Thermal depolymerization of alginate in the solid state

A new method of introduction carboxyl groups to chitosan sulfate by the acylation reaction between hydroxyethyl chitosan sulfates and butane dioic anhydride in homogeneous solution was used to obtain carboxybutyrylated hydroxyethyl chitosan sulfates. The structures of the derivatives were characterized by element analysis, FT-IR, ¹³C-NMR, and gel permeation chromatography. The content and position of the carboxyl groups could be controlled favorably. Their anticoagulant activity was determined for human plasma with respect to activated partial thromboplastin time (APTT), thrombin time (TT), and prothombin time (PT). The introducing of carboxyl groups to amino groups greatly prolonged the APTT and TT. The best result occurred when the degree of substitution of the carboxyl groups was about 0.4/unit that prolonged APTT and TT with about 5 and 1.5 times compared to that of the uncarboxylated hydroxyethyl chitosan sulfates; another conclusion is that introducing of carboxyl groups into N,O-position gave better results than that just into N-positions. Low S% chitosan sulfate and 6-O-desulfated chitosan sulfate showed little anticoagulant activity but their N,O-carboxybutyrylated derivatives (0.6/unit ds) showed increased APTT or TT, while their N-carboxybutyrylated derivatives (0.6/unit ds) gave no improvement. Generally, the introducing of carboxyl groups could not increase PT in spite of the position introduced.     

Generation of anti-factor Xa active, 3-O-sulfated glucosamine-rich sequences by controlled desulfation of oversulfated heparins.

In the framework of a project aimed at generating heparin-like sulfation patterns and biological activities in biotechnological glycosaminoglycans, different approaches have been considered for simulating the alpha(1-->4)-linked 2-O-sulfated L-iduronic acid (IdoA2SO(3))-->N,6-O-sulfated D-glucosamine (GlcNSO(3)6SO(3)) disaccharide sequences prevalent in mammalian heparins. Since the direct approach of sulfating totally O-desulfated heparins, taken as model compounds for C-5-epimerized sulfaminoheparosan (N-deacetylated, N-sulfated Escherichia coli K5 polysaccharide), preferentially afforded heparins O-sulfated at C-3 instead than at C-2 of the iduronate residues, leading to products with low anticoagulant activities, the problem of re-generating a substantial proportion of the original IdoA2SO(3) residues was circumvented by performing controlled solvolytic desulfation (with 9:1 v/v DMSO-MeOH) of extensively sulfated heparins. The order of desulfation of major residues of heparin GlcN and IdoA and of the minor one D-glucuronic acid was: GlcNSO(3)>GlcN6SO(3)>IdoA3SO(3) congruent with GlcA2SO(3) congruent with GlcN3SO(3)>IdoA2SO(3) congruent with GlcA3SO(3). Starting from a 'supersulfated' low-molecular weight heparin, we obtained products with up to 40% of iduronate residues O-sulfated exclusively at C-2 and up to 40% of their glucosamine residues O-sulfated at both C-6 and C-3. Upon re-N-sulfation, these products displayed an in vitro antithrombotic activity (expressed as anti-factor Xa units) comparable with those of current low-molecular weight heparins.     

Fibroblast growth factor-2 antagonist activity and angiostatic capacity of sulfated Escherichia coli K5 polysaccharide derivatives

The angiogenic basic fibroblast growth factor (FGF2) interacts with tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs) in endothelial cells. Here, we report the FGF2 antagonist and antiangiogenic activity of novel sulfated derivatives of the Escherichia coli K5 polysaccharide. K5 polysaccharide was chemically sulfated in N- and/or O-position after N-deacetylation. O-Sulfated and N,O-sulfated K5 derivatives with a low degree and a high degree of sulfation compete with heparin for binding to 125I-FGF2 with different potency. Accordingly, they abrogate the formation of the HSPG.FGF2.FGFR ternary complex, as evidenced by their capacity to prevent FGF2-mediated cell-cell attachment of FGFR1-overexpressing HSPG-deficient Chinese hamster ovary (CHO) cells to wild-type CHO cells. They also inhibited 125I-FGF2 binding to FGFR1-overexpressing HSPG-bearing CHO cells and adult bovine aortic endothelial cells. K5 derivatives also inhibited FGF2-mediated cell proliferation in endothelial GM 7373 cells and in human umbilical vein endothelial (HUVE) cells. In all these assays, the N-sulfated K5 derivative and unmodified K5 were poorly effective. Also, highly O-sulfated and N,O-sulfated K5 derivatives prevented the sprouting of FGF2-transfected endothelial FGF2-T-MAE cells in fibrin gel and spontaneous angiogenesis in vitro on Matrigel of FGF2-T-MAE and HUVE cells. Finally, the highly N,O-sulfated K5 derivative exerted a potent antiangiogenic activity on the chick embryo chorioallantoic membrane. These data demonstrate the possibility of generating FGF2 antagonists endowed with antiangiogenic activity by specific chemical sulfation of bacterial K5 polysaccharide. In particular, the highly N,O-sulfated K5 derivative may provide the basis for the design of novel angiostatic compounds.     

The structure-anticoagulant activity relationships of sulfated lacquer polysaccharide: effect of carboxyl group and position of sulfation

Regiospecific oxidation of the primary hydroxyl groups in lacquer polysaccharide (LPL, Mw 6.85 x 10(4)) and its NaIO4 oxidation derivatives (LPLde) to C-6 carboxy groups was achieved with NaOCl in the presence of Tempo and NaBr. Sulfate groups were incorporated into the oxidated polysaccharides using Py.SO3 complex as a reagent. Reactivity of polysaccharide hydroxyl group was C-6 > C-2 > C-4. Sulfate groups were mainly linked to the second hydroxy at C-2 in the products. The results of APTT assay showed after incorporation of carboxyl groups into lacquer polysaccharides, the intrinsic coagulation pathway was promoted, and all sulfated polysaccharides had very weak anticoagulant activity within the scope of studied DS (0.39-1.11). These indicated that carboxyl groups and sulfate groups had the synergistic action. At the same time, the anticoagulant activity increased very slowly with the DS in the second hydroxy. This indicated that 6-O-SO3- in the side chains took an important role in the anticoagulant activity.     

Chemically sulfated Escherichia coli K5 polysaccharide derivatives as extracellular HIV-1 Tat protein antagonists

The HIV-1 transactivating factor (Tat) acts as an extracellular cytokine on target cells, including endothelium. Here, we report about the Tat-antagonist capacity of chemically sulfated derivatives of the Escherichia coli K5 polysaccharide. O-sulfated K5 with high sulfation degree (K5-OS(H)) and N,O-sulfated K5 with high (K5-N,OS(H)) or low (K5-N,OS(L)) sulfation degree, but not unmodified K5, N-sulfated K5, and O-sulfated K5 with low sulfation degree, bind to Tat preventing its interaction with cell surface heparan sulfate proteoglycans, cell internalization, and consequent HIV-LTR-transactivation. Also, K5-OS(H) and K5-N,OS(H) prevent the interaction of Tat to the vascular endothelial growth factor receptor-2 on endothelial cell (EC) surface. Finally, K5-OS(H) inhibits alphav beta3 integrin/Tat interaction and EC adhesion to immobilized Tat. Consequently, K5-OS(H) and K5-N,OS(H) inhibit the angiogenic activity of Tat in vivo. In conclusion, K5 derivatives with distinct sulfation patterns bind extracellular Tat and modulate its interaction with cell surface receptors and affect its biological activities. These findings provide the basis for the design of novel extracellular Tat antagonists with possible implications in anti-AIDS therapies.     

