Anticoagulant activity of fucoidans from brown algae

The anticoagulant activity of polysaccharide fucoidans from 11 species of brown algae was studied. The anticoagulant activity was measured by the activated partial thromboplastin time (APTT), prothrombin time, and thrombin time. Inhibitory action of these fucoidans significantly varied from one species to another. Fucoidans from Laminaria saccharina and Fucus distichus exhibited high anticoagulant activity, while fucoidans from Cladosiphon okamuranus and Analipus japonicus were almost inactive. Other fucoidans exhibited intermediate inhibitory activity. The inhibitory effect of fucoidans on thrombin and factor Xa was investigated in the presence or in the absence of natural thrombin inhibitor, antithrombin III (AT III). In contrast to the best-studied anticoagulant, heparin, most of these fucoidans inhibited thrombin in the absence of AT III. In the presence of AT III the inhibitory effect of fucoidans considerably increased. In contrast to heparin, fucoidans weakly influenced factor Xa activity in the presence of AT III and their inhibitory effect was not observed in the absence of AT III. There was no correlation between the anticoagulant activities of this series of fucoidans and their anti-inflammatory action, studied earlier. It is suggested that these two types of fucoidan activities depend on different structural features of fucoidans. Results of this study demonstrate a possibility of preparation of fucoidans with high anti-inflammatory activity but low anticoagulant activity. Anticoagulant activity of the fucoidans did not exhibit direct dependence on the content of fucose, the other neutral sugars and sulfates; no dependence was also found between the anticoagulant activity and the structure of the backbone of their molecules.     

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.     

Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans?

We attempted to identify the specific structural features in sulfated galactans and sulfated fucans that confer anticoagulant activity. For this study we employed a variety of invertebrate polysaccharides with simple structures composed of well-defined units of oligosaccharides. Our results indicate that a 2-O-sulfated, 3-linked alpha-L-galactan, but not a alpha-L-fucan with a similar molecular size, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaced thrombin. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked alpha-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units. The presence of 2-O-sulfated fucose residues always had a deleterious effect on anticoagulant activity. Overall, our results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.     

A sulfated fucan from the brown alga Laminaria cichorioides has mainly heparin cofactor II-dependent anticoagulant activity

The major acidic polysaccharide from the brown alga Laminaria cichorioides is a complex and heterogeneous sulfated fucan. Its preponderant structure is a 2,3-disulfated, 4-linked alpha-fucose unit. The purified polysaccharide has a potent anticoagulant activity, as estimated by APTT assay ( approximately 40 IU/mg), which is mainly mediated by thrombin inhibition by heparin cofactor II. It also accelerates thrombin and factor Xa inhibition by antithrombin but at a lower potency. Sulfated fucan from L. cichorioides is a promising anticoagulant polysaccharide and a possible alternative for an antithrombotic compound due to its preferential heparin cofactor II-dependent activity.     

Role of the antithrombin-binding pentasaccharide in heparin acceleration of antithrombin-proteinase reactions. Resolution of the antithrombin conformational change contribution to heparin rate enhancement.

