts commercial LMW-heparins are sold by the of glucuronic residues, has a very high potency of their anti-Xa units. The anticloting activity by the USP assay (320 biodisponibility is also measured by this activity. IU/mg) and an anti-Xa activity similar to normal Regarding the structural differences for activity heparins [37,55,56]. 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 Injection Minutes after mg/kg route injection 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 similar to that of heparin. Similar to the mollusk responsible for the binding site of antithrombin heparin these molecules contain only glucuronic led to the synthesis of a pentasaccharide acid in their structures suggesting that iduronic GlcNR(6-OSO3)-GlcA-GlcNSO3(3,6-di-OSO3)- acid may play a minor role for the anti-Xa IdoA(2-OSO3)-GlcNSO3(6-OSO3) (where R activity. Supporting this suggestion, another represents either a sulfate or an acetyl group and - heparin-like compound, a OSO3 represents an O-sulfate/ester sulfate group, prepared from the giant African snail Achatina with locations of O-sulfate groups indicated in fulica, which has a repeating disaccharide parentheses) with high affinity for antithrombin structure of →4)-2-deoxy-2-acetamido-α-D- [50,57]. This first chemically defined compound glucopyranose (1→ pentasaccharide→4)-2- with anti-Xa activity is now in clinical trials [58]. sulfo-α-L-idopyranosyl- uronic acid (1→ was Several attempts to modify preexisting chemically modified and tested for its glycosaminoglycans for increase of anticloting pharmacological activity [61]. After N- activity are being performed. As an example, O- deacetylation, acharan sulfate was N-sulfonated sulfation of sulfaminoheparosan, a using either chlorosulfonic acid-pyridine or glycosaminoglucuronan with the structure→4)-β- sulfur trioxide-trimethylamine complex. The D-GlcA(1→4)- β−D-GlcNSO(3)(-)-(1→, sulfate level in these products ranged from 22 to obtained by N-deacetylation and N-sulfation of 24%(w/w), significantly less than that of the capsular polysaccharide from E. coli K5 has heparin (36%, w/w) whereas the molecular an increased anti-Xa activity. Some of the weight of both N-sulfoacharan sulfates were products contained the trisulfated aminosugar comparable with that of heparin. "In vitro" GlcNSO(3)(-)3,6SO(3)(-), which is a marker anticoagulant activity showed that N- component of the pentasaccharide sequence sulfoacharan sulfate derivatives were through which heparin binds to antithrombin moderately active for the inhibition of thrombin [59,60]. Depending on the reaction conditions, and neither product showed any measurable the products showed different proportions of anti-factor Xa activity. The differences in the components with high affinity for antithrombin. activities of N-sulfoacharan sulfates produced A high-affinity subtraction, with 36 KDa, was by these two methods are probably ascribable to shown to cause conformational changes in the a small level of concomitant O-sulfonation molecule very similar to those induced by high- obtained when using chlorosulfonic acid- affinity heparin. The anti-Xa activity was 170 pyridine. units/mg, similar to that of the third international Analyzing all this data, we can conclude heparin standard and markedly higher than that glycosaminoglycans with high iduronic activities of previously described heparin residues are not crucial for the anticoagulant analogues. Another preparation, of 13 KDa, activity of the compounds. Molecules composed exhibited an anti-Xa activity of 70 units/mg. exclusively of iduronic acid 2-O-sulfate have These findings suggest that the modified bacterial weak activities, whereas molecules that contain polysaccharide interacts with antithrombin and both iduronic acid 2-O sulfate, iduronic acid and promotes its anticoagulant action in a manner small amounts of glucuronic acid, such as 14 heparin, or mixed amounts of glucuronic and This activity was absent in the heparan sulfate iduronic acids (mollusk heparin) possess high from the adjacent smooth muscle cells of anticloting and anti-Xa activity. These results arterial vessel [66]. also suggest that a proper combination of these When heparin is given to a patient, the elements might furnish the ideal antithrombotic is possibly one of the sites of agent. action for the compound. Endothelial cells from Opposite to the antithrombotics described rabbit aorta [68] and human umbilical cord above, a heparan sulfate (molecules that contain a exposed to heparin increase the synthesis of the small region similar to heparin) derived from antithrombotic heparan sulfate present at the pancreas with negligible anti-IIa and anti-Xa cell surface, as well as the one released to the activity (< 5 I.U./mg) is a potent antithrombotic medium [69]. As shown in Fig. 9, this effect is "in vivo" (Table 4) measured by a variety of also elicited by LMW-heparins [70,71]. Heparin methods including vena cavae ligature [62,63]. was fragmented with heparinase from Furthermore, it was shown that this heparan Flavobacterium heparinum [45]. The different sulfate has also a potent inhibitory effect of fragments were also tested as elicitors of the arterial thrombosis. The bovine lung heparan synthesis of endothelial heparan sulfate [70]. It sulfate has a low "in vivo" antithrombotic activity was shown that the minimum structural in both venous (Table 4) and arterial vessels [64]. requirement to produce the enhancement in the This variation of activity of heparan sulfates is synthesis of the antithrombotic heparan sulfate due to the findings that the structure of these is a pentasulfated tetrasaccharide (Fig. 9). N- compounds varies according to tissue and species desulfation of heparin completely abolishes the of origin [64,65]. Another example of stimulatory activity. Other sulfated compounds, antithrombotic compounds "in vivo" without "in such as lactobionic acids, that consist of sulfated vitro" activities is a sulfated fucan isolated from lactose linked by a sequence from 3 to 12 the brown algae Spatoglossum schroderi [Hugo carbons, a cyclic octaphenol-octasulfonic acid A. O. Rocha, personal communication]. These (compound Y), dextran sulfate, oversulfated last results cast some doubts that the "in vivo" chondroitin sulfates, a fucan from brown antithrombotic activity of heparin, LMW- seaweed [70-72] and other compounds that heparins and other heparinoids is mainly related possess antithrombotic activity also increase the with the inhibition of Factor -Xa. synthesis of the endothelial antithrombotic heparan sulfate (Fig. 9). EFFECT OF HEPARIN AND OTHER The increased synthesis of heparan sulfate ANTITHROMBOTIC COMPOUNDS ON chains is observed when the cells are exposed to VASCULAR ENDOTHELIAL CELLS heparin and other structurally unrelated We have so far discussed the possible site of antithrombotic agents. This lead to the action of these drugs in the protease network of hypothesis that the antithrombotic activity of coagulation. As previously mentioned, the vessel these compounds "in vivo" could be related, at wall is another site of action for antithrombotic least in part, to the increased production of this compounds. In the eighties Colburn and peculiar heparan sulfate by endothelial cells. In Buonassisi [66] have shown that an endothelial favor of this hypothesis are the findings that the cell line in culture shows blood compatibility, heparin-tetrasaccharide and compound Y which that is, their surface does not promote clotting. are antithrombotic agents "in vivo" exhibit a Thus, when the surface of endothelial cells negligible activity "in vitro" upon the serine- changes, there is a chance of thrombus formation. proteases of the coagulation cascade as One of the compounds present at the cell surface previously observed for bovine pancreas and extracellular matrix of endothelial cells is a heparan sulfate. heparan sulfate proteoglycan. This heparan A protein of 47 KDa that binds heparin and sulfate, which has been totally sequenced [67], other antithrombotic agents with high affinity has shown some heparin sequences and possesses has been isolated from the proteins of the antithrombotic activity in different models [66]. endothelial cell surface (79). Fractionation of 15 the surface proteins by heparin-affinity heparins in animal experiments as well as in reveals the enrichment of two is less pronounced or equal to heparin major protein bands (47 and 28 KDa) that are [73-77]. These differences could be related to present in very small amounts in the crude cell the structural differences of the LMW-heparins surface extracts (Fig 10A). The binding of the used [54, 78]. The neutralization of the bleeding proteins with heparin and GL522 in the presence produced by LMW-heparins is still under study. of 0.5M NaCl reveals that besides the 47 and 28 In two bleeding models in rats, failed KDa several proteins bind with [125I]-heparin and to reverse the bleeding activity of these [14C]-GL522 whereas only a 47KDa protein bind heparins. with heparin in the presence of 1M NaCl (Fig. Cruz and coworkers in the earlier sixties 10B). Likewise, the 47 KDa is the only protein have shown that the antihemostatic effect of that binds [14C]-GL522 in the presence of 1 M heparin was independent of blood cells like NaCl (Fig. 10C). platelets and was related to special structures of the damaged tissue [80]. Thus, when heparin MECHANISM OF THE HEMORRHAGIC was applied topically to skin wounds it ACTIVITY OF HEPARIN AND LMW- produced enhanced bleeding from small vessels HEPARINS and . [81, 82]. This antihemostatic activity persisted even after extensive washing The main drawback in heparin and of the preparation with saline solutions, heparinoids antithrombotic therapy is their suggesting that heparin molecule binds to a hemorrhagic activity. Thus, several reports have receptor of the wound resulting in the shown that heparin, LMW-heparins and other uncontrollable hemorrhage. Among the several sulfated antithrombotics, except for bovine proteins tested to counteract the inhibition of pancreas heparan sulfate and a sulfated fucan hemostasis produced by heparin, namely, isolated from brown alga, produce bleeding in plasma and serum proteins, tropomyosin and some patients. The extent of bleeding of LMW- actin, only non denaturated myosin was able to 16

LMW-HEPASE pK 10169

Op 386

CY 222

CY 216 HEPARIN MOLECULAR WEIGHT CONTROL LOW LOW HEPARINS (100 µg/ml) 0 500 1000 1500 2000 2500 CS GLYCOSAMINOGLYCANS HS (cpm/µg cell protein) ml)

/ HEPARIN N-DESULFATED HEPARIN SULEPAROID LW 10082 LW 10282 DEXTRAN SULFATE GENTS (100 µg (100 GENTS A GAGPS MPS LACTOBIONIC ACID COMPOUND Y CONTROL

0 1000 2000 3000 4000

ANTITHROMBOTIC GL YCOSA MIN OGL YCAN S (cpm/ µg cell prot ein)

NONE

HEPARIN

TETRA- 6S HEPARIN HEPARIN TETRA- 5S ASACCHARIDES µg/ml) (100 TETRA- 4S

TETR 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 extensive washing with saline. This effect, the phosphates tested, such as UTP, nevertheless, could be reversed by ATP and/or ITP, GTP, CTP, AMP, only ATP and ADP at myosin [81, 85]. Table 6 shows that the low concentrations (10-5 M) were able to bleeding produced by the LMW-heparins and -4 dislodge the residual heparin bound to the well as heparin are totally reversed by 10 M of receptor counteracting its inhibitory activity [81]. ATP. A significant reduction of bleeding caused by heparin was also observed in patients subjected to 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

Fig. (10). Isolation of a 47KDa protein with high Compound Dosis Bleeding affinity for heparin at the surface of endothelial (µg/ml) potency cells. GL-522, octaphenoloctasulfonic acid, Saline ATP x compound Y. 10-4 M Heparin 200 5.3 0.7 "In vitro" experiments have shown that Heparin 400 10.3 1.0 heparin inhibits competitively the hydrolysis of LMW-heparin1 200 2.5 0.3 ATP by myosin ATPase [81, 83, 84]. These LMW-heparin1 400 4.2 0.4 combined results suggested that heparin was LMW-heparin2 200 2.0 0.2 binding to a myosin-like molecule of the smooth 2 muscular cells inhibiting the contractility of the LMW-heparin 400 3.8 0.3 LMW-heparin3 200 2.3 0.1 vessels and thus producing the increase of 3 bleeding. The minimum structural requirement of LMW-heparin 400 4.1 0.5 the heparin molecule capable of producing bleeding was a heparin-derived disaccharide with 1, enoxaparin; 2, dalteparin; 3, nadroparin. a sulfate at the C-6 position of the hexosamine residue [85]. IN SEARCH FOR AN IDEAL ANTITHROMBOTIC. REVERSION OF THE ANTIHEMOSTATIC ACTIVITY BY ATP Contrasting with cancer, a significant reduction of death caused directly or indirectly As described above heparin binds to the by thrombosis has been observed in the last ten wounded tissue in such a way that its years [1]. This was probably due to the 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 18

