A novel allosteric pathway of thrombin inhibition: Exosite II mediated potent inhibition of thrombin by chemo-enzymatic, sulfated dehydropolymers of 4-hydroxycinnamic acids

Thrombin and factor Xa, two important pro-coagulant proteinases, can be regulated through direct and indirect inhibition mechanisms. Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties (Monien, B. H., Henry, B. L., Raghuraman, A., Hindle, M., and Desai, U. R. (2006) Bioorg. Med. Chem. 14, 7988-7998). To better understand their mechanism of action, we studied the direct inhibition of thrombin, factor Xa, factor IXa, and factor VIIa by CDSO3, FDSO3, and SDSO3, three analogs of sulfated DHPs. All three sulfated DHPs displayed a 2-3-fold preference for direct inhibition of thrombin over factor Xa, whereas this preference for inhibiting thrombin over factor IXa and factor VIIa increased to 17-300-fold, suggesting a high level of selectivity. Competitive binding studies with a thrombin-specific chromogenic substrate, a fluorescein-labeled hirudin peptide, bovine heparin, enoxaparin, and a heparin octasaccharide suggest that CDSO3 preferentially binds in or near anion-binding exosite II of thrombin. Studies of the hydrolysis of H-D-hexahydrotyrosol-Ala-Arg-p-nitroanilide indicate that CDSO3 inhibits thrombin through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. Overall, designed sulfated DHPs appear to be the first molecules that bind primarily in the region defined by exosite II and allosterically induce thrombin inhibition. The molecules are radically different in structure from all the current clinically used anticoagulants and thus represent a novel class of potent dual thrombin and factor Xa inhibitors.     

Interaction of Antithrombin with Sulfated, Low Molecular Weight Lignins: OPPORTUNITIES FOR POTENT, SELECTIVE MODULATION OF ANTITHROMBIN FUNCTION*