The synthetic antithrombin-binding heparin pentasaccharide and a full-length heparin of approximately 26 saccharides containing this specific sequence have been compared with respect to their interactions with antithrombin and their ability to promote inhibition and substrate reactions of antithrombin with thrombin and factor Xa. The aim of these studies was to elucidate the pentasaccharide contribution to heparin's accelerating effect on antithrombin-proteinase reactions. Pentasaccharide and full-length heparins bound antithrombin with comparable high affinities (KD values of 36 +/- 11 and 10 +/- 3 nM, respectively, at I 0.15) and induced highly similar protein fluorescence, ultraviolet and circular dichroism changes in the inhibitor. Stopped-flow fluorescence kinetic studies of the heparin binding interactions at I 0.15 were consistent with a two-step binding process for both heparins, involving an initial weak encounter complex interaction formed with similar affinities (KD 20-30 microM), followed by an inhibitor conformational change with indistinguishable forward rate constants of 520-700 s-1 but dissimilar reverse rate constants of approximately 1 s-1 for the pentasaccharide and approximately 0.2 s-1 for the full-length heparin. Second order rate constants for antithrombin reactions with thrombin and factor Xa were maximally enhanced by the pentasaccharide only 1.7-fold for thrombin, but a substantial 270-fold for factor Xa, in an ionic strength-independent manner at saturating oligosaccharide. In contrast, the full-length heparin produced large ionic strength-dependent enhancements in second order rate constants for both antithrombin reactions of 4,300-fold for thrombin and 580-fold for factor Xa at I 0.15. These enhancements were resolvable into a nonionic component ascribable to the pentasaccharide and an ionic component responsible for the additional rate increase of the larger heparin. Stoichiometric titrations of thrombin and factor Xa inactivation by antithrombin, as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the products of these reactions, indicated that pentasaccharide and full-length heparins similarly promoted the formation of proteolytically modified inhibitor during the inactivation of factor Xa by antithrombin, whereas only the full-length heparin was effective in promoting this substrate reaction of antithrombin during the reaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4.

Oligosaccharides of well-defined molecular size were prepared from heparin by nitrous acid depolymerization, affinity chromatography on immobilized antithrombin III (see footnote on Nomenclature) and gel chromatography on Sephadex G-50. High affinity (for antithrombin III) octa-, deca-, dodeca-, tetradeca-, hexadeca- and octadeca-saccharides were prepared, as well as oligosaccharides of larger size than octadecasaccharide. The inhibition of Factor Xa by antithrombin III was greatly accelerated by all of these oligosaccharides, the specific anti-Factor Xa activity being invariably greater than 1300 units/mumol. The anti-Factor Xa activity of the decasaccharide was not significantly decreased in the presence of platelet factor 4, even at high platelet factor 4/oligosaccharide ratios. Measurable but incomplete neutralization of the anti-Factor Xa activities of the tetradeca- and hexadeca-saccharides was observed, and complete neutralization of octadeca- and larger oligo-saccharides was achieved with excess platelet factor 4. The octa-, deca-, dodeca-, tetradeca- and hexadeca-saccharides had negligible effect on the inhibition of thrombin by antithrombin III, whereas specific anti-thrombin activity was expressed by the octadeca-saccharide and by the larger oligosaccharides. An octadecasaccharide is therefore the smallest heparin fragment (prepared by nitrous acid depolymerization) that can accelerate thrombin inhibition by antithrombin III. The anti-thrombin activities of the octadecasaccharide and larger oligosaccharides were more readily neutralized by platelet factor 4 than were their anti-Factor Xa activities. These findings are compatible with two alternative mechanisms for the action of platelet factor 4, both involving the binding of the protein molecule adjacent to the antithrombin III-binding site. Such binding results in either steric interference with the formation of antithrombin III-proteinase complexes or in displacement of the antithrombin III molecule from the heparin chain.     

Anticoagulant activities in salivary glands of tabanid flies

Tabanid flies are telmophages (pool feeders), taking frequent and rapid bloodmeals from many different individual hosts. To investigate how they accomplish this intermittent feeding strategy, we examined the anticoagulant activities in salivary gland extracts (SGE) from 19 species representing six genera: Atylotus, Chrysops, Haematopota, Heptatoma, Hybomitra and Tabanus (Diptera: Tabanidae). Standard coagulation screen assays were used to determine thrombin time, prothrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. SGE of most species (except Chrysops spp.) considerably prolonged human plasma clotting time in a dose-dependent manner, and showed potent and specific antithrombin activity in the chromogenic substrate assay. Heptatoma pellucens displayed the strongest anticoagulant activity. Specific anti-factor Xa activity in tabanid SGE was not detected. Electrophoretic profiles of SGE proteins differed between genera and species. Overall, the results suggest that tabanids have evolved at least two antihaemostatic strategies.     

A 2-sulfated, 3-linked alpha-L-galactan is an anticoagulant polysaccharide

Marine alga is an abundant source of sulfated polysaccharides with potent anticoagulant activity. However, several attempts to identify the specific structural features in these compounds, which confer the biological activity, failed due to their complex, heterogeneous structure. We isolated and characterized several sulfated alpha-L-galactans and sulfated alpha-L-fucans from marine invertebrates. In contrast to the algal fucans and galactans, these invertebrate polysaccharides have a simple structure, composed of well-defined units of oligosaccharides. We employed two of these compounds to elucidate their structure-anticoagulant action relationship. Our results indicate that a 2-sulfated, 3-linked alpha-L-galactan, but not an alpha-L-fucan, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaces thrombin. Thus, the anticoagulant activity of sulfated galactan and sulfated fucan is not merely a consequence of their charge density. The interaction of these polysaccharides with coagulation cofactors and their target proteases are specific. Identification of specific structural requirements in sulfated galactans and sulfated fucans necessary for interaction with coagulation cofactors is an essential step for a more rational approach to develop new anticoagulant and antithrombotic drugs.     

Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum

An anticoagulant was isolated from a marine green alga, Codium cylindricum. The anticoagulant was composed mainly of galactose with a small amount of glucose, and was highly sulfated (13.1% as SO Na). The anticoagulant properties of the purified anticoagulant were compared with that of heparin by assays of activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) using normal human plasma. The anticoagulant showed similar activities with heparin, however, weaker than heparin. On the other hand, the anticoagulant did not affect PT even at the concentration at which APTT and TT were strongly prolonged. The anticoagulant did not potentiate antithrombin III (AT III) and heparin cofactor II (HC II), thus the anticoagulant mechanism would be different from that of other anticoagulants isolated so far from the genus Codium.     

The anticoagulant properties of mast cell product, chondroitin sulphate E

The anticoagulant potency in vitro of chondroitin sulphate E has been found to be similar to that of the heparinoids. In purified systems chondroitin sulphate E was shown to be principally an activator of heparin cofactor II. Maximum acceleration of heparin cofactor II:thrombin interaction was 185-fold (9.3 X 10(7) M-1 min-1), antithrombin III:thrombin interaction was 11-fold (4.16 X 10(6) M-1 min-1) and antithrombin III:factor Xa was 146-fold (3.86 X 10(6) M-1 min-1). Chondroitin sulphate E was observed to prolong the thrombin clotting time of fibrinogen in the absence of antithrombin III and heparin cofactor II. The effect appeared to be related to interference in thrombin:fibrinogen interaction rather than in fibrin monomer polymerization.     

Effect of polyanetholesulphonic acid and xylan sulphate on antithrombin III activity.

Both polyanetholesulphonic acid and xylan sulphate prolonged the partial thromboplastin clotting time of plasma. The anticoagulant effect of both compounds was reduced following pre-incubation of plasma with antiserum specific for antithrombin III. Polyanetholesulphonic acid was more effective than xylan sulphate in inhibiting thrombin-initiated clotting of plasma, and potentiated antithrombin III inhibition of both thrombin and Xa. Xylan sulphate was more effective in potentiating antithrombin III inhibition of Xa than of thrombin. These differential effects of xylan sulphate on different blood serine proteases are discussed in terms of the antithrombin III-mediated anticoagulant activity of heparin.     

Kinetic analysis of various heparin fractions and heparin substitutes in the thrombin inhibition reaction

Kinetic characteristics of several heparin preparations and substitute heparins were determined to help understand the bases for activity differences. Several materials were highly active in factor Xa inhibition and the reaction rate at constant factor Xa concentration appeared to be predicted by the extent of intrinsic antithrombin III fluorescence change induced by the polysaccharide. Heparin fractions of different molecular weight and affinity for antithrombin III showed similar kinetic parameters in catalysis of the thrombin-antithrombin III reaction when these parameters were expressed on the basis of antithrombin III-binding heparin. The latter was determined by stoichiometric titration of the antithrombin III fluorescence change by the heparin preparation. However, the various heparin fractions showed very different specific activities per mg of total polysaccharide. This indicated that functional heparin molecules had similar kinetic properties regardless of size or antithrombin III-binding affinity and is possible because the Km for antithrombin III is determined by diffusion rather than by binding affinity. Substitute heparins and depolymerized heparin were poor catalysts for thrombin inhibition, due at least partially to their affinity for thrombin. This latter binary interaction inhibits thrombin reaction in the heparin-catalyzed reaction.     