1000

p< 0.08p< 0.005 p>0.1 800

600

400

200

0 BLOOD VOLUME IN THE DRAINS (ml) CONTROLS 10 µM 50 µM 100 µM 0.1 mg/ml ATP 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 hemorrhagic activity suggest that a proper popularized the use of antithrombotic therapy structural modification of heparin, LMW- with sulfated . Nevertheless, heparins or other sulfated polysaccharides these compounds have an unwanted effect, without hemorrhagic activity is worth which is the production of hemorrhage, which pursuing. Alternatively, ATP or ATP could be serious in some patients. Thus, a derivatives that bind to the smooth muscle search for this class of compounds without cells but are resistant to ATPases could be hemorrhagic activity is actively being used to displace heparin and its fragments pursued. The heparan sulfate from bovine from the purinergic receptor. These pancreas and the sulfated fucan from brown compounds would be used to decrease the algae that are potent antithrombotic hemorrhage of patients caused by these compounds "in vivo" and devoid of compounds.

19

1000

p< 0.08p< 0.005 p>0.1 800

600

400

200

0 BLOOD VOLUME IN THE DRAINS (ml) CONTROLS 10 µM 50 µM 100 µM 0.1 mg/ml ATP 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 that bind to the smooth muscle cells popularized the use of antithrombotic but are resistant to ATPases could be therapy with sulfated polysaccharides. used to displace heparin and its Nevertheless, these compounds have an fragments from the purinergic unwanted effect, which is the receptor. These compounds would be production of hemorrhage, which could used to decrease the hemorrhage of be serious in some patients. Thus, a patients caused by these compounds. search for this class of compounds without hemorrhagic activity is REFERENCES actively being pursued. The heparan sulfate from bovine pancreas and the [1] National Vital Statistics Reports sulfated fucan from brown algae that 2003; 51:4-9 are potent antithrombotic compounds [2] McLean J. The discovery of heparin. "in vivo" and devoid of hemorrhagic Circulation 1959; 19:75-8 activity suggest that a proper structural [3] Silva ME, Dietrich CP. The modification of heparin, LMW- structure of heparin Characterization heparins or other sulfated of the products formed from heparin polysaccharides without hemorrhagic be the action of a heparinase and a activity is worth pursuing. heparitinase from Flavobacterium Alternatively, ATP or ATP derivatives 20 heparinum. J Biol Chem 1975; 250: mollusca. J Amer Chem Soc 1956; 6841-6 78: 5874-8 [4] Cassaro CMF, Dietrich CP. [12] Rahemtulla F, Løvtrup S. The Distribution of sulfated comparative biochemistry of mucopolysaccharides in invertebrates. invertebrate mucopolysaccharides-V J Biol Chem 1977; 252: 2254-61 Mollusca. Comp Biochem Physiol [5] Toledo OMS, Dietrich CP. Tissue B 1976; 53: 15-8 specific distribution of sulfated [13] Ehlich P. Beiträge zur Kenntnis der mucopolysaccharides in mammals. granulierten Bindegewebszellen und Biochim Biophys Acta 1977; 498 : der eosinophilen Leukocythen. Arch 114-22 Anat Physiol 1879: 166-9 [6] Barlow GH, Coen LJ, Mozen MM. A [14] Holmgren H, Willander O. biological chemical physical Beitrag zur Kenntnis der Chemie und comparison of heparin from different Funktion der Ehrlichschen mammalian species. Biochim Mastzellen. Z Mikrosk Anat Forsch Biophys Acta 1964; 83: 272-7 1937; 42: 242-78 [7] Hashimoto K, Matsuno M, Yosizawa [15] Jorpes E, Holmgren H, Willander Z, Shibata T . A dextrorotatory O. Ueber das Vorkommen von polysaccharide of very high Heparin in den Gefässwänden und in anticoagulant