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Bioconjugate Chem. 2004, 15, 162−167

Dendritic Polyglycerol Sulfates as New Heparin Analogues and Potent Inhibitors of the Complement System Holger Tu¨rk,† Rainer Haag,*,† and Susanne Alban*,‡ Freiburg Materials Research Center, Albert-Ludwigs-University of Freiburg, Stefan-Meier-Strasse 21, 79104 Freiburg, Germany, and Pharmaceutical Institute, Christian-Albrechts-University of Kiel, 24118 Kiel, Germany. Received March 26, 2003; Revised Manuscript Received October 20, 2003

Due to several limitations of heparin, a widely used antithrombotic drug, there is large interest to develop alternatives. The aim of the presented study was to produce fully synthetic highly branched heparin mimetics. For this purpose, a new type of ‘treelike’ polysulfated polymers based on dendritic polyglycerol was synthesized. An efficient synthetic approach has been chosen to prepare several polyglycerol sulfates with different molecular weights as well as a polyglycerol carboxylate analogue and to evaluate them for their anticoagulant and anticomplementary activities. In contrast to the nonderivatized and the carboxylated polyglycerols, the polyglycerol sulfates prolong the activated partial thromboplastin time (APTT) and thrombin time (TT) and inhibit both the classical (CCA) and alternative complement activation (ACA). Whereas their anticoagulant activity in the APTT and in the TT amounts to 5.7-8.1% and 15.7-33.6%, respectively, of that of unfractionated heparin (UFH), their CCA and ACA inhibitory activity is 13.4-23.9 and 2.7-3.7 times, respectively, higher. In contrast to sulfated polysaccharides, the activities are not clearly dependent on the molecular weight, which might be due to the globular 3D-structure of the dendritic molecules. Due to the coherence between coagulation, complement activation and inflammation in the pathophysiology of numerous diseases, polyglycerol sulfates with both anticoagulant and anticomplementary activities represent promising candidates for the development of potential drugs.

INTRODUCTION

Although the glycosaminoglycan heparin has been the drug of choice in the prevention and treatment of thromboembolic disorders for nearly 70 years, there is large interest to find alternatives to both unfractionated heparin (UFH1) and low molecular weight heparins (LMWH). Beside well-documented clinical limitations (1), some disadvantages of heparin are based on its natural origin (2). It still has to be isolated from mammalian organs, which implies a potential risk of contamination with pathogens such as prions. Because of the increased use of heparin, especially LMWH, there is a growing shortage of the raw material. Moreover, heparin is a polydisperse mixture of molecules with different chain length and chemical structure (3, 4). Numerous parameters, ranging from the animals providing heparin up to the last purification step of the product, influence its respective * To whom correspondence should be addressed. R.H.: e-mail [email protected].: [email protected]. † Albert-Ludwigs-University of Freiburg. Present address: Department of Chemistry, University of Dortmund, Otto-HahnStr. 6, 44227 Dortmund, Germany. Fax: +49-231-755-6148. E-mail: [email protected]. Internet: www.polytree.de. ‡ Pharmaceutical Institute, Christian-Albrechts-University of Kiel. 1 Abbreviations: ACA, alternative complement activation; APTT, activated partial thromboplastin time; CCA, classical complement activation; DC, concentration for doubling the baseline coagulation time; dc, degree of carboxylation; DMF, N,N-dimethylformamide; ds, degree of sulfation; IC50, inhibitory concentration 50%; LMWH, low molecular weight heparins; PG, polyglycerol; PPP, citrated human platelet poor pool plasma; TT, thrombin time; UFH, unfractionated heparin.

composition resulting in wide chemical and thus also activity variations between different heparin preparations. Recent developments such as fondaparinux (5), the first selective synthetic factor Xa inhibitor, and ximelagatran (6), the first orally available direct thrombin inhibitor, circumvent many problems related to heparin. However, these specifically acting new antithrombotics lack the other biological activities, which are characteristic for heparin and other natural sulfated polysaccharides, e.g., complement inhibiting (7), antiinflammatory (8, 9), antiangiogenic (10), antimetastatic (11), antiatherosclerotic (12), antiproliferative (13), antiadhesive (14), and antiviral effects (15). These additional actions can contribute to the overall therapeutic benefit of heparin in some cases (11). For example, heparin-coated cardiopulmonary bypass circuits have an improved biocompatibility showing better clinical outcomes (16). Therefore, heparin analogues with a similar or even improved action profile, but lacking the disadvantages of this animal product, are of interest. Beside partial synthetic sulfated linear polysaccharides (17), fully synthetic sulfated linear polymers (18), which are produced independently of any suitable starting carbohydrate, may represent promising heparin mimetics. Recently, a new type of polysulfated heparin analogue based on branched polysaccharides has been described to possess a much higher anticoagulant activity than their linear counterparts (19). However, the accessibility of branched polysaccharides is problematic due to limited natural sources. In the present study, we describe a new synthetic approach to highly branched polysulfated heparin analogues based on dendritic polyglycerol (20-22). Several polyglycerol sulfates with different molecular weights as