Antithrombin, a major regulator of coagulation and angiogenesis, is known to interact with several natural sulfated polysaccharides. Previously, we prepared sulfated low molecular weight variants of natural lignins, called sulfated dehydrogenation polymers (DHPs) (Henry, B. L., Monien, B. H., Bock, P. E., and Desai, U. R. (2007) J. Biol. Chem. 282, 31891–31899), which have now been found to exhibit interesting antithrombin binding properties. Sulfated DHPs represent a library of diverse noncarbohydrate aromatic scaffolds that possess structures completely different from heparin and heparan sulfate. Fluorescence binding studies indicate that sulfated DHPs bind to antithrombin with micromolar affinity under physiological conditions. Salt dependence of binding affinity indicates that the antithrombin-sulfated DHP interaction involves a massive 80–87% non-ionic component to the free energy of binding. Competitive binding studies with heparin pentasaccharide, epicatechin sulfate, and full-length heparin indicate that sulfated DHPs bind to both the pentasaccharide-binding site and extended heparin-binding site of antithrombin. Affinity capillary electrophoresis resolves a limited number of peaks of antithrombin co-complexes suggesting preferential binding of selected DHP structures to the serpin. Computational genetic algorithm-based virtual screening study shows that only one sulfated DHP structure, out of the 11 present in a library of plausible sequences, bound in the heparin-binding site with a high calculated score supporting selectivity of recognition. Enzyme inhibition studies indicate that only one of the three sulfated DHPs studied is a potent inhibitor of free factor VIIa in the presence of antithrombin. Overall, the chemo-enzymatic origin and antithrombin binding properties of sulfated DHPs present novel opportunities for potent and selective modulation of the serpin function, especially for inhibiting the initiation phase of hemostasis.Antithrombin (AT),³ a plasma glycoprotein and a member of the serpin superfamily of proteins, is a major regulator of the coagulation cascade. Its primary targets are thrombin, factor Xa (fXa), and factor IXa (fIXa) (1). It has also been suggested to inhibit several other coagulation enzymes (2–6), albeit with much weaker inhibitory efficiency. Antithrombin alone is a rather poor inhibitor of factors IIa, Xa, and IXa and requires the presence of heparin to exhibit its full anticoagulant potential.Heparin is a highly sulfated polysaccharide that greatly enhances the rate of AT inhibition of these enzymes under physiological conditions (1). This acceleration forms the basis for heparin''s use as an anticoagulant for the past several decades. Yet heparin is associated with bleeding complications and suffers from a number of other limitations. In addition, the animal origin of the drug is also a cause for concern as suggested by recent incidences of oversulfated chondroitin sulfate contaminating unfractionated heparin (UFH) preparations and resulting in numerous deaths (7–9). Although low molecular weight heparins (LMWHs) are superior to UFH with respect to therapeutic complications, the iatrogenic bleeding risk is not completely eliminated. Likewise, fondaparinux, or the minimal antithrombin binding pentasaccharide sequence (H5), is also associated with bleeding (10, 11) and lacks an effective antidote to reverse excessive anticoagulation.The major reason for the limitations of UFH and LMWH therapies is the presence of numerous negative charges on each polymeric chain. UFH and LMWH are linear co-polymers of glucosamine and uronic acid residues that are decorated with numerous sulfate groups generating a massive polyanion (12, 13). This polyanion is capable of interacting with a large number of plasma proteins and proteins present on cells lining the vasculature, which likely induce many of the UFH and LMWH complications (14, 15). Fondaparinux displays a much better pharmacological profile primarily because of its limited number of sulfate and carboxylate groups.To design better anticoagulants that are less polyanionic and more hydrophobic than UFHs and LMWHs, we recently prepared low molecular weight variants of lignin, called sulfated dehydropolymers (DHPs) (Fig. 1), as functional mimetics of heparin. These designed molecules were prepared in a simple two-step chemo-enzymatic process involving enzymatic coupling of 4-hydroxycinnamic acids followed by the chemical sulfation of the resulting DHPs (16). In terms of structural polydispersity and heterogeneity, sulfated DHPs are similar to LMWHs. Sulfated DHPs are composed of many oligomeric chains of varying lengths and contain different inter-monomeric linkages such as β-O-4 and β-5. Yet sulfated DHPs are completely unlike LMWHs with respect to the nature of their backbone. In contrast to the highly anionic scaffold of the heparins, sulfated DHPs possess a highly aromatic scaffold with fewer anionic groups (Fig. 1). In fact, in terms of structure, sulfated DHPs are unlike any other class of anticoagulant investigated to date, including the heparins, the coumarins, the hirudins, the peptidomimetics, and the small molecule direct inhibitors. Functionally, the sulfated DHPs display plasma and blood anticoagulation similar to that of LMWHs (17, 18). Yet mechanistically, the sulfated DHPs were found to exhibit a novel mechanism of anticoagulation involving an exosite II-mediated allosteric inhibition of thrombin (17). The DHPs represent the first example of an exclusive exosite II-dependent inactivation of the catalytic function of thrombin.Open in a separate windowFIGURE 1.A representative structure of sulfated DHPs. CDSO3, FDSO3, and SDSO3 were chemo-enzymatically synthesized in two steps from the corresponding starting 4-hydroxycinnamic acid monomers, caffeic acid (CA), ferulic acid (FA), or sinapic acid (SA). The average molecular mass of CDs, FDs, and SDs was in the range of 3,000–4,000 Da. Two types of common linkages present in sulfated DHPs include the β-O-4 and β-5 linkages (shown as shaded ovals).In this work we study the interaction of sulfated DHPs with AT at a molecular level. This study reveals that the indirect antithrombin-mediated pathway may contribute to the inhibition efficiency of sulfated DHPs, thus realizing molecules that can utilize both the direct and indirect inhibition pathways. These studies uncover the ability of a specific sulfated DHP to induce potent antithrombin inhibition of free fVIIa. The antithrombin-mediated effects originate from the sulfated DHPs binding to the heparin-binding site (HBS) of the serpin through non-ionic forces that contribute more than 80% of the total binding energy. Our work supports the idea that aromatic scaffolds, which exhibit hydrophobic and hydrophilic nature, can be designed to target the HBS of antithrombin for modulation of its inhibitory functions.     

