Structural peculiarity and antithrombin binding region profile of mucosal bovine and porcine heparins.

The major compositional differences between bovine mucosal heparin (BMH) and the currently employed porcine mucosal heparin (PMH) have been reported to essentially consist of reduced 6-O-sulfation of the glucosamine residues in BMH and somewhat lower 2-O-sulfation of the iduronate residues in PMH. The present work is based on direct comparison of several BMH and PMH commercial preparations. A combined study by 2D (heteronuclear single quantum coherence, HSQC) NMR and ion-pair reversed-phase high performance liquid chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) on the heparins, extended to the analysis of their heparinases digests and fractions separated by affinity chromatography on antithrombin (AT), confirmed the previously reported lower degree of 6-O-sulfation and showed lower 3-O-sulfated glucosamine content in BMH. More detailed studies allowed the identification of structural variants of AT-binding region (ATBR) structural variants, showing higher content of the N-sulfated components in BMH than in PMH.

Re-visiting the structure of heparin.

The sulfated polysaccharide heparin has been used as a life-saving anticoagulant in clinics well before its detailed structure was known. This mini-review is a survey of the evolution in the discovery of the primary and secondary structure of heparin. Highlights in this history include elucidation and synthesis of the specific sequence that binds to antithrombin, the development of low-molecular-weight heparins currently used as antithrombotic drugs, and the most promising start of chemo-enzymatic synthesis. Special emphasis is given to peculiar conformational properties contributing to interaction with proteins that modulate different biological properties.

Glycosaminoglycans: anticoagulant and nonanticoagulant actions: a short history of symposia held at villa vigoni.

Heparin, a sulfated polysaccharide belonging to the family of glycosaminoglycans, was discovered in the beginning of the 20th century and was initially identified as a procoagulant isolated from liver tissue. After the first application in patients approximately 30 years later, further purification identified the major as well as minor, but important, component units of the complex chain mixtures constituting heparin and the multiplex actions became a scientific challenge recently. A series of "Glycosaminoglycan symposium-anticoagulant and nonanticoagulant actions" developed over the past 20 years and focused on this topic has published research data in three issues of Seminars in Thrombosis & Hemostasis and in several other international scientific journals. The latest developments on the methods of analysis, the synthesis, the degradation by heparanases and the nonanticoagulant effects in tumor growth, in anti-inflammatory diseases, and in Alzheimer diseases as presented in the 21st symposium are summarized in the present overview on the occasion of the 40th anniversary of the journal with special reference to the journal's founding Editor in Chief, Eberhard F. Mammen.

Structural features of glycol-split low-molecular-weight heparins and their heparin lyase generated fragments.

Periodate oxidation followed by borohydride reduction converts the well-known antithrombotics heparin and low-molecular-weight heparins (LMWHs) into their "glycol-split" (gs) derivatives of the "reduced oxyheparin" (RO) type, some of which are currently being developed as potential anti-cancer and anti-inflammatory drugs. Whereas the structure of gs-heparins has been recently studied, details of the more complex and more bioavailable gs-LMWHs have not been yet reported. We obtained RO derivatives of the three most common LMWHs (tinzaparin, enoxaparin, and dalteparin) and studied their structures by two-dimensional nuclear magnetic resonance spectroscopy and ion-pair reversed-phase high-performance liquid chromatography coupled with electrospray ionization mass spectrometry. The liquid chromatography-mass spectrometry (LC-MS) analysis was extended to their heparinase-generated oligosaccharides. The combined NMR/LC-MS analysis of RO-LMWHs provided evidence for glycol-splitting-induced transformations mainly involving internal nonsulfated glucuronic and iduronic acid residues (including partial hydrolysis with formation of "remnants") and for the hydrolysis of the gs uronic acid residues when formed at the non-reducing ends (mainly, in RO-dalteparin). Evidence for minor modifications, such as ring contraction of some dalteparin internal aminosugar residues, was also obtained. Unexpectedly, the N-sulfated 1,6-anhydromannosamine residues at the enoxaparin reducing end were found to be susceptible to the periodate oxidation. In addition, in tinzaparin and enoxaparin, the borohydride reduction converts the hemiacetalic aminosugars at the reducing end to alditols. Typical LC-MS signatures of RO-derivatives of individual LMWH both before and after digestion with heparinases included oligosaccharides generated from the original antithrombin-binding and "linkage" regions.

Heparin dodecasaccharide containing two antithrombin-binding pentasaccharides: structural features and biological properties.

The antithrombin (AT) binding properties of heparin and low molecular weight heparins are strongly associated to the presence of the pentasaccharide sequence AGA*IA (A(NAc,6S)-GlcUA-A(NS,3,6S)-I(2S)-A(NS,6S)). By using the highly chemoselective depolymerization to prepare new ultra low molecular weight heparin and coupling it with the original separation techniques, it was possible to isolate a polysaccharide with a biosynthetically unexpected structure and excellent antithrombotic properties. It consisted of a dodecasaccharide containing an unsaturated uronate unit at the nonreducing end and two contiguous AT-binding sequences separated by a nonsulfated iduronate residue. This novel oligosaccharide was characterized by NMR spectroscopy, and its binding with AT was determined by fluorescence titration, NMR, and LC-MS. The dodecasaccharide displayed a significantly increased anti-FXa activity compared with those of the pentasaccharide, fondaparinux, and low molecular weight heparin enoxaparin.

Unravelling structural information from complex mixtures utilizing correlation spectroscopy applied to HSQC spectra.

The first use of statistical correlation spectroscopy to extract chemical information from 2D-HSQC spectra, termed HSQC correlation spectroscopy (HSQCcos), is reported. HSQCcos is illustrated using heparin, a heterogeneous polysaccharide, whose diverse composition causes signals in HSQC spectra to disperse. HSQCcos has been used to probe the chain modifications that cause this effect and reveals hitherto unreported structural details. An interesting finding was that the signal for position 2 of trisulfated glucosamine [N-, 3-O-, and 6-O-sulfated] (A*) is bifurcated, owing to the presence of A* residues in both the "normal" antithrombin binding site and also at the nonreducing end of the molecule, which is reported in intact heparin for the first time. The method was also applied to investigating the environment around other rare sequences/disaccharides, suggesting that the disaccharide; 2-O-sulfated iduronic acid linked to 6-O-sulfated N-glucosamine, which contains a free amine at position 2, is adjacent to the heparin linkage region. HSQCcos can extract chemically related signals from information-rich spectra obtained from complex mixtures such as heparin.

An unusual antithrombin-binding heparin octasaccharide with an additional 3-O-sulfated glucosamine in the active pentasaccharide sequence.

The 3-O-sulfation of N-sulfated glucosamine is the last event in the biosynthesis of heparin/heparan sulfate, giving rise to the antithrombin-binding pentasaccharide sequence AGA*IA, which is largely associated with the antithrombotic activity of these molecules. The aim of the present study was the structural and biochemical characterization of a previously unreported AGA*IA*-containing octasaccharide isolated from the very-low-molecular-mass heparin semuloparin, in which both glucosamine residues of the pentasaccharide moiety located at the non-reducing end bear 3-O-sulfate groups. Two-dimensional and STD (saturation transfer difference) NMR experiments clearly confirmed its structure and identified its ligand epitope binding to antithrombin. The molