WO1991015217A1

WO1991015217A1 - Anticoagulant oligosaccharides

Info

Publication number: WO1991015217A1
Application number: PCT/US1991/002371
Authority: WO
Inventors: Douglas M. Tollefsen; Margaret M. Maimone
Original assignee: Washington University
Priority date: 1990-04-06
Filing date: 1991-04-05
Publication date: 1991-10-17
Also published as: AU7742591A

Abstract

Low molecular weight polysaccharides related to dermatan sulfate have been shown to be the effective minimal structures to prevent or reduce coagulation in warm blooded animals.

Description

ANTICOAGULANT OLIGOSACCHARIDES

Technical Field

The invention relates to the inhibition of coagulation using polysaccharide inhibitors. More particularly, it concerns dermatan sulfate-related oligosaccharides which have anticoagulant activity.

Background Art A number of naturally occurring polysaccharides have been used as anticoagulants. Heparin is the most commonly employed, but the anticoagulant activity of dermatan sulfate is also well known. While heparin consists mainly of alternating idouronic acid and glucosamine residues which are partially^" sulfated, and acts on antithrombin III. A pentapeptide "minimum structure" has been shown to bind and stimulate ATIII; however, an extended form is required to complex both ATIII and thrombin. The pentapeptide is of the formula: GlcNAc(6S)-(alpha 1,4)-GluA-(beta 1,4)-GlcNS(3S,6S)-(alpha l,4)-Ido(2S)-(beta 1,4)-GlcNS(6S) , or the nonreducing terminal residue may be GlcnS(δS).

Abbreviations used are as follows: A glucosamine residue is represented by GlcNH; if the amino group is acetylated the abbreviation is GlcNAc; if the amino group is sulfated the abbreviation if GlcNS. Galac- tosamine and its acetylated and sulfated forms are cor¬ respondingly represented as GalNH, GalNAc and GalNS. Idouronic acid is represented by IdoA and glucuronic acid by GlcA. All of the foregoing may be sulfated at one or more hydroxyls; the position of the sulfate will be indicated after the name designation by the number of the hydroxyl and "S" in parentheses. Thus, GlcNS(6S) represents N-sulfated glucosamine which is further sulfated in the 6 position, GlcNS(3S,6S) indicates N-sulfated glucosamine which is also sulfated in both the 3 and 6 positions. In the depolymerization products described below, the oligosaccharides which result have ring contractions at the nonreducing end. The ring contraction obtained from galactosamine is a talose residue which is abbreviated herein ATal. The products are often also treated with reducing agents resulting in the formation of a talitol residue at this position which is abbreviated ATal_D. Sulfation of these residues is indicated in a manner corresponding to that set forth above; thus, a talose residue sulfated in the 4 position would be ATal(4S). The corresponding depolymerization procedure wherein the reducing terminus corresponds to a glucosamine residue would result in a ring contraction of an anhydromannose, abbreviated AMan, and the corresponding reduced form would be designated AMan....

Dermatan sulfate consists of alternating glucuronic or idouronic acid residues conjugated to N- acetylgalactosamine (GalNAc) residues and acts through the intermediate heparin cofactor II (heII) as described below. As is heparin, dermatan sulfate is a heterogeneous structure resulting from the varying degrees of 0- sulfation_r and, also, to a greater extent than heparin, as a result of the inclusion of the two types of uronic acid residues. As stated above, it is known that dermatan sulfate exerts its anticoagulant effect by stimulating the effect of heparin cofactor II (hell). Heparin cofactor II is a human plasma protein that normally inhibits thro bin. Dermatan sulfate encourages this normal inhibition of thrombin by hell. Although extensive work has resulted in the localization of the anticoagulant effect of heparin to the pentasaccharide structure described above, low molecular weight substitutes for dermatan sulfate have not hitherto been disclosed. Clearly a low molecular weight counter¬ part for the natural product dermatan sulfate permits control of the composition utilized, as well as permitting the use of a synthetic rather than a biologically derived product. This eliminates the danger of contamination with infectious agents associated with the use of biologically derived products .

The present invention describes low molecular weight oligosaccharides related to dermatan sulfate which greatly stimulate thrombin inhibition by heparin cofactor II.

Disclosure of the Invention

The invention is directed to low molecular weight oligosaccharides which bind heparin cofactor II. These compounds have the formula:

wherein R is H, alkyl(l-6C), or a monosaccharide or reduced monosaccharide residue. One aspect of the inven¬ tion is directed to the pentasaccharide and hexasaccharide compounds of formula 1 per se. The invention is also directed to low molecular weight oligosaccharides which are extended forms of the pentasaccharide minimum structure shown above having no more than an additional 10 saccharide residues; preferably no more than 6 saccharide residues. These extensions, which may be either at the reducing or nonreducing end of the compound of formula 1 shown are also useful as anticoagulants .

In another aspect, the invention is directed to a method to effect anticoagulant activity in a subject which comprises administering to the subject an effective amount of the compound of formula 1 or its extended forms as defined above.

In still another aspect, the invention is directed to pharmaceutical compositions in which the compound of formula 1 or its extended forms are active ingredients .

Brief Description of the Drawings

Figure 1 is a flow chart showing the preparation of the compounds of the invention from dermatan sulfate. Figure 2 shows size fractionation of oligosaccharides obtained by hydrazine/hydrazine sulfate treatment of dermatan sulfate.

Figure 3 shows the percentage of oligomer which is bound to a hcII-Sepharose column as a function of size. Figure 4 shows the elution pattern of depolymerization products of dermatan sulfate from a gel filtration column.

Figure 5 shows the elution pattern of labeled dermatan sulfate hexasaccharides from an hell affinity column.

Modes of Carrying Out the Invention

The compounds of formula 1 shown above and their extended forms are effective anticoagulants and are believed to behave in the manner analogous to dermatan sulfate by stimulating the activity of hell. Because they bind to hell, affinity chromatography using hell as a ligand can also be used to purify these oligosaccharides . Preferred embodiments of the compound of formula

1 include those wherein R is a monosaccharide. Preferably the monosaccharide is either a sulfated galactosamine residue or is a ring contracted form thereof—i.e., a sulfated anhydrotalose. The ring contracted form can also be reduced—i.e., a sulfated anhydrotalitol. A particularly preferred set of embodiments are the hexasaccharides of the formulas:

IdoA(2S)-GalNAc(4S)-IdθA(2S)-GalNAc(4S)-IdoA(2S)-ATal(4S) ; IdoA(2S)-GalNAc(4S)-IdoA(2S)-GalNAc(4S)-IdoA(2S)- ATal_R(4S); and IdoA(2S)-GalNAc(4S)-IdoA(2S)-GalNAc(4S)- IdoA(2S)-GalNAc(4S) .

When the representations using the abbreviations above are used, it will be understood that the linkage between IdoA and GalNAc residues is alpha(l,3) and that between GalNAc and IdoA is beta(1,4). The oligosaccharides of the invention can be prepared from complete nitrous acid digestion of partially N-de-acetylated dermatan sulfate in a procedure analogous to that described in the examples below. Following hydrolysis, the fragments are size separated, and the ac- tive portion of the hexasaccharide fraction is recovered using affinity chromatography on hell. A flow chart which outlines the steps in this procedure is shown in Figure 1.

More flexibility is obtained if the hexasaccharides are obtained synthetically. Standard methods for synthesis of the hexasaccharides of the inven¬ tion are known in the art. If prepared synthetically, embodiments wherein R is a GalNAc or N-sulfated galactosamine residue are preferred. If synthetic methods are used, additional saccharide extensions of the required minimum pentasaccharide unit can also be obtained.

Alternate size fractions from the nitrous acid digestion can also be used so long as they contain the required pentasaccharide.

The compounds of the invention are employed in conventional procedures for administration of anticoagulants. Generally, anticoagulant compositions are administered systemically, usually by injection. Injec¬ tion can be intravenous, intramuscular, subcutaneous, or intraperitoneal. In addition, systemic administration can be accomplished using transmembrane techniques wherein the compositions containing the compounds of the invention employ agents which enhance the transfer of materials across, for example, the nasal membrane. If properly formulated, the compounds may also be administered orally. Suitable formulations are found, for example, in Remington's Pharmaceutical Science (latest edition); Mack Publishing Company, Easton, PA.

The dose to be administered depends on the severity of the condition, the nature of the subject, the compound chosen and the judgment of the attending physi- cian or veterinarian. Administration can be in a single dose, or may involve repeated continuous dosage.

The following examples are intended to il¬ lustrate but not to limit the invention.

Preparation A

Materials Dermatan sulfate (chondroitin sulfate B from porcine skin) and Sephadex G-25-80 were purchased from Sigma. The dermatan sulfate (100 mg/ml H„0) was treated with an equal volume of nitrous acid at pH 1.5 (prepared as described by Shively and Conrad (1976)) for 10 min at room temperature to degrade contaminating heparin. The material was then neutralized with 1 M Na-CO,, dialyzed against H^O, and assayed for uronic acid (Bitter and Muir, 1962). In contrast to the untreated material, the nitrous acid-treated dermatan sulfate was not active in the antithrombin-mediated thrombin inhibition assay. Bio-Gel P-10 was purchased from Bio-Rad; CNBr-activated Sepharose from Pharmacia; tosyl=Gly-Pro~Arg-p_-nitroanilide (Chromozym TH) from Boehringer Mannheim; [U- 14C]D-glucose

(310 mCi/mmole) from ICN; and [ H]NaBH₄ (5-15 Ci/mmole) from NEN. [14C]glucitol was prepared by NaBH. reduction of [U- 14C]glucose. Human heparin cofactor II and thrombin were purified from plasma as previously described (Blinder et al. , 1989) .

Preparation B

Preparation of Dermatan Sulfate Disaccharide and Monosaccharide Standards

Dermatan sulfate disaccharide standards were prepared using methods described by Shaklee and Conrad

(1986). Disaccharides produced by complete chemical cleavage of dermatan sulfate as described below were fractionated on a Micropak AX-5 column, and the peaks were assigned tentative structures based on their peak size and elution position compared to published data (Shaklee and

Conrad, 1986). The peaks representing GlcA-ATal--.( 6S) ,

GlcA-ATal_R(4S) , IdoA-ATal_R(4S) , and IdoA2S-ATal_R(4S) were pooled, desalted and subjected to paper electrophoresis to confirm the degree of sulfation. The disaccharides were also subjected to acid hydrolysis for various time periods, and the products formed were analyzed by paper chromatography as described below. The hydrolysis time courses were compared to published data (Shaklee and

Conrad, 1986). The paper electrophoresis and paper chromatography data taken together confirmed the tentative structure assignments.

Additional standards were isolated from preparative paper chromatography of the above standards after acid hydrolysis for specific periods of time: GlcA- ATal_. from a 1 h hydrolysis of GlcA-ATal_D(4S) ; IdoA-ATal_n and ATal_.(4S) from a 0.5 h hydrolysis of IdoA-ATal^( 4S) ; and Tal_ from a 4 h hydrolysis of IdoA-ATal_D(4S) . Follow- ing elution from the paper, a sample of each standard was run on paper electrophoresis to confirm the expected degree of sulfation of each. Preparation C

Paper Electrophoresis and Paper Chromatography

Paper electrophoresis and paper chromatography were run as described by Shaklee and Conrad (1986). Paper electrophoresis was run on Whatman 3MM paper (3 x 57 cm lanes) in formic acid/acetic acid/H^O (2:7:70, by vol., pH

1.7) for 2 h at 1500 volts. About 5000 cpm of

3 [ H]disaccharides were spotted per lane. Descending paper chromatography was run in two systems: system 1 on Whatman no. 1 paper (3 x 57 cm lanes) developed in 1- butanol/acetic acid/1 M NH-. (3:2:1, by vol.) and system 2 on Whatman P81 paper (cellulose phosphate) developed in ethyl acetate /pyridine/5 mM boric acid (3:2:1, by vol.) .

About 4000 cpm of acid hydrolysis samples were spotted per lane. After development, the papers were cut into 1 cm segments which were counted for radioactivity.

Preparation D Acid Hydrolysis Time Course The disulfated [ H]disaccharide (100,000 cpm) was mixed with [ 14C]glucitol (72,000 cpm) and dried in a 6 x 50 mm glass tube. The sample was dissolved in 0.5 M

H~S0., covered with a layer of mineral oil, and heated at

100°C. Aliquots (8 ul) were removed at various time points, cooled on ice, and neutralized with 7 ul of 1 M

Na_^CO.,. A portion of each aliquot was analyzed by paper chromatography in systems 1 and 2. The migration

3 distances of the [ H]products relative to the [ 14C]glucitol internal standard was compared to those of standard disaccharides run simultaneously.

Preparation E Assay Method for Stimulation of hell Inhibition of Thrombin The rate of inhibition of thrombin by hell in the presence of dermatan sulfate oligosaccharides was determined by incubating 10 ul of unlabeled NaBH. reduced oligosaccharides (0-3400 ug/ l H₂0) in 80 ul of 0.15 M NaCl, 0.01 M Tris-HCl, 1 mg/ml polyethylene glycol, pH 7.5 (TS/PEG) with 5 ul of 400 nM hell and 5 ul 342 nM thrombin (added last) in a disposable polystyrene cuvette. After a 60-s incubation at room temperature, 500 ul of 95 uM Chromozym TH in TS/PEG was added, and the absorbance at 405 nm was measured continuously for 100 s. The rate of change of absorbance was proportional to the concentration of active thrombin remaining in the incubation.

Example 1 Nitrous Acid Depolymerization of Dermatan Sulfate

Dermatan sulfate oligosaccharides were prepared using a hydrazinolysis/deamination reaction sequence (Hopwood and Muller, 1983; Shaklee and Conrad, 1986). Heparin-free dermatan sulfate (60 mg) was dried in a 4 ml Reacti-Vial (Pierce Chemical Co.) and 4 ml of anhydrous hydrazine containing 40 mg of hydrazine sulfate was added. The vial was capped and heated in a dry bath at 100 C for 1 h (partial N-deacetylation) or 10 h (complete N- deacetylation) . The sample was cooled in ice and dried under a stream of N«. H„0 (12 ml) was added, and the pH was adjusted to <7 with acetic acid. The sample was dialyzed against 15 [0.15??] M NaCl followed by H₂0 and then lyophilized. To produce nitrous acid at pH 4 in situ, the N-deacetylated dermatan sulfate was dissolved in 1.5 ml 1 M H₂S0₄, and 3.75 ml of 5.5 M NaN0₂ was added. After 30 min at room temperature, the sample was cooled on ice, and the pH was raised to 8.5 with 1 M Na^CO-..

Example 2 Reduction of Dermatan Sulfate Oligosaccharides

Unreduced dermatan sulfate oligosaccharides were incubated with 0.5 M NaBH₄ in 0.2 M Na₂C0₃, pH 10.2 (10 ul/mg oligosaccharide) or [ H]NaBH. in the same buffer (4- 5 mCi/mg oligosaccharide) for 2 h at 0 C. In the H reduction, unlabeled 0.5 M NaBH. (10 ul/mg oligosaccharide) was added after 1 h. At the end of the incubation period, excess NaBH. and [ 3H]NaBH. was destroyed in a fume hood by dropwise addition of 2 M H₂S0. until no more bubbling occurred. The samples were dried under a stream of N«, resuspended in H-0, and neutralized with 1 M a^CC before gel filtration chromatography. To

H label hexasaccharides alone, the unreduced fragments were size-fractionated by gel filtration chromatography, and then the hexasaccharide peak fractions were pooled, desalted, dried and resuspended in 200 ul of 1 M sodium

3 carbonate, pH 9.0. The [ H]NaBH. reduction was.then car¬ ried out as above.