Structural basis for inhibition promiscuity of dual specific thrombin and factor Xa blood coagulation inhibitors

BACKGROUND: A major current focus of pharmaceutical research is the development of selective inhibitors of the blood coagulation enzymes thrombin or factor Xa to be used as orally bioavailable anticoagulant drugs in thromboembolic disorders and in the prevention of venous and arterial thrombosis. Simultaneous direct inhibition of thrombin and factor Xa by synthetic proteinase inhibitors as a novel approach to antithrombotic therapy could result in potent anticoagulants with improved pharmacological properties. RESULTS: The binding mode of such dual specific inhibitors of thrombin and factor Xa was determined for the first time by comparative crystallography using human alpha-thrombin, human des-Gla (1--44) factor Xa and bovine trypsin as the ligand receptors. The benzamidine-based inhibitors utilize two different conformations for the interaction with thrombin and factor Xa/trypsin, which are evoked by the steric requirements of the topologically different S2 subsites of the enzymes. Compared to the unliganded forms of the proteinases, ligand binding induces conformational adjustments of thrombin and factor Xa active site residues indicative of a pronounced induced fit mechanism. CONCLUSION: The structural data reveal the molecular basis for a desired unselective inhibition of the two key components of the blood coagulation cascade. The 4-(1-methyl-benzimidazole-2-yl)-methylamino-benzamidine moieties of the inhibitors are able to fill both the small solvent accessible as well as the larger hydrophobic S2 pockets of factor Xa and thrombin, respectively. Distal fragments of the inhibitors are identified which fit into both the cation hole/aromatic box of factor Xa and the hydrophobic aryl binding site of thrombin. Thus, binding constants in the medium-to-low nanomolar range are obtained against both enzymes.     

From selective substrate analogue factor Xa inhibitors to dual inhibitors of thrombin and factor Xa. Part 3

Highly potent and selective substrate analogue factor Xa inhibitors were obtained by incorporation of non-basic or modestly basic P1 residues known from the development of thrombin inhibitors. The modification of the P2 and P3 amino acids strongly influenced the selectivity and provided potent dual factor Xa and thrombin inhibitors without affecting the fibrinolytic enzymes. Several inhibitors demonstrated excellent anticoagulant efficacy in standard clotting assays in human plasma.     

First steps in the direction of synthetic,allosteric, direct inhibitors of thrombin and factor Xa

Designing non-saccharide functional mimics of heparin is a major challenge. In this work, a library of small, aromatic molecules based on the sulfated DHP scaffold was synthesized and screened against thrombin and factor Xa. The results reveal that (i) selected monomeric benzofuran derivatives inhibit the two enzymes, albeit weakly; (ii) the two enzymes recognize different structural features in the benzofurans studied suggesting significant selectivity of recognition; and (iii) the mechanism of inhibition is allosteric. The molecules represent the first allosteric small molecule inhibitors of the two enzymes.     

Heterocyclic thrombin inhibitors. Part 2: quinoxalinone derivatives as novel,potent antithrombotic agents

Quinoxalinone derivatives as prototypes of dual thrombin and factor Xa inhibitors have been discovered. Nanomolar inhibition of both coagulation enzymes resulted in very potent antithrombotic activity in vitro.     

A simple,general approach of allosteric coagulation enzyme inhibition through monosulfated hydrophobic scaffolds

Allosteric inhibition of coagulation enzymes offers the advantage of controlled inhibition. In this study, a small library of mono sulfated indole and benzothiazole based molecules was synthesized and screened against the panel of coagulation proteases. The results reveal that selected molecules inhibit the thrombin, factor Xa and factor XIa with moderate potency. Compound 6a was found to have an allosteric mode of inhibition against thrombin. Plasma clotting assays suggest that selected inhibitors 14b, 14c and 14d prolong both prothrombin and activated partial thromboplastin time. Overall, this work presents the newer class of allosteric inhibitors of thrombin and factor XIa with improved aqueous solubility profile.     

Heparinoid-active two sulfated polysaccharides isolated from marine green algae Monostroma nitidum

Two sulfated polysaccharides WF1 and WF3 were isolated from marine green algae Monostroma nitidum, and their structural characteristics were determined. Anticoagulant activities of WF1 and WF3 were evaluated by assays of the activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT), antithrombin and anticoagulation factor Xa activities. The results showed that WF1 and WF3 had similar high contents of rhamnose, whereas their sulfate contents, sulfation positions, molecular sizes and linkage patterns of rhamnose residues were different. The bioassay results demonstrated that WF1 and WF3 had high anticoagulant activities, and were potent thrombin inhibitors mediated by heparin cofactor II, especially WF3. They also hastened thrombin and coagulation factor Xa inhibition by potentiating antithrombin III, but at a lower effectiveness. The differences of anticoagulant activities between WF1 and WF3 were directly due to their structural features discrepancy.     