10.1021/bc034044j CCC: $27.50 © 2004 American Chemical Society Published on Web 12/06/2003

Dendritic Polyglycerol Sulfates as New Heparin Analogues

Bioconjugate Chem., Vol. 15, No. 1, 2004 163

Table 1. Characterization of Polyglycerol Sulfates 2a-c and Polyglycerol Carboxylate 3

polyglycerol derivative

polymer core DPn

sulfate 2a sulfate 2b sulfate 2c carboxylate 3

1a 1b 1c 1d

32 66 133 106

Mn of the degree of Mn of the polymer functional- polyglycerol corea derivativec izationb [g/mol] [%] [g/mol] 2500 5000 10 000 8000

85 79 84 26

5500 10 500 21 700 10 300

a Determined from NMR and/or GPC (DMF). b 2a-c: Degree of sulfation (ds) obtained from elemental analysis; 3: degree of carboxylation (dc) obtained from titration (see Experimental Procedures and Supporting Information). c Calculated using the Mn of the polyglycerol core and the experimental degree of functionalization.

well as a polyglycerol carboxylate analogue have been prepared and evaluated for their anticoagulant and anticomplementary activities. EXPERIMENTAL PROCEDURES

Materials. Polyglycerols (PG) 1a (Mn ) 2500 g/mol, Mw/Mn ) 1.6), 1b (Mn ) 5000 g/mol, Mw/Mn ) 1.6), 1c (Mn ) 10 000 g/mol, Mw/Mn ) 1.8) and 1d (Mn ) 8000 g/mol, Mw/Mn ) 2.0) were prepared as described previously, using 1,1,1-tris(hydroxymethyl)propane (TMP) as initiator (22). SO3/pyridine complex and sodium chloroacetate were purchased from Fluka (Buchs, Switzerland). Both reagents were used without further purification. The solvent N,N-dimethylformamide (DMF, p.a. quality, purchased from Roth, Karlsruhe, Germany) was dried over CaH2 and stored over molecular sieve 4 Å prior to use. Dialysis (regenerated cellulose tubing, SpectraPore 6/Roth) in distilled water was performed in a 5 L beaker, changing the solvent three times over a period of 48 h. Unfractionated heparin (UFH) of porcine mucosal origin (147 USP-U/mg, purchased from Sigma, Deisenhofen, Germany) was used as reference substance in the coagulation and complement assays. Analysis. 1H NMR and 13C NMR spectra were recorded in D2O at concentrations of 100 mg/mL on a Bruker ARX 300 spectrometer operating at 300 and 75.4 MHz, respectively. IR measurements were performed on a Bruker IFS 88 FT-IR spectrometer using a KBr disk. The degree of sulfation (ds) (Table 1) of compounds 2a-c was determined by elemental analysis. The amount of carboxylate groups in compound 3 was determined by a common titration method (23): 50 mg polyglycerol carboxylate (dried over P2O5) dissolved in 20 mL of distilled water was stirred with 2 g of Lewatit K1131 (H+) resin. The solution of the polyglycerol carboxylic acid was filtered and subsequently titrated with 0.01 N NaOH (1 mL steps), and the pH was recorded by conductivity measurements. The equivalence point was evaluated by extrapolation of the two branches of the titration curve. The degree of carboxylation (dc) (Table 1) was then calculated using the following eq 1 (for derivation, see Supporting Information):

dc )

(A‚Mn)/(m - AB) × 100 (%) DPn + fc

(1)

with A ) consumption of NaOH (mmol) corresponding to the inflection point; Mn ) average molecular weight of the polyglycerol core; m ) dry weight of the polyglycerol carboxylate (50 mg); B ) equiv weight corresponding to one additional C2HO2Na unit (80.02 g/mol); DPn ) degree of polymerization of the polyglycerol core; fc ) functionality of the initiator molecule TMP (fc ) 3).