Example 3 Gel Filtration Chromatography Dermatan sulfate oligosaccharides (reduced or unreduced) were mixed with two standards (200 ul blue

14 dextran (20 mg/ml H₂0) and [ C]glucose (20,000 cpm/ul)) and loaded onto a P-10 column (1.5 x 198 cm) equilibrated in 10% ethanol containing 1 M NaCl. The fragments were eluted with the same solvent at a flow rate of 9-12 ml/h, and 2-ml fractions were collected. A portion of each fraction was assayed for uronic acid by the carbazole method (Bitter and Muir, 1962), and the radioactivity in each fraction was determined by liquid scintillation counting. Blue dextran was detected by absorbance at 540 run. Desalting of dermatan sulfate oligosaccharides was carried out on a Sephadex G-25-80 column (1.5 x 57 cm) run in 7% propanol. Fractions of 3 ml were collected, and the radioactivity and conductivity of each was determined. Fractions containing the desalted oligosaccharides were pooled and dried. The elution pattern of the Sephadex G- 25 column is shown generally in Figure 2. The effect of varying the time of hydrolysis is also indicated in the Figure. The open circles indicate the results when dermatan sulfate was treated with hydrazine/hydrazine sulfate for one hour; the closed circles for ten hours prior to cleavage with nitrous acid at pH 4. As shown in Figure 2, treatment for ten hours results almost completely in a preparation of disaccharides when cleaved with nitrous acid.

Figure 3 shows the percentage binding of dermatan sulfate oligosaccharides labeled with tritium to hcII-Sepharose. 100,000 cpm were applied to a 1 x 5.4 cm. column of hcII-Sepharose equilibrated in 50 mM NaCl, 50 M Tris-Hcl pH 7.4 and the column was washed with the same buffer. The bound oligosaccharides were eluted with a linear sodium chloride gradient. A 500 ml aliquot of each fraction was counted and percent cpm in the bound peaks was determined. As shown in Figure 3, 81.7% of the intact dermatan sulfate bound to the column; decreasing percent¬ ages of binding were found as the number of monosaccharide units decreased. The smallest unit which bound in any appreciable percentage was the hexasaccharide.

Example 4 Affinity Chromatography An hcII-Sepharose 4B was prepared as described for AT-Sepharose by Hook et al. (1976). Briefly, 48 mg of hell in 12 ml of coupling buffer (50 mM NaCl, 100 mM NaHC0₃, pH 8.2) was mixed with 600 mg (138 mg/ml 100 mM NaHCO-., pH 8.2) of N-acetylated heparin, prepared as described by Danishefsky and Steiner (1965), to block free amino groups in the glycosaminoglycan binding sites of the hell molecules. CNBr-activated Sepharose was combined with the hcll/heparin solution and rotated end over end for 18 h at 4°C The hcII-Sepharose was treated with 1.3 ml of 1 M ethanolamine for 2 h, to block unreacted coupling sites, and washed repeatedly with coupling buffer and acetate buffer (50 mM NaCl, 100 mM sodium acetate pH

4.0). Final resuspension was in column buffer (50 mM

NaCl, 10 mM Tris-HCl, pH 7.4).

[ H]dermatan sulfate hexasaccharides (100,000 cpm = ~0.2 nmole) prepared as in Example 3 were diluted in

500 ul of column buffer, loaded onto the hcII-Sepharose column and washed with ~30 ml of the same buffer at a flow rate of 28.6 ml/h. Bound fragments were eluted at the. same flow rate with an 80-ml linear gradient from 0.05 to 2 M NaCl n 50 mM Tris-HCl, pH 7.4. Fractions of 2 ml were collected and the radioactivity in each was determined.

In the preparation of large quantities of high affinity hexasaccharide, 1.7 mg of unfractionated hexasaccharides was applied to the column, and the material which flowed through during the wash

(flowthrough-1) was desalted, concentrated and reapplied to the column. The material which bound in this second run (bound-2) was eluted by the same gradient as above.

These elution patterns are shown in Figure 4.