activity newly isolated den Augen Ein Beitrag zur from the 's lung intestine. Physiologie der Ehrlichschen Tohoku J Exp Med 1963; 81: 93-5 Mastzellen. Z Mikrosk Anat Forsch [8] Nader HB, Takahashi HK, Straus AH, 1937; 42: 279-301 Dietrich CP. Selective distribution of [16] Jaques LB Debnath AK. heparin in mammals: conspicuous Simultaneous evaluation of tissue presence of heparin in lymphoid heparin mast cells in small tissue tissues. Biochim Biophys Acta 1980; samples. Am J Physiol 1970; 219: 627: 40-8 1155-60 [9] Medeiros GF, Mendes A, Castro [17] Nader HB, Straus AH, Takahashi RAB, Baú EC, Nader HB, Dietrich HK , Dietrich CP. Selective CP. (2000) Distribution of sulfated appearance of heparin in mammalian glycosaminoglycans in the animal tissues during development. Biochim kingdom: widespread occurrence of Biophys Acta 1982; 714: 292-297 heparin-like compounds in [18] Csaba G, Toro I , Modo K. invertebrates. Biochim Biophys Acta (1962) The behaviour of the thymus 2000; 1475: 287-294 in conditions associated with tissue [10] Cavalcante MCM, Allodi S, proliferation. Acta Anatom 1962; 48 Valente AP, Straus AH, Takahashi : 114-21 HK , Mourao PA S, et.al. Occurrence [19] Ginsburg H, Lagunoff D. The "in of heparin in the invertebrate Styela vitro" differentiation of mast cells. J plicata (Tunicata) is restricted to cell Cell Biol 1967; 48 :685-97 layers facing the outside environment - [20] Ishizaka T, Okudaira H, Mauser An ancient role in defense? J Biol LE, Ishizaka K. Development of rat Chem 2000; 275: 36189-96 mast cells "in vitro" I- Differentiation [11] Burson SL, Fahrenbach MJ, of mast cells from thymus cells. J Frommhagen LH, Riccardi BA, Immunol 1976; 116: 747-754 Brown RA, Brockman JA, et al. [21] Burnet FM. The probable Isolation purification of mactins relationship of some or all mast cells heparin-like from to the T-cell system. Cell Immunol 1977; 30: 358-60 21 [22] Selye H. The mast cells. 1965; fraction from mast cell tumors. J Butterworth Washington D C Biol Chem 1967; 242: 2301-5 [23] Straus AH, Nader HB, Dietrich [35] Lindahl U. Enzymes involved in the CP. Absence of heparin or heparin like formation of the compounds in mast cell-free tissues structure of heparin. Meth Enzymol animals. Biochim Biophys Acta 1982; 1972; 28:676-84 Academic Press. 717: 478 -85 New York [24] Keller R, Hess, MW Riley, JF. [36] Nader HB, Marx W, Spolter L. Mast cells in the skin of normal Glycosaminoglycans of some mouse hairless athymic mice. Experientia mastocytomas. Biochim Biophys 1979; 32: 171-2 Acta 1980; 631: 463-78 [25] Wlodarski K. Mast cells in the pinna [37] Dietrich CP, Paiva JF, Moraes of Balb/c "nude" (nu/nu) CT, Takahashi HK, Porcionatto heterozygotes (nu/+) mice. Experientia MA, Nader HB. Isolation 1976; 32: 1591-2 characterization of a heparin with [27] Kitamura Y, Go S, Hatanaka K. high anticoagulant activity from Decrease of mast cells in W/Wv their Anomalocardia brasiliana. Biochim increase by bone marrow Biophys Acta 1985; 843: 1-7 transplantation. Blood 1978; 52: 447- [37] Enerbäck L, Kolset SO, Kusche 52 M, Hjerpe A, Lindahl U. (1985) [28] Kitamura Y Go S. Decreased Glycosaminoglycans in ral mucosal production of mast cells in S1/S1d mast-cells. Biochem J 1985; 227: anemic mice, Blood 1979; 53: 492-7 661-8 [29] Marshall JS, Kawabori S, Nielsen [38] Nader HB, Dietrich CP. Natural L , Bienenstock J. (1994) occurrence possible biological role Morphological functional- of heparin in Heparin: Chemical characteristics of peritoneal mast-cells Biological Properties Clinical from young-rats. Cell Tissue Res Applications (D A Lane U Lindahl 1994; 276: 565-570 eds) Edward Arnold Publishers. 