Synthesis of Polyglycerol Sulfates 2a-c. The sulfation of the polyglycerols was performed according to the method described by Alban et al. (24). To a stirred solution of 5.0 g of polyglycerol (1a, 1b, 1c) (67.5 mmol OH-groups) in 25 mL DMF was added dropwise a solution of 10.75 g (67.5 mmol) SO3/pyridine complex in 67.5 mL of DMF over 4 h at 60 °C under an argon atmosphere. After the reaction mixture was stirred for additional 2 h at 60 °C and 18 h at room temperature, 50 mL of distilled water was added. To the aqueous solution was added 1 M NaOH immediately until a pH of 11 was reached. Concentration in vacuo gave the crude product which was further purified by dialysis in water. After evaporation of the solvent, the polyglycerol sulfates 2a-c were obtained as pale yellow solids which were further dried over P2O5. Yields: 7.49 g (2a); 8.96 g (2b); 7.01 g (2c). 1H NMR (300 MHz, D2O): δ (ppm) 0.98 [t, 3H,CH3CH2C(CH2O)3-PG-OSO3Na],1.48[m,2H,CH3CH2C(CH2O)3-PG-OSO3Na], 3.40-4.00 [m, CH3CH2C(CH2O)3PG-OSO3Na], 4.19, 4.33, 4.38 [PG-OSO3Na], 4.72 [PGOCH2CH(OSO3Na)CH2OSO3Na]. 13C NMR (D2O, 75.4 MHz): δ (ppm) 66.9, 67.6, 68.2, 69.4, 70.3, 75.8, 77.2, 78.3 [PG-OSO3Na]. IR (KBr): ν (cm-1) 3470 [OH], 2930 [CH], 1260 [SdO], 780 [C-O-S]. Sulfur content from elemental analysis: 2a: 15.38% S, 2b: 14.28% S, 2c: 15.20% S. No degradation of the polyglycerol core could be observed by 1H NMR spectroscopy (see Supporting Information). Synthesis of Polyglycerol Carboxylate 3. To a stirred solution of 23.0 g of polyglycerol 1d (310.5 mmol OH-groups) in 124.2 g of 30 wt % NaOH (931.5 mmol NaOH) was added dropwise a solution of 108.5 g (931.5 mmol) sodium chloroacetate in 130 mL of distilled water over 30 min. The reaction mixture was heated to 80 °C for 16 h and then cooled to room temperature, neutralized with 2 N hydrochloric acid and concentrated in vacuo. The crude product was purified by dialysis in distilled water. Upon evaporation of the solvent, 12.2 g of the polyglycerol carboxylate 3 were obtained as a colorless solid which was then dried over P2O5. 1H NMR (D2O, 300 MHz): δ (ppm) 0.89 [t, 3H, CH3CH2C(CH2O)3-PG-CH2CO2Na], 1.39 [m, 2H, CH3CH2C(CH2O)3-PG-CH2CO2Na], 3.35-3.90 [m, PG-CH2CO2Na], 3.98, 4.09 [s, 2H, PGOprim-CH2CO2Na, PG-Osec-CH2CO2Na]. 13C NMR (D2O, 75.4 MHz): δ (ppm) 60.9, 62.7, 68.9, 70.3, 72.2, 78.0, 79.5 [PG-CH2CO2Na],77.1[PG-CH2CO2Na],177.8[PG-CH2CO2Na]. IR (KBr): ν (cm-1) 3500 [OH], 2870 [CH], 1610 [C(dO)O-], 1110 [C-O-C]. Titration of carboxylic acid groups with 0.01 N NaOH (consumption corresponds to the inflection point): 13.7 mL (mean value, which corresponds to a dc of 26%). Anticoagulant Activity. The anticoagulant activity of the polyglycerol derivatives was evaluated by classical coagulation assays. All assays were performed using citrated human platelet poor pool plasma (PPP) pooled from at least eight healthy adults. PPP was obtained according to the guidelines for preparing citrated plasma for hemostaseological analyses (25) and stored at -70 °C until use. Freshly thawed PPP was supplemented with dilution series of the polyglycerols 1, the polyglycerol derivatives 2, 3 and UFH in 0.9% sodium chloride resulting in final concentrations ranging between 0.1 and 100 µg/mL. The clotting times in seconds were recorded with a Kugel coagulometer KC 10 (Amelung, Lemgo, Germany). All assays were performed in duplicate and repeated at least three times on different days (n ) 6). For comparison with the standard UFH, the concentrations needed for doubling the baseline coagulation time (DC) were determined.