Isolation of a pure octadecasaccharide with antithrombin activity from an ultra-low-molecular-weight heparin

Heparin and low-molecular-weight heparins (LMWHs) are anticoagulant drugs that mainly inhibit the coagulation cascade by indirectly interacting with factor Xa and factor IIa (thrombin). Inhibition of factor Xa by antithrombin (AT) requires the activation of AT by specific pentasaccharide sequences containing 3-O-sulfated glucosamine. Activated AT also inhibits thrombin by forming a stable ternary complex of AT, thrombin, and a polysaccharide (requires at least an 18-mer/octadeca-mer polysaccharide). The full structure of any naturally occurring octadecasaccharide sequence has yet to be determined. In the context of the development of LMWH biosimilars, structural data on such important biological mediators could be helpful for better understanding and regulatory handling of these drugs. Here we present the isolation and identification of an octadecasaccharide with very high anti-factor Xa activity (∼3 times higher than USP [U.S. Pharmacopeia] heparin). The octadecasaccharide was purified using five sequential chromatographic methods with orthogonal specificity, including gel permeation, AT affinity, strong anion exchange, and ion-pair chromatography. The structure of the octadecasaccharide was determined by controlled enzymatic sequencing and nuclear magnetic resonance (NMR). The isolated octadecasaccharide contained three consecutive AT-binding sites and was tested in coagulation assays to determine its biological activity. The isolation of this octadecasaccharide provides new insights into the modulation of thrombin activity.     

Anticoagulant properties of sulphated polysaccharides and a proteoglycan from Codium fragile ssp. atlanticum

A high molecular weight sulphated (18.4%) proteoglycan was isolated from extracts of Codium fragile ssp. atlanticum by molecular exclusion chromatography on Sepharose 2B. Ion exchange chromatography, using Sepharose CL-6B, of lower molecular weight components eluted from the Sepharose 2B column gave two major products with sulphate contents of 10.2% and 7.5%, respectively. Anticoagulant activities of each of the three products were assessed using coagulation techniques and chromogenic substrate assays. An increase in anticoagulant effect was demonstrated by increasing concentration and sulphate content of each algal component. The mechanism of anticoagulant action was shown to be, principally, anti-thrombin in character due to potentiation of heparin cofactor II and antithrombin III activity. Although the anticoagulant substances described are unlikely to be used as antithrombotic therapeutic agents, they have uses as biomedical reagents for investigation of the processes of thrombin inhibition.     

Sulfated polysaccharide purified from <Emphasis Type="BoldItalic">Ecklonia cava</Emphasis> accelerates antithrombin III-mediated plasma proteinase inhibition

Surface plasmon resonance is an important technique for studying molecular interactions and was used to investigate the molecular interaction of anticoagulant sulfated polysaccharides purified from an enzymatic hydrolysate of the brown alga Ecklonia cava (ECA) with blood coagulation factors. In a direct binding assay, binding affinity between ECA/antithrombin III (ATIII) and activated blood coagulation factors was in the order: factor VIIa (FVIIa) > factor Xa (FXa) > thrombin (FIIa); kinetic analysis determined K _D values of ECA for FVIIa, FXa, and FIIa of 15.1, 45.0 and 65.0 nM, respectively. Therefore, ECA strongly and selectively (FVII, FX, and FII) enhanced ATIII-mediated coagulation factor inhibition in both the extrinsic and common coagulation pathways. This may contribute to its high anticoagulant activity in vitro. The low cytotoxicity of ECA to venous endothelial cell line (ECV-304) also expands its value in future in vivo studies. However, to utilize it as a model for novel anticoagulant agents, its possible interference with other anticoagulant mechanisms must be addressed.     