1989; [30] Pejler G, Berg L. Regulation of rat London pp81-96 mast-cell protease-1 activity - protease [39] Santos EA (1997) Heparina do inhibition is prevented by heparin molusco Anomalocardia brasiliana: proteoglycan. Eur J Biochem 1995; A. Natureza dos resíduos de ácidos 233: 192-9 urônicos. B. Distribuição em tecidos [31] Amihai D, Trachtenberg S, Terkel e correlação com mastócitos. Tese de J , Hammel I. (2001) The structure of Doutorado ; 1997. Escola Paulista de mast cell secretory granules in the Medicina São Paulo SP, Brazil blind mole rat (Spalax ehrenbergi). J [40] Santos EA , Menezes LR, Pereira Struct Biol 2001; 136: 96-100 NML, Andrade GPV, Nader HB, [32] Dunn TB, Potter MJ. A Dietrich CP. Mast cells are present transplantable mast-cell in in epithelial layers of different tissues the mouse. J Natl Cancer Inst 1957; of the mollusk Anomalocardia 18: 587-601 brasiliana "In situ" characterization [33] Furth J, Hagen P, Hirsch EI. of heparin a correlation of heparin Transplantable in the histamine concentration. Histochem mouse containing histamine heparin J; 2003 (in press) 5-hydroxy- triptamine. Proc Soc [41] Jaques LB. (1980) Heparins- Exptl Biol Med 1957;95 :824-28 Anionic Polyelectrolyte Drugs. 35 Pharmacol Rev 1980; 31: 99-166 [34] Silbert JE. Incorporation of SO4 endogenous heparin by a microsomal 22 [42] Casu B, Guerrini M, Naggi A, [49] Casu B, Lindahl U. Structure Torri G, De-Ambrosi L, Boveri G, biological interactions of heparin et al. (1996) Characterization of heparan sulfate. Adv. Carbohyd sulfation patterns of beef pig mucosal Chem Biochem 2001; 57: 159-206 heparins by nuclear magnetic [50] Choay J, Petitou M, Lormeau JC, resonance spectroscopy. Sinay P, Casu B, Gatti G. Structure- Arzneimittelforschung 1996; 46: 472-7 activity relationship in heparin - a [43] HB Nader, CP Dietrich. Extraction synthetic pentasaccharide with high- characterization of heparins In affinity for anti-thrombin-iii eliciting Analytical techniques to evaluate the high anti-factor-xa activity. Biochem structure function of natural Biophys Res Commun 1983; 116: polysaccharides glycosaminoglycans. 492-9 Ed Nicola Volpi. Research Sign Post [51] Nader HB, McDuffie NM, 2002; 23-9 Dietrich CP. (1974) Heparin [44] Nader HB, Porcionatto MA, fractionation by electrofocusing: Moraes CT, Dietrich CP. (1990) Presence of 21 components of Purification substrate specificity of different molecular weights. heparinase heparitinase I heparitinase Biochem Biophys Res Commun II from Flavobacterium heparinum. J 1974; 57: 488-93 Biol Chem 1990; 265: 6807-13 [52] McDuffie NM, Nader HB, Dietrich [45] Nader HB, Chavante SF, Santos CP. Electrophocusing of heparin EA, Oliveira FW, Paiva JF, Fractionation of heparin into 21 Jerônimo SMB, et al. (1999) Heparan components distinguishable from sulfates heparins: similar compounds other acidic mucopolysaccharides performing the same functions in Biopolymers 1975; 14: 1473-86 vertebrates invertebrates? Brazil J [53] Dietrich CP, Nader HB, and Med Biol Res 1999; 32: 529-38 McDuffie NM. (1975) [46] Dietrich CP, Paiva JF, Castro Electrophocusing of heparin RAB, Chavante SF, Jeske W, Presence of 21 monomeric dimeric Fareed J, et al Structural features molecular species in heparin anticloting activities of a novel natural preparations. An Acad Brasil Ci low molecular weight heparin from the 1975; 47: 301-9 shrimp Penaeus brasiliensis. Biochim [54] Dietrich CP, Shinjo SK, Moraes FA , Biophys Acta 1999; 1428: 273-83 Castro RAB, Mendes A, Nader HB. [47] Lindahl U, Backstrom G, Thunberg Structural features