164 Bioconjugate Chem., Vol. 15, No. 1, 2004

Tu¨rk et al.

Scheme 1. Synthesis of Polyanionic Polyglycerol Derivatives (sulfates 2a-c, Carboxylate 3)a

a The depicted structures have a degree of branching of ca. 60% and are only small idealized fragments of the large dendritic polymer cores 1a-d.

Activated Partial Thromboplastin Time (APTT). An amount of 100 µL of supplemented plasma was incubated with 100 µL of Pathromtin (Dade Behring, Marburg, Germany) for 120 s at 37 °C. The coagulation was started by adding 100 µL of 0.025 M calcium chloride solution (Dade Behring, Marburg, Germany). Thrombin Time (TT). An amount of 100 µL supplemented plasma and 100 µl diethylbarbiturate-acetate buffer solution (pH 7.6) (Dade Behring, Marburg, Germany) was incubated at 37 °C for 60 s. The coagulation was started by adding 100 µL of 3.0 IU/mL TestThrombin (bovine) in diethylbarbiturate-acetate buffer solution (pH 7.6) (Dade Behring, Marburg, Germany). Anticomplementary Activity. The influence of the polyglycerol derivatives on both classical complement activation (CCA) and alternative complement activation (ACA) was determined by complement-induced hemolysis assays using a microtiter plate technique as recently described by Alban et al. (26). The assay principle is based on the lysis of antibody-sensitized sheep (for the classical complement activation) and rabbit erythrocytes (for the alternative complement activation) by the remaining active complement proteins contained in human pooled serum and the photometric measurement of the released hemoglobin. All assays were performed in triplicate and repeated at least one time on a different day (n ) 6). For comparison with the standard UFH, the concentrations reducing the baseline (i.e., in the absence of test compounds) hemolysis by 50% (IC50) were determined. RESULTS AND DISCUSSION

A. Synthesis and Characterization of Dendritic Polyanions. Polyglycerol 1 is a readily available welldefined polymer with dendritic (treelike) branching obtained by controlled anionic polymerization of glycidol (20-22). The degree of branching of 1 (60%) is lower than that of perfect glycerol dendrimers (100%) (27). Nevertheless, the physicochemical properties are very similar (28b). The molecular weight (1000-30 000 g mol-1) and hence the degree of polymerization (DP) can be easily tailored by the monomer/initiator ratio and narrow polydispersities are obtained (typically < 2.0) (22). Due to the biocompatible properties of the aliphatic polyether

polyols in general (e.g., polysaccharides, poly(ethylene glycol)s), similar properties are expected for polyglycerol (21). In addition, oligoglycerols (2-10 monomer units) have been studied in detail with respect to their toxicological properties and have been approved as food and pharma additives (28, 29). Dendritic polyglycerol 1 should therefore be well suited as a highly functional support for drugs and for the generation of dendritic polyanions (polysulfates and polycarboxylates) as highly branched heparin analogues. The synthesis of the polyglycerol sulfates was carried out by modifying a method established for the sulfation of β-1,3-glucans (24) using dendritic polyglycerols (20) with different molecular weights (1a-c) as core polymers and the SO3/pyridine complex as sulfation reagent in dry DMF as solvent (Scheme 1). For all examples the concentration of the SO3/pyridine complex in DMF as well as its molar ratio (SO3 per OH groups) were kept constant. The polyglycerol sulfates (2a-c) were obtained in good yields (50-75%) and high purities (>98% according to 1H NMR) after dialysis in distilled water. Using a SO3/pyridine concentration equimolar to the OH groups, about 85% of all the free OH groups have been sulfated (Table 1). This high degree of sulfation shows that polyglyerols are much more accessible to sulfation than polysaccharides (24). The synthesis of the polyglycerol carboxylate was performed using the well-known preparation of carboxymethylcellulose from cellulose (30). In close analogy to this method, polyglycerol 1d was treated with aqueous sodium hydroxide solution and the reagent sodium chloroacetate at elevated temperature (80 °C) in order to obtain polyglycerol carboxylate 3 (Scheme 1). Although 3-fold excess of sodium chloroacetate was used, only a moderate carboxylate content (26%) could be achieved (Table 1). However, for biological evaluation this should be sufficient, since most of these carboxylate groups will be on the terminal groups of the dendritic macromolecule. Detailed analysis (see experimental procedures) of all starting materials 1a-d and products 2a-c, 3 by NMR, IR, elemental analysis, and titration confirmed the structure and degree of functionalization of these dendritic polyanionic heparin analogues. The molecular weights of the unfunctionalized polyglycerols have been