An anticoagulant proteoglycan from the marine green alga, Codium pugniformis

An anticoagulant isolated from the marine green alga Codium pugniformis was composed mainly of glucose with minor amounts of arabinose and galactose. It was highly sulfated (326 μg mg^-1 polysaccharide) and contained protein(52 μg mg^-1 polysaccharide) and was thus a proteoglycan. The anticoagulant properties of the purified proteoglycan were compared with those of heparin by studying the activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time(TT) using normal human plasma. The proteoglycan showed similar activities to heparin, but was weaker than heparin. On the other hand, the proteoglycan did not affect PT even at the concentration at which APTT and TT were prolonged. The anticoagulation mechanism of this proteoglycan was due to the direct inhibition of thrombin and the potentiation of antithrombin III. This revised version was published online in August 2006 with corrections to the Cover Date.     

The role of the COOH-terminal region of antithrombin III. Evidence that the COOH-terminal region of the inhibitor enhances the reactivity of thrombin and factor Xa with the inhibitor.

To elucidate the role of the COOH-terminal region of antithrombin III, we studied the effects of synthetic peptides corresponding to its sequence on the amidolytic and proteolytic activities of thrombin and Factor Xa in the presence or absence of the inhibitor, antithrombin III. The peptides ANRPFLVFI and IIFMGRVANP corresponding to residues Ala404 to Ile412 and Ile420 to Pro429, respectively, blocked the inhibition by antithrombin III. The effect of IIFMGRVANP was reduced in the presence of heparin. Both peptides at a concentration of 1 mM blocked complex formation between antithrombin III and thrombin or Factor Xa. The two peptides, particularly IIFMGRVANP, directly enhanced the amidolytic activity of thrombin and Factor Xa on the synthetic substrate Boc-Ala-Gly-Arg-MCA (where Boc is t-butoxycarbonyl and MCA is 4-methylcoumarin), which corresponds to residues P3-P1 of the reactive site of antithrombin III, and also on other substrates due to increased Vmax. IIFMGRVANP also shortened the thrombin-induced fibrinogen clotting time, whereas ANRPFLVFI inhibited the thrombin-catalyzed activation of protein C both in the presence and absence of thrombomodulin. The direct effect of ANRPFLVFI and IIFMGRVANP on thrombin was confirmed by enhancement of the incorporation of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide into thrombin. These findings suggest that the COOH-terminal region of antithrombin III interacts with thrombin and Factor Xa to increase the reactivity of the enzyme, which may enhance acyl-bond formation between the inhibitor and the enzyme.     

Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin

Heparin activates the primary serpin inhibitor of blood clotting proteinases, antithrombin, both by an allosteric conformational change mechanism that specifically enhances factor Xa inactivation and by a ternary complex bridging mechanism that promotes the inactivation of thrombin and other target proteinases. To determine whether the factor Xa specificity of allosterically activated antithrombin is encoded in the reactive center loop sequence, we attempted to switch this specificity by mutating the P6-P3' proteinase binding sequence excluding P1-P1' to a more optimal thrombin recognition sequence. Evaluation of 12 such antithrombin variants showed that the thrombin specificity of the serpin allosterically activated by a heparin pentasaccharide could be enhanced as much as 55-fold by changing P3, P2, and P2' residues to a consensus thrombin recognition sequence. However, at most 9-fold of the enhanced thrombin specificity was due to allosteric activation, the remainder being realized without activation. Moreover, thrombin specificity enhancements were attenuated to at most 5-fold with a bridging heparin activator. Surprisingly, none of the reactive center loop mutations greatly affected the factor Xa specificity of the unactivated serpin or the several hundred-fold enhancement in factor Xa specificity due to activation by pentasaccharide or bridging heparins. Together, these results suggest that the specificity of both native and heparin-activated antithrombin for thrombin and factor Xa is only weakly dependent on the P6-P3' residues flanking the primary P1-P1' recognition site in the serpin-reactive center loop and that heparin enhances serpin specificity for both enzymes through secondary interaction sites outside the P6-P3' region, which involve a bridging site on heparin in the case of thrombin and a previously unrecognized exosite on antithrombin in the case of factor Xa.     