Structure and anticoagulant activity of a sulfated galactan from the red alga, Gelidium crinale. Is there a specific structural requirement for the anticoagulant action?

Marine red algae are an abundant source of sulfated galactans with potent anticoagulant activity. However, the specific structural motifs that confer biological activity remain to be elucidated. We have now isolated and purified a sulfated galactan from the marine red alga, Gellidium crinale. The structure of this polysaccharide was determined using NMR spectroscopy. It is composed of the repeating structure -4-alpha-Galp-(1-->3)-beta-Galp1--> but with a variable sulfation pattern. Clearly 15% of the total alpha-units are 2,3-di-sulfated and another 55% are 2-sulfated. No evidence for the occurrence of 3,6-anhydro alpha-galactose units was observed in the NMR spectra. We also compared the anticoagulant activity of this sulfated galactan with a polysaccharide from the species, Botryocladia occidentalis, with a similar saccharide chain but with higher amounts of 2,3-di-sulfated alpha-units. The sulfated galactan from G. crinale has a lower anticoagulant activity on a clotting assay when compared with the polysaccharide from B. occidentalis. When tested in assays using specific proteases and coagulation inhibitors, these two galactans showed significant differences in their activity. They do not differ in thrombin inhibition mediated by antithrombin, but in assays where heparin cofactor II replaces antithrombin, the sulfated galactan from G. crinale requires a significantly higher concentration to achieve the same inhibitory effect as the polysaccharide from B. occidentalis. In contrast, when factor Xa instead of thrombin is used as the target protease, the sulfated galactan from G. crinale is a more potent anticoagulant. These observations suggest that the proportion and/or the distribution of 2,3-di-sulfated alpha-units along the galactan chain may be a critical structural motif to promote the interaction of the protease with specific protease and coagulation inhibitors.     

Studies on the different modes of action of the anticoagulant protease inhibitors DX-9065a and Argatroban. I. Effects on thrombin generation

The present study began with mathematical modeling of how inhibitors of both factor Xa (fXa) and thrombin affect extrinsic pathway-triggered blood coagulation. Numerical simulation demonstrated a stronger inhibition of thrombin generation by a thrombin inhibitor than a fXa inhibitor, but both prolonged clot time to a similar extent when they were given an equal dissociation constant (30 nm) for interaction with their respective target enzymes. These differences were then tested by comparison with the real inhibitors DX-9065a and argatroban, specific competitive inhibitors of fXa and thrombin, respectively, with similar K(i) values. Comparisons were made in extrinsically triggered human citrated plasma, for which endogenous thrombin potential and clot formation were simultaneously measured with a Wallac multilabel counter equipped with both fluorometric and photometric detectors and a fluorogenic reporter substrate. The results demonstrated stronger inhibition of endogenous thrombin potential by argatroban than by DX-9065a, especially when coagulation was initiated at higher tissue factor concentrations, while argatroban appeared to be slightly less potent in its ability to prolong clot time. This study demonstrates differential inhibition of thrombin generation by fXa and thrombin inhibitors and has implications for the pharmacological regulation of blood coagulation by the anticoagulant protease inhibitors.     

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.     

Effects of dihydropyridines on calcium release from the isolated membrane complex consisting of the transverse tubule and sarcoplasmic reticulum.

We have investigated a) the effects of the dihydropyridines (DHPs) nifedipine and nimodipine on depolarization-induced (T-tubule-mediated) Ca2+ release in the vesicles consisting of the complex of the T-tubule and SR, and b) the binding of [3H]nimodipine to these vesicles. These DHPs inhibited the slow but not the fast phase of depolarization-induced release, both of which are mediated via the T-tubule. The DHPs have no effect on caffeine-induced release in which T-tubules are not involved. There are two classes of DHP binding sites: one, with high affinity and small capacity, and another, exhibiting low affinity and a much larger capacity. The inhibition paralleled the low affinity binding of DHP with no correlation with the high affinity binding. These results suggest that the low affinity DHP binding sites located probably in the DHP receptor, rather than the high affinity DHP binding site, are responsible for the inhibition of e-c coupling.     