Influence of functional groups on the in vitro anticoagulant activity of chitosan sulfate

A new method for the chemical modification of chitosan sulfate was used to prepare N-propanoyl-, N-hexanoyl- and N,O-quaternary substituted chitosan sulfate. Structural analysis by elemental analysis, FTIR, 13C NMR, and 1H NMR spectroscopy, and gel-permeation chromatography showed that these methods could conveniently be used for the introduction of functional groups. The influences of the acyl or quaternary groups on the anticoagulant activity of the polysaccharides were studied with respect to activated partial thromboplastin time (APTT) thrombin time (TT), and prothrombin time (PT). The propanoyl and hexanoyl groups increased the APTT activity, and the propanoyl groups also increased the TT anticoagulant activity slightly, while the N,O-quaternary chitosan sulfate showed only a slight TT coagulant activity.     

Biosynthesis of heparin. O-sulfation of D-glucuronic acid units

Incubation of a microsomal fraction from murine mastocytoma, with UDP-[1-3H]GlcA, UDP-GlcNAc, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS), yielded labeled, N-sulfated polysaccharides, in which most of the incorporated O-sulfate groups were located at C2 of L-iduronic acid and at C6 of D-glucosamine units. Analysis by anion-exchange high pressure liquid chromatography of disaccharides, generated by deaminative cleavage of these polysaccharides, revealed that, in addition, an appreciable portion of the -GlcNSO3-HexA-GlcNSO3- sequences in the intact polymers contained O-sulfated (at C2 or C3) D-glucuronic acid units. Calculations based on such compositional analysis of the N- and O-sulfated biosynthetic product, isolated by chromatography on DEAE-cellulose, showed that glucuronosyl 2/3-O-sulfate accounted for approximately 12% of the total incorporated O-sulfate groups. With [35S]PAPS (at a low total PAPS concentration) as an alternative source of label, the sulfated glucuronic acid residues were again detectable, albeit in much smaller amounts (1.8% of the total O-sulfate groups). Incorporation of label from UDP-[5-3H]GlcA was retained by the O-sulfated glucuronic acid units, thus demonstrating that these components had in fact been formed by sulfation of glucuronic acid residues and not by "back epimerization" of sulfated iduronic acid units. Structural analysis of polysaccharide intermediates at various stages of biosynthetic polymer modification, separated by ion-exchange chromatography, showed O-sulfation of glucuronic and iduronic acid units to appear simultaneously and before the 6-O-sulfation of glucosamine residues.     

Effect of substitution pattern on 1H, 13C NMR chemical shifts and 1J(CH) coupling constants in heparin derivatives

1H, 13C NMR chemical shifts and 1J(CH) coupling constants were measured for derivatives of heparin containing various sulfation patterns. 1H and 13C chemical shifts varied considerably after introducing electronegative sulfate groups. Chemical shifts of protons linked to carbons changed by up to 1 ppm on substitution with O- and N-sulfate or acetyl groups. Differences up to 10 ppm were detected for 13C chemical shifts in substituted glucosamine, but a less clear dependence was found in iduronate. 1J(CH) values formed two groups, corresponding to either sulfation or non-sulfation at positions 2 and 3 of glucosamine. O-sulfation caused increases up to 6 Hz in 1J(CH) and N-sulfation decreases up to 4 Hz. N-acetylation gave similar 1J(CH) values to N-sulfation. At positions 2 and 3 of iduronate the trend was less marked; 1J(CH) for O-sulfated positions usually increasing. Introduction of sulfate groups influences chemical shift and 1J(CH) values at the position of substitution, but also at more remote positions. 1J(CH) at the glycosidic linkage positions varied between free-amino and N-sulfated compounds, by up to 9 Hz. These results and changes in chemical shift values suggest that iduronate residues and the glycosidic linkages are affected, indicating overall conformational change. This may have important implications for biological activities.     

New cross-linked and sulfated derivatives of partially deacetylated hyaluronan: synthesis and preliminary characterization

Partial chemical deacetylation of hyaluronan (HA) has been carried out using known procedures and carefully controlled experimental conditions in order to minimize chain degradation. The sample described herein (deHA) has a degree of deacetylation of about 17%, which corresponds to what required for its further use, but a molecular weight of about 1/25 with respect to the native, starting material. Chemical gels have been prepared with different degrees of cross-linking by means of a Ugi multicomponent condensation reaction involving aqueous deHA, formaldehyde, and cyclohexylisocyanide: the gels are mechanically stable and exhibit good water uptake strongly dependent on the extent of cross-linking, as expected. deHA samples have also been selectively N-sulfated or O-sulfated: the former exhibit anticoagulant properties well exceeding those of the latter and not too inferior to heparin.     

Synthesis and anticoagulant activity of the quaternary ammonium chitosan sulfates

Quaternary ammonium chitosan sulfates with diverse degrees of substitution (DS) ascribed to sulfate groups between 0.52 and 1.55 were synthesized by reacting quaternary ammonium chitosan with an uncommon sulfating agent (N(SO₃Na)₃) that was prepared from sodium bisulfite (NaHSO₃) through reaction with sodium nitrite (NaNO₂) in the aqueous system homogeneous. The structures of the derivatives were characterized by FTIR, ¹H NMR and ¹³C NMR. The factors affecting DS of quaternary ammonium chitosan sulfates which included the molar ratio of NaNO₂ to quaternary ammonium chitosan, sulfated temperature, sulfated time and pH of sulfated reaction solution were investigated in detail. Its anticoagulation activity in vitro was determined by an activated partial thromboplastin time (APTT) assay, a thrombin time (TT) assay and a prothrombin time (PT) assay. Results of anticoagulation assays showed quaternary ammonium chitosan sulfates significantly prolonged APTT and TT, but not PT, and demonstrated that the introduction of sulfate groups into the quaternary ammonium chitosan structure improved its anticoagulant activity obviously. The study showed its anticoagulant properties strongly depended on its DS, concentration and molecular weight.     

Oligomers of glycamino acid

Glycamino acids, a family of sugar amino acids, are derivatives of C-glycosides that possesses a carboxyl group at the C-1 position and an amino group replacing one of the hydroxyl groups at either the C-2, 3, 4, or 6 position. We have prepared a series of glucose-type glycamino acids as monomeric building blocks: these are derivatives of 2-NH(2)-Glc-beta-CO(2)H 1, 3-NH(2)-Glc-beta-CO(2)H 2, 4-NH(2)-Glc-beta-CO(2)H 3, and 6-NH(2)-Glc-beta-CO(2)H 4 and constructed four types of homo-oligomers, beta(1-->2)-linked I, beta(1-->3)-linked II, beta(1-->4)-linked III, and beta(1-->6)-linked IV, employing the well-established N-Boc and BOP strategy. CD and NMR spectral studies of these oligomers suggested that only the beta(1-->2)-linked homo-oligomer possessed a helical structure that seems to be predetermined by the linkage position. Homo-oligomers with beta(1-->2)-linkages I and beta(1-->6)-linkages IV were also subjected to O-sulfation, and these O-sulfated oligomers were found to be able, in a linkage-specific manner, to effectively inhibit L-selectin-mediated cell adhesion, HIV infection, and heparanase activity without the anticoagulant activity associated with naturally occurring sulfated polysaccharides such as heparin.     

Preparation and characterization of NADP derivatives alkylated at 2'-phosphate and 6-amino groups

Reaction of NADP with 3-propiolactone at pH 6 gave new NADP derivatives carboxyethylated at the 2'-phosphate or 6-amino group, or both: 2'-O-(2-carboxyethyl)phosphono-NAD (I), N6-(2-carboxyethyl)-NADP (II), and 2'-O-(2-carboxyethyl)phosphono-N6-(2-carboxyethyl)-NAD (III). Their structures were assigned on the basis of ultraviolet, 1H-NMR and 31P-NMR spectra, and also treatment with nucleotide pyrophosphatase or alkaline phosphatase. Carbodiimide-promoted reaction of derivative I with 1,2-diaminoethane gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-NAD (IV); derivative III gave 2'-O-[N-(2-aminoethyl)carbamoylethyl]phosphono-N6-[N-(2-aminoethyl ) carbamoylethyl]-NAD (IV). The same reaction of derivative II, on the other hand, gave a mixture of N6-[N-(2-aminoethyl)carbamoylethyl]-NADP (Va) and its 3'-phosphate isomer (Vb). The mixture was converted to Va via the 2',3'-cyclic derivative (Vc). Their structures were assigned on the basis of ultraviolet and 1H-NMR spectra, and also treatment with alkaline phosphatase or 3'-nucleotidase. All the NADP derivatives obtained in this work could be reduced with yeast glucose-6-phosphate dehydrogenase.     

Characterization of anti-heparan sulfate phage display antibodies AO4B08 and HS4E4

Heparan sulfates (HS) are linear carbohydrate chains, covalently attached to proteins, that occur on essentially all cell surfaces and in extracellular matrices. HS chains show extensive structural heterogeneity and are functionally important during embryogenesis and in homeostasis due to their interactions with various proteins. Phage display antibodies have been developed to probe HS structures, assess the availability of protein-binding sites, and monitor structural changes during development and disease. Here we have characterized two such antibodies, AO4B08 and HS4E4, previously noted for partly differential tissue staining. AO4B08 recognized both HS and heparin, and was found to interact with an ubiquitouys, N-, 2-O-, and 6-O-sulfated saccharide motif, including an internal 2-O-sulfate group. HS4E4 turned out to preferentially recognize low-sulfated HS motifs containing iduronic acid, and N-sulfated as well as N-acetylated glucosamine residues. Contrary to AO4B08, HS4E4 did not bind highly O-sulfated structures such as found in heparin.     

Syntheses of the methyl glycosides of the repeating units of chondroitin 4- and 6-sulfate

3,4,6-Tri-O-acetyl-D-galactal was transformed into methyl 6-O-acetyl-2-azido-4-O-benzyl-2-deoxy-beta-D-galactopyranoside and its 4-O-acetyl-6-O-benzyl analogue, each of which was glycosylated with activated, O-acetylated derivatives of methyl D-glucopyranosyluronate. The resulting beta-(1----3)-linked disaccharide derivatives were each reductively N-acetylated, hydrogenolysed, O-sulfated, and saponified to afford the disodium salts of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-4-O-sulfo-beta-D-galactopyranoside and the 6-O-sulfo analogue. D-Galactal was also transformed into activated derivatives of 2-azido-3,6-di-O-benzyl-2-deoxy-D-galactopyranose and their 3,4-di-O-benzyl analogues with various substituents at O-4 and O-6. These glycosyl donors were condensed with 6-O-protected derivatives of methyl 2,3-di-O-benzyl-beta-D-glucopyranoside to give the beta-(1----4)-linked disaccharide derivatives, which were selectively deprotected, then oxidised at C-6 of the gluco unit, reductively N-acetylated, selectively deprotected, O-sulfated at C-4 or C-6 of the galacto unit, and hydrogenolysed to give the disodium salts of methyl 4-O-(2-acetamido-2-deoxy-4-O-sulfo-beta-D-galactopyranosyl)-beta-D- glucopyranosiduronic acid and the 6-O-sulfo analogue.     