Inhibition of factor X and factor V activation by dermatan sulfate and a pentasaccharide with high affinity for antithrombin III in human plasma

There is evidence that by catalyzing thrombin inhibition, several glycosaminoglycans can inhibit the thrombin-mediated amplification reactions of coagulation and thereby delay prothrombin activation. The two amplification reactions can apparently be catalysed by endogenously generated factor Xa and thrombin. This study provides evidence which suggests that on a molar basis, an agent which can only catalyse thrombin inhibition is approximately 10 times more effective than an agent which can only catalyse factor Xa inhibition in their ability to inhibit intrinsic prothrombin activation. We determined the concentrations of each of heparin, dermatan sulfate and a pentasaccharide with high affinity for antithrombin III, to delay intrinsic prothrombin activation for at least 15s. Heparin catalyses both thrombin and factor Xa inhibition; dermatan sulfate catalyses only thrombin inhibition, while the pentasaccharide only catalyses factor Xa inhibition. Efficient prothrombin activation, which coincided with both factor X activation and factor V proteolysis, was first observed 45s after CaC12 was added to contact-activated plasma. Heparin (approximately 0.1 microM) prolonged by at least 30 s the time required for the activation of the three clotting factors to begin. The minimum concentrations of the pentasaccharide and dermatan sulfate to delay the activation of prothrombin, factors X and V were approximately 50 microM and approximately 5 microM, respectively. Thus, each anticoagulant could inhibit intrinsic prothrombin activation only when it inhibited activation of both factors X and V. A combination of approximately 5 microM pentasaccharide and approximately 0.05 microM dermatan sulfate similarly delayed the activation of all three clotting factors. Thus, while catalysis of thrombin inhibition is a more effective pathway than catalysis of factor Xa inhibition for delaying prothrombin activation, the simultaneous catalysis of thrombin and factor Xa inhibition can synergistically improve the ability of a sulfated polysaccharide to delay prothrombin activation.     

Structural requirements for the neutralization of heparin-like saccharides by complement S protein/vitronectin

S protein, a major inhibitor of the assembly of the membrane attack complex of complement, has recently been shown to be identical to the serum spreading factor vitronectin. It also neutralizes the anticoagulant activities of heparin. We have studied the structural requirements for the heparin neutralizing properties of S protein/vitronectin using heparin, heparan sulfate, and heparin oligosaccharides with well defined anticoagulant specificities. The abilities of heparin fractions, Mr 7,800-18,800, with high affinity for antithrombin, and of the International Heparin Standard, to accelerate the inactivation of thrombin and Factor Xa by antithrombin were readily neutralized by S protein/vitronectin. Binding and neutralization of heparin by S protein/vitronectin was inhibited by heparin with low affinity for antithrombin, indicating that S protein/vitronectin can interact with a region on the heparin chain that might serve as a proteinase binding site. S protein/vitronectin efficiently neutralized oligosaccharides of Mr 2,400-7,200, unlike the two other physiologically occurring heparin neutralizing proteins histidine-rich glycoprotein and platelet factor 4. Furthermore, S protein/vitronectin neutralized the anti-Factor Xa activity of a synthetic pentasaccharide comprising the antithrombin-binding sequence of heparin. High molar excess of a synthetic tridecapeptide corresponding to part (amino acids 374-359) of the proposed glycosaminoglycan binding domain of S protein/vitronectin neutralized high affinity heparin and some oligosaccharides, but failed to neutralize the synthetic antithrombin-binding pentasaccharide. Like platelet factor 4, but unlike histidine-rich glycoprotein, S protein/vitronectin readily neutralized the anticoagulant activities of heparan sulfate of Mr approximately 20,000. These findings suggest that S protein/vitronectin may interact through its glycosaminoglycan binding domain(s) with various functional domains of the heparin (heparan sulfate) molecule, including the antithrombin-binding pentasaccharide sequence. Furthermore, the results suggest that S protein/vitronectin may be a physiologically important modulator of the anticoagulant activity of heparin-like material on or near the vascular endothelium.