Example 5

Ion-Exchange High Performance Liquid Chromatography

Samples of material bound in the hcII-Sepharose column were then applied to ion-exchange HPLC. The

3 procedure was conducted as follows: [ H]hexasaccharide samples (~20,000 cpm) in H₂0 were injected onto a 4.6 x

250 mm Partisil 10 SAX column (Whatman) equilibrated in

3 0.04 M KH₂P0₄. The [ H]hexasaccharides were eluted with a linear gradient from 0.04 to 1.0 M KH₂P0₄ over 2 h at a flow rate of 1 ml/min. Radioactivity was measured by a

Flo-one/Beta Radioactivity Flow Detector (Radiomatic

3 Instruments). [ H]disaccharides (2000-7000 cpm) were injected onto a 4 x 300 mm Micropak AX-5 column (Varian) equilibrated in 10 mM KH-PC . The disaccharides were eluted by isocratic steps of 10 (20 min, 40 (50 min), 120

(40 min), and 200 (15 min) mM KH₂P0₄ at a flow rate of 1 ml/min. Radioactivity was measured by the flow detector described above.

Figure 5 shows the elution pattern of the hexasaccharide fractions . Panel A shows the elution pat- tern for unfractionated hexasaccharides; Panel B shows the flow through fraction from hcII-Sepharose chromatography; Panel C shows the bound fraction and Panel D shows the fraction bound on the second pass. Elution was with a linear potassium phosphate gradient. As shown in Figure 5, the majority of the unfractionated hexasaccharides elute as a group of peaks at 0.4 M potassium phosphate while smaller peaks elute at higher concentrations. It was concluded that since more than 90% of the disaccharides in the source dermatan sulfate are monosulfated most of the hexasaccharides should have three sulfates, and the major peak was as¬ signed to this condition. Peaks eluting at higher salt concentrations were assumed to have 4, 5 and 6 sulfates per hexasaccharide respectively. Panel B shows that the flow through fraction from the hell column lacks the more highly sulfated peaks. On the other hand, the first bound pool from the affinity column eluted as a major peak at the group 6 position indicating the presence of 6 sulfates in the hexasaccharide. A more complex elution pattern was obtained from the second bound pool .

(The chain length of the material eluted from the first bound portion was verified by P-10 gel filtra¬ tion. )

Example 6 Analysis of the High Affinity Hexasaccharide

The eluted first bound fraction was character¬ ized by depolymerization into disaccharides using the hydrazinolysis/deamination reaction sequence and the products were divided into equal portions for reduction. One sample was reduced with unlabeled sodium borohydride and the other with triteated borohydride. As the reducing end of the disaccharide had already been labeled, only the disaccharides from the second group could be certain all to contain label. Analysis of the disaccharide mixture showed that the hexasaccharide contains a single disacchride repeated three times, which disaccharide is disulfated.

The high affinity [³H]hexasaccharide (100,000 cpm) was dried in a 100 ul Reacti-Vial (Pierce) , and 20 ul of anhydrous hydrazine containing 0.2 mg of hydrazine sulfate was added. The sample was heated at 100 C for 1 h (partial N-deacetylation) or 10 h (complete N- deacetylation) , cooled on ice, dried under a steam of N_, and lyophilized overnight. The sample was then incubated with 20 ul of nitrous acid at pH 4 (prepared by mixing 2.5 ml of 5.5 M NaN0₂ with 1 ml of 1 M H₂S0₄ (Shively and Conrad, 1970)) for 30 min at room temperature, and cooled on ice. The pH was adjusted to 8.5 with 3-10 ul of 0.2 M Na_^CO-., pH 10.2. One-half of the sample was reduced with 10 ul of 0.5 M NaBH₄ in 0.2 M Na₂C0₃, pH 10.2 for 2 h at 0°C. The other half of the sample was reduced with 10 ul (1 mCi) of [³H]NaBH₄ in 0.2 M a₂C0₃, pH 10.2 for 0.5 h at 0°C followed by 5 ul of 0.5 M NaBH₄ for an additional 1.5 h at 0 C. Excess NaBH. was destroyed in a hood by the addition of 5 ul of 3 M H₂S0₄. Following 3 rounds of dry¬ ing under a stream of N₂ and redissolving in 200 of H₂0, the samples were neutralized with 5-10 ul of 1 M Na^CO-. and desalted. The components of the disaccharide depolymerization product of the hexasaccharide prepared above were subjected to anion exchange chromatography on MicroPak AX-5 and elution by isocratic steps which separate all disaccharides found in porcine skin dermatan sulfate. The disaccharides which are the product of the hexasaccharide of the invention elute as a single peak corresponding to IdoA(2S)-ATal (4S) .

The disaccharides were also subjected to paper electrophoresis as described in preparation C above and compared to disaccharide standards containing 0, 1, or 2 sulfates. The results showed that the disaccharides obtained from the high-affinity hexasaccharide contained 2 sulfates.

The location of the sulfates was verified by analysis of data obtain by acid hydrolysis of the disaccharides as described in preparation D. Analysis of the results as compared to standards locates the positions of the sulfates as the 2 position of IdoA and the 4 posi¬ tion of ATal_R. Finally, the high affinity hexasaccharide was tested in the assay method for stimulation of hell as set forth in preparation E and found to be active.

References

Bitter, T., et al. , Anal Biochem (1962) 4.:340-344. Blinder, M.A. , et al. , J Biol Chem (1989)

264:5128-5133.

Conrad, H.E., Biochem J (1980) 191:355-363. Danishefsky, I., et al . , Biochim Biophys Acta (1965) 101:37-45. Hook, M. , et al., FEBS Lett (1975) 66:90-93.

Hopwood, J.J., et al. , Carb Res (1983) 122:227-239.

Shaklee, P.N., et al . , Biochem J (1984) 217:187-197. Shaklee, P.N., et al. , Biochem J (1986)

235:225-236.

Shively, J.E., et al . , Biochemistry (1976) 15_:3932-3942.

Shively, J.E., et al. , Biochemistry (1970) 9:33-43.

Claims

A compound of the formula

wherein R is H, alkyl(l-βC) or a monosaccharide or reduced monosaccharide residue, in purified and isolated form.

2. The compound of claim 1 wherein R is H.

3. The compound of claim 1 wherein R is a monosaccharide or reduced monosaccharide residue.

4. The compound of claim 3 wherein the monosaccharide residue is a sulfated talose residue.

5. The compound of claim 3 wherein the monosaccharide residue is a sulfated GalNAc residue.

6. The compound of claim 3 wherein the monosaccharide residue is a sulfated talitol residue.

7. The compound of claim 1 which is selected from the group consisting of

IdoA(2S)-GalNAc( 4S)-IdoA(2S)-GalNAc(4S)- IdoA(2S)-ATal(4S) ; IdoA(2S)-GalNAc( 4S) -IdoA(2S)-GalNAc( 4S)-

IdoA(2S)-ATal_R(4S) ; and IdoA(2S)-GalNAc(4S)-IdoA(2S)-GalNAc(4S) IdoA(2S)-GalNAc(4S) .

8. A compound of the formula

wherein each X is independently an oligosaccharide of 1-10 sugar residues.

9. The compound of claim 10 wherein the sugar residues of X are sulfated.

10. A pharmaceutical composition useful in preventing or diminishing coagulation in a subject, which composition comprises an amount of the compound of claim 1 effective to prevent or reduce coagulation in admixture with a pharmaceutically acceptable excipient.

11. A pharmaceutical composition useful in preventing or diminishing coagulation in a subject, which composition comprises an amount of the compound of claim 9 effective to prevent or reduce coagulation in admixture with a pharmaceutically acceptable excipient.

12. A method to prevent or reduce coagulation in a warm blooded animal which method comprises administering to an animal in need of such treatment an amount of the compound of claim 1 effective to reduce or prevent coagulation or a pharmaceutical composition thereof.

13. A method to prevent or reduce coagulation in a warm blooded animal which method comprises administering to an animal in need of such treatment an amount of the compound of claim 9 effective to reduce or prevent coagulation or a pharmaceutical composition thereof.