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.     

Amido-(propyl and allyl)-hydroxybenzamidines: development of achiral inhibitors of factor Xa

The design, synthesis and SAR of amido-(propyl and allyl)-hydroxybenzamidine coagulation factor Xa inhibitors is described. These achiral inhibitors are selective for fXa vis a vis structurally related serine proteases and are readily prepared in 6-7 linear steps. The most potent member 9j (fXa Ki = 0.75 nM) is selective (>1000-fold) and an effective anticoagulant in mammalian plasma.     

Synthesis and structure-activity relationship of new analogues of antistasin.

Intensive investigation connected with the development of new anticoagulant agents for the treatment of cardiovascular diseases was carried out. Direct and specific inhibition of thrombin and Factor Xa-like serine proteases in the coagulation cascade has been the focus of many efforts to design novel anticoagulants over the past decade. This work reports the synthesis and biological activity of new anticoagulant peptide analogues of natural isoforms 2 and 3 of antistasin. In addition they include different tripeptide sequences in their molecules, which are highly active inhibitors of different serine proteases such as plasmin, trypsin, thrombin and Factor Xa.     

Isolation and partial purification of a novel anticoagulant from arteries of human umbilical cord

An anticoagulant fraction was isolated from the homogenate of human umbilical cord arteries, using Sephadex gel filtration and DEAE-Sephacel chromatography. Analysis with dodecyl sulfate/polyacrylamide gel electrophoresis and inactivation studies using proteolytic enzymes indicate that the anticoagulant activity is associated with a polypeptide with an apparent Mr of 32 000. The anticoagulant inhibits thromboplastin as well as factor Xa induced clotting but does not affect thrombin initiated fibrin formation. The anticoagulant inhibits the activation of prothrombin by the complete prothrombinase complex, by phospholipid bound factor Xa but not by free factor Xa. The inhibition is instantaneous and independent of the incubation time over the whole range of concentrations tested. Therefore, the anticoagulant is unlikely to be a phospholipase or a protease. Its action does not resemble that of the plasma protease inhibitors, but it probably interferes with the phospholipid--clotting factor interactions.     

Antithrombin-mediated anticoagulant activity of sulfated polysaccharides: different mechanisms for heparin and sulfated galactans

We investigated the mechanisms of anticoagulant activity mediated by sulfated galactans. The anticoagulant activity of sulfated polysaccharides is achieved mainly through potentiation of plasma cofactors, which are the natural inhibitors of coagulation proteases. Our results indicated the following. 1) Structural requirements for the interaction of sulfated galactans with coagulation inhibitors and their target proteases are not merely a consequence of their charge density. 2) The structural basis of this interaction is complex because it involves naturally heterogeneous polysaccharides but depends on the distribution of sulfate groups and on monosaccharide composition. 3) Sulfated galactans require significantly longer chains than heparin to achieve anticoagulant activity. 4) Possibly, it is the bulk structure of the sulfated galactan, and not a specific minor component as in heparin, that determines its interaction with antithrombin. 5) Sulfated galactans of approximately 15 to approximately 45 kDa bind to antithrombin but are unable to link the plasma inhibitor and thrombin. This last effect requires a molecular size above 45 kDa. 6) Sulfated galactan and heparin bind to different sites on antithrombin. 7) Sulfated galactans are less effective than heparin at promoting antithrombin conformational activation. Overall, these observations indicate that a different mechanism predominates over the conformational activation of antithrombin in ensuring the antithrombin-mediated anticoagulant activity of the sulfated galactans. Possibly, sulfated galactan connects antithrombin and thrombin, holding the protease in an inactive form. The conformational activation of antithrombin and the consequent formation of a covalent complex with thrombin appear to be less important for the anticoagulant activity of sulfated galactan than for heparin. Our results demonstrate that the paradigm of heparin-antithrombin interaction cannot be extended to other sulfated polysaccharides. Each type of polysaccharide may form a particular complex with the plasma inhibitor and the target protease.     

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.     

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.