Dendritic Polyglycerol Sulfates as New Heparin Analogues

Bioconjugate Chem., Vol. 15, No. 1, 2004 165

Table 2. Anticoagulant and Anticomplementary Activities of the Polyglycerol Derivatives

polyglycerol Mn (theoret) derivative [g/mol] UFH 1b 2a 2b 2c 3

5000 5500 10 500 21 700 10 300

anticoagulant activity, DC [µg/mL]a

anticompelementary activity, IC50 [µg/mL]b

APTT

TT

CCA

ACA

0.99 inactivec 17.3 12.3 14.9 inactivec

1.21 inactivec 7.70 3.60 5.10 inactivec

33.9 inactivec 2.53 1.42 1.56 inactivec

406 inactivec 110 116 149 inactivec

a DC [µg/mL] corresponds to the concentration doubling the baseline coagulation time in the APPT and TT assay. b IC50 [µg/ mL] corresponds to the concentration reducing the baseline hemolysis by 50%. c Inactive ) not determinable due to missing activity up to 100 µg/mL in APTT and TT and up to 250 µg/mL in CCA and ACA.

calculated from 1H NMR data after precipitation (see Supporting Information). The obtained values agreed with the theoretical adjusted MW in this pseudo-living anionic ring-opening polymerization process (see ref 22). Molecular weights of these highly branched polyglycerols obtained from GPC are systematically lower due to the use of linear calibration standards (e.g. poly(ethylene oxide)). During sulfation, no depolymerization of the polyglycerol backbone has been observed as demonstrated by 2-fold dialysis and 1H NMR analysis of the product 2a. By comparison of the relative integrals of incorporated starter unit and polyglycerol backbone identical proton ratios were observed as in the starting material 1a (see Supporting Information). This allowed us to calculate the molecular weight of the products 2a-c and also 3 (degradation is not possible under alkalic reaction conditions) with the functionalization data of elemental analysis or titration measurements, respectively (cf. Table 1). B. Biological Evaluation (APTT, TT, CCA, ACA). The anticoagulant activity of the polyglycerol derivatives was investigated by the classical coagulation assays APTT and TT using UFH as reference compound. The APTT determines the intrinsic coagulation, i.e., an influence on Factor XIIa, XIa, IXa, VIIIa, kallikrein, and high molecular weight kininogen. Further, effects on FVa and FXa, and moderately also on thrombin and fibrinogen can be detected. The thrombin time, i.e., the coagulation time after adding an excess of thrombin, is the assay for the last step of coagulation, i.e., the thrombin-mediated fibrin formation. Whereas the unfunctionalized polyglycerols (1a-d) as well as the polyglycerol carboxylate 3 did not influence the coagulation time, the three polyglycerol sulfates 2a, 2b, and 2c concentration-dependently prolonged both APTT and TT. As determined from their DC (Table 2), their APTT-activities amounted to 5.7% (2a), 8.1% (2b), and 6.6% (2c) of that of UFH (Figure 1). Regarding their relative TT-activities of 15.7% (2a), 33.6% (2b), and 23.7% (2c), it appears that the polyglycerol sulfates interfere rather with the last coagulation step than with the intrinsic coagulation activation. However, further studies will be needed to identify their detailed mode of anticoagulant action. Comparing the three compounds, which actually differ only in their molecular weight, especially in the TT assay, the activity of 2b with a medium molecular weight is significantly the highest, followed by 2c and finally 2a with the lowest molecular weight. These findings are in contrast to the correlation between the anticoagulant activity and the molecular weight of sulfated polysaccharides (31-34). This might be due to the different

Figure 1. Anticoagulant activities of the polyglycerol derivatives in the APTT and TT assay in relation to UFH as calculated from the DC [µg/mL]. The activity of UFH corresponds to 100%.

Figure 2. Anticomplementary activities of the polyglycerol derivatives in the CCA and ACA assay in relation to UFH as calculated from the IC50 [µg/mL]. The activity of UFH corresponds to 1.0.

three-dimensional structure and the higher density of sulfate groups; whereas a longer chain length of flexible polysaccharide molecules increases the probability of interactions with biomolecules, the globular conformation of polyglycerols may be hindering above a certain size. On the other hand, as demonstrated by 2a, already a relatively low molecular weight is sufficient for pronounced activity, which is supposed to be advantageous with respect to a potential in vivo application. Similar to the coagulation assays, both the unfunctionalized polyglycerols 1a-d and the polyglycerol carboxylate 3 were completely inactive in the complementinduced hemolysis assays. They also did not show any complement activating effect as it is known for numerous unsulfated polysaccharides (26). All three polyglycerol sulfates, however, concentration-dependently inhibited the hemolysis induced by classical (CCA) as well as alternative (ACA) complement activation. Like some sulfated polysaccharides (35), they turned out to be superior to UFH particularly in the CCA assay (Table 2). As calculated from the IC50 values, they were 13 (2a), 24 (2b), and 22 times (2c) more active than UFH (Figure 2). In the ACA assay, the factor ranged only between 2.7 and 3.7. In view of the inactivity of polyglycerol carboxylate 3 in both the CCA and ACA assay, sulfate groups are essential to any effect and cannot be replaced by other negatively charged residues (i.e., carboxylates). In the CCA assay, there is again no clear molecular weight dependency of the activities of 2a, 2b, and 2c, whereby in contrast to the anticoagulant activity 2b is only slightly more active than 2c. But in the ACA assay, the inverse order of activities applies: 2c with the highest molecular weight revealed a considerable weaker effect

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than 2b and 2a. Consequently, the biological effects of polyglycerol sulfates on diverse biological systems, e.g., CCA and ACA, seem to be favored by different structural characteristics. In relation to UFH, whose ratio of the anticomplementary to the anticoagulant activity is set to 1.0, the ratio of the anticomplementary- (CAA) to the anticoagulant activity (APTT or TT) of the polyglycerol sulfates amounts to about 300 or 80, respectively. The therapeutic utilization of the relatively weak anticomplementary activity of UFH is limited by the potential bleeding risk (7). Referring to this, polyglycerol sulfates exhibit an improved efficacy-risk ratio so that efficient anticomplementary doses may be combined with moderate anticoagulant activities, which do not induce bleeding. CONCLUSIONS

For the development of synthetic heparin alternatives without the characteristic disadvantages of this animalderived product, but having a similar activity profile, dendritic polyglycerols have been functionalized with negatively charged sulfate and carboxylate groups. A major advantage of these fully synthetic polyglycerol sulfates is their straightforward preparation, which can be easily scaled up. In contrast to inactive nonderivatized and carboxylated polyglycerols, polyglycerol sulfates turned out to exhibit both anticoagulant and anticomplementary activities. Whereas their anticoagulant activity is lower than that of heparin, they show manyfold stronger anticomplementary effects. A clear molecular weight dependence could not be detected with this limited set of accessible polymer sizes, but the respective activities turned out to depend on the individual structure. Consequently, it should be possible to produce compounds with specific action profiles. Due to the cross-linking between coagulation and inflammation (including complement activation) in the pathophysiology of, for example, sepsis or cardiovascular diseases (36, 37), polyglycerol sulfates with both anticoagulant and antiinflammatory activities represent promising candidates for the development of corresponding drugs. To evaluate the therapeutic potential of this new class of heparin analogues, further studies on the structure-activity-relationships and investigations of the complete action profile and the mechanisms of actions are in progress. ACKNOWLEDGMENT

H.T. and R.H. would like to acknowledge Prof. Rolf Mu¨lhaupt for his generous support and thank Dr. Be´la Ola´h, Bernhard Siegel, and Katrin Armbruster for their synthetic support in this project. S.A. is grateful to Gabriele Brunner for her excellent technical assistance at the testing of the compounds. R.H. is indebted to the Deutsche Forschungsgemeinschaft, the Fonds der chemischen Industrie, and Dr. Otto-Ro¨hm Geda¨chtnisstiftung for financial support. LITERATURE CITED (1) Hirsh, J., Warkentin, T. E., Shaughnessy, S. G., Anand, S. S., Halperin, J. L., Raschke, R., Granger, C., Ohman, E. M., and Dalen, J. E. (2001) Heparin and Low-Molecular-Weight Heparin: Mechanisms of Action, Pharmacokinetics, Dosing, Monitoring, Efficacy, and Safety. Chest 119, 64S-94S. (2) Demir, M., Iqbal, O., Dietrich, C. P., Hoppensteadt, D., Ahmad, S., Daud, A. N., and Fareed, J. (2001) Anticoagulant and antiprotease effects of a novel heparinlike compound from shrimp (Penaeus brasiliensis) and its neutralization by heparinase I. Clin. Appl. Thromb. Hemostasis 7, 44-52.

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