Chemical modification,characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides

A natural lacquer polysaccharide with complex branches was separated into two fractions, LPH (MW 16.9x10(4)) and LPL (MW 6.85x10(4)). Results of 13C NMR and FT-IR indicated they had the same structure. The treatment of LPL with sodium periodate led to a partial cut-off of side chains with 4-O-methyl-D-glucuronic acid in the terminal. These polysaccharides were sulfated in the presence of Py*SO3/DMSO. Depending on the reaction conditions, the products showed a different degree of sulfation (DS) ranging from 0.57 to 1.57 and different molecular weights ranging from 1.71x10(4) to 3.49x10(4). FT-IR analysis showed the equatorial primary OH at O-6 and the axial secondary OH at O-4 were sulfated. Activated partial thromboplastin time (APTT), prothrombin time and thrombin time (TT) assays showed the sulfated polysaccharides could prolong APTT and TT, but not TP. These activities strongly depended on the DS, the molecular weights (MW) and the branching structure of polysaccharides. DS of above 0.8 was essential for anticoagulant activity. The anticoagulant activity increased with the DS and the molecular weights. The molecular weights played a more important role. The branching structure of polysaccharides increased the activities. In our studies, the sulfated polysaccharides with the DS of 1.15 and the highest MW of 3.49x10(4) had the best blood anticoagulant activities.     

New D-modified androstane derivatives as aromatase inhibitors.

Starting from a 16-oximino derivative of 5-androstene the newly-synthesized 16-oximino-17-hydroxy-17-substituted derivatives 2-4 gave by the Beckmann fragmentation reaction the corresponding D-seco derivatives 6-9. Besides, in the case of the 17-hydroxy-17-methyl-16-oximino derivative 2, as a result of the rearrangement, the hydrolysis product 5 of the 16-oximino group with the opposite configuration at the C-17 was obtained. By the Oppenauer oxidation and/or by dehydration of 7 with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), the corresponding derivatives 12, 13, and 14 were obtained. The structures of 6 and 12 were unambiguously proved by the appropriate X-ray structural analysis. Kinetic analysis for anti-aromatase activity showed that compound 12 expressed the highest inhibition in the denucleated rat ovarian fraction in comparison to other androstene derivatives (IC(50) was 0.42 microM). In comparison to aminoglutethimide (AG) activity, it was 3.5 times lower. The inhibition was competitive, with K(i) of 0.27 microM. Introduction of additional units of unsaturation (compounds 13 and 14) in D-seco derivatives did not increase anti-aromatase activity.     

Preparation and anticoagulant activity of N-succinyl chitosan sulfates

In order to develop a promising substitute for heparin, N-succinyl chitosan (NSC) was chemically modified by sulfating agent N(SO(3)Na)(3), which were synthesized with sodium bisulfite and sodium nitrite in aqueous solution. The N-succinyl chitosan sulfates (NSCS) products were characterized by infrared spectroscopy (FT-IR) and (13)C NMR. The degree of substitution (DS) of NSCS depended on the ratio of sulfating agent to N-succinyl chitosan, reaction temperature, reaction time and pH of sulfation agent. N-succinyl chitosan sulfates with DS of 1.97 were obtained under optimal conditions. The in vitro coagulation assay of NSCS was determined by activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) assays. The results showed that NSCS obviously prolonged APTT. The anticoagulant activity strongly depended on DS, molecular weight (M(w)) and concentration of NSCS. The anticoagulant activity of NSCS promoted with the increase of DS and concentration, and NSCS exhibited the best anticoagulant activity with the M(w) of 1.37×10(4).     

Mannich bases of scutellarein as thrombin-inhibitors: Design,synthesis, biological activity and solubility

Two series of 8-aminomethylated derivatives were prepared by Mannich reaction of scutellarein (2) with appropriate aliphatic amines, alicyclic amines and formaldehyde. All the compounds were tested for their thrombin inhibition activity through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). The antioxidant activities of these target products were assessed by 1,1-diphenyl-2-picrylhydrazyl radical 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay using 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) assay method and the solubility were assessed by ultraviolet (UV). The results showed that morpholinyl aminomethylene substituent derivative (3d) demonstrated stronger anticoagulant activity, better water solubility and good antioxidant activity compared with scutellarein (2), which warrants further development as a agent for ischemic cerebrovascular disease treatment.     

Anticoagulant activity of synthetic hirudin peptides

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.