bleeding activity L, Leder I G. Evidence for a 3-O of commercial LMW-heparins: sulfated D-glucosamine residue in the Neutralization by ATP protamine. antithrombin-binding sequence of Sem Thromb Hemost 1999; 25: 43- heparin Proc Natl Acad Sci USA 50 1980; 77: 6551-5 [55] Pejler G, Danielsson A , Bjork I, [48] Olson ST, Bjork I, Sheffer R, Lindahl U, Dietrich CP, Nader HB Craig PA, Shore JD, Choay J. Structure antithrombin-binding Role of the antithrombin-binding properties of heparin isolated from pentasaccharide in heparin acceleration the clams Anomalocardia brasiliana of antithrombin-proteinase reactions - and Tivela mactroides. J Biol Chem resolution of the antithrombin 1987; 262: 1413-21 conformational change contribution to [56] Dietrich CP, Nader HB, Paiva JF, heparin rate enhancement. J Biol Tersariol ILS. Santos EA. Holme Chem 1992; 267: 12528-38 KR. et al. Heparin in molluscs: Chemical enzymatic degradation 23 13C 1H nmr spectroscopical evidences AH. Structural diferences of heparan for the maintainance of the structure sulfates according to the tissue and through evolution. Int J Biol Macromol species of origin. Biochem Biophys 1989; 11: 361-366 Res Commun 1983; 111:865 -71 [57] Lindahl U, Thunberg L , Backstrom [65] Dietrich CP, Tersariol ILS, Toma L, G, Riesenfeld J, Nordling K, Moraes CT Porcionatto MA, Oliveira Bjork I. Extension structural FW and Nader HB. Sequencing of variability of the antithrombin-binding heparan sulfate: Identification of sequence in heparin. J Biol Chem variable and constant 1984; 259: 12368-76 regions in eight heparan sulfates from [58] Walenga JM, Jeske WP, Samama different origins Cell Molecul Biol MM, Frapaise FX, Bick RL, Fareed 1998; 44: 417-29 J. : a synthetic heparin [66] Colburn P, Buonassisi V. Anti- pentasaccharide as a new clotting activity of endothelial cell antithrombotic agent. Exp Opin cultures heparan sulfate Invest Drugs 2002; 11: 397-407 proteoglycans. Biochem Biophys [59] Casu B, Grazioli G, Razi N, Guerrini Res Commun 1982; 104: 220-227 M, Naggi A, Torri G, et al. Heparin- [67] Nader HB, Dietrich CP, Buonassisi like compounds prepared by chemical V, Colburn P. Heparin sequences in modification of capsular the heparan sulfate chains of an polysaccharide from Escherichia-coli endothelial cell proteoglycan. Proc K5. Carbohyd Res 1994; 263: 271-84 Natl Acad Sci USA 1987; 84: 3565- [60] Naggi A, Torri G, Casu B, Oreste P, 69 Zoppetti G, Li JP, et al. Toward a [68] Buonassisi V, Venter JC. Hormone biotechnological heparin through neurotransmitter receptors in an combined chemical enzymatic established vascular endothelial cell modification of the Escherichia coli K5 line. Proc Natl Acad Sci USA polysaccharide. Semin Thromb 1976;73:1612-16 Hemost 2001; 27: 437-43 [69] Nader HB, Buonassisi V, Colburn P, [61] Wu SJ, Chun MW, Shin KH, Toida Dietrich CP. Heparin stimulates the T, Park Y, Linhardt RJ, et al. synthesis modifies the sulfation Chemical sulfonation anticoagulant pattern of heparan sulfate activity of acharan sulfate. Thromb proteoglycan from endothelial cells. J Res 1998; 92: 273-81 Cell Physiol 1989;140:305-310 [62] Bianchini P, Osima B, Parma B, [70] Pinhal MAS, Santos IAN, Silva IF, Nader HB, Dietrich CP. (1985) Lack Dietrich CP, Nader HB. Stimulation of correlation between "in vitro" "in of the synthesis of an antithrombotic vivo" antithrombotic activity of heparin heparan sulfate from endothelial cells fractions related compounds: Heparan by heparin its fragments. sulfate as an antithrombotic agent "in Thrombosis Haemostasis 1995;74: vivo". Thromb Res 1985; 40: 597-607 1169-1174 [63] Bianchini P, Osima B, Parma B, [71] Pinhal MAS, Walenga JM, Jeske Dietrich CP, Takahashi HK, Nader W, Hoppensteadt D, Dietrich CP, HB Structural studies "in vivo" "in Fareed J, et al. Antithrombotic agents vitro" pharmacological activities of stimulate the synthesis modify the heparin fractions fragments prepared sulfation pattern of a heparan sulfate by chemical enzymic proteoglycan from endothelial cells. depolimerization. Thromb Res 1985; Thromb Res 1994;74:143-153 40: 49-58 [72] Nader HB, Pinhal MAS, Baú EC, [64] Dietrich CP, Nader HB and Straus Castro RAB, Medeiros GF, 24 Chavante SF, et al. Development of [80] Cruz WO. The significance of a new heparin-like compounds other smooth muscle component in antithrombotic drugs and their hemostasis. Proc Soc Exptl Biol Med interaction with vascular endothelial 1965;119: 876-883 cells. Brazil J Med Biol Res 2001;34: [81] Cruz WO, Dietrich CP. 699-709 Antihemostatic effect of heparin [73] Bagge L, Wahlberg T, Holmer E, counteracted by adenosine Tydén H, Nyström SO, Malm T. triphosphate. Proc Soc Exptl Biol Low-molecular-weight heparin Med 1967;126: 420-426 (Fragmin) versus heparin for [82] Nader HB Dietrich CP. Effect of anticoagulation during heparitin sulfate fractions on cardiopulmonary bypass in open heart hemostasis Proc Soc Exptl Biol Med surgery using a pig model. Blood 1974;146: 504-508 Coagul Fibrinol 1994;5:265-272 [83] Nader HB, Tersariol ILS, Dietrich [74] Colwell CW Jr.Recent advances in the CP. Antihemostatic activity of use of low molecular weight heparins heparin disaccharides as prophylaxis for deep vein obtained by thrombosis. Orthopedics 1994;17: 5-7 chemical enzymatic fragmentation: [75] Haas S, Flosbach CW (1993) Reversal of the hemorrhagic activity Prevention of postoperative by ATP myosin Thromb Res thromboembolism with Enoxaparin in 1989;54: 207-214 general surgery: a German multicenter [84] Tersariol ILS, Dietrich CP, Nader trial. Sem Thromb Hemost 1993;19: HB. Uncoupling of actomyosin 164-173 ATPase by heparin its fragments [76] Hirsh J. Comparison of the relative Eur J Biochem 1997;245: 40-46 efficacy safety of low molecular [85] Dietrich CP, Tersariol ILS, Silva RG, weight heparin unfractionated heparin Bianchini P, Nader HB. Dependence for the treatment of venous thrombosis. of the C-6 sulfate of the glucosamine Haemostasis 1996; 26: 189-198 moiety and 1-4 glycosidic linkage of [77] Martineau P, Tawil N. Low-molecular- heparin disaccharides for production weight heparins in the treatment of of hemorrhage. Reversal of the deep-vein thrombosis. Ann antihemostatic activity of heparin Pharmacother 1998;32: 588-598 their fragments by ATP and myosin. [78] Fareed J, Jeske W, Hoppensteadt D, Sem Thromb Hemost 1991;17: 65-73 Clarizio R, Walenga JM. Low- [86] Garcia-Jr HV, Buffolo E, Nader HB. molecular-weight heparins: Dietrich CP (1994) Reduction of pharmacologic profile product blood loss produced by heparin after differentiation. Amer J Cardiol topical application of adenosine 1998;82: 3L-10L triphosphate in cardiopulmonary by- [79] Pinhal, MAS, Trindade, E, Fareed, J, pass operations. Ann Thorac Surg Dietrich, CP and Nader, HB Heparin 1994;57: 956-959. and a cyclic octaphenol-octasulfonic [87] Nader HB, Tersariol ILS and Dietrich acid (GL-522-Y-1) bind with high CP. Structural requirements of affinity to a 47KDa protein from heparin disaccharides responsible for vascular endothelial cell surface and hemorrhage: Reversion of the stimulate the synthesis and structural antihemostatic effect by ATP. FASEB changes of an antithrombotic heparan J 1989; 3: 2420-24 sulfate.Thromb Res 2001; 103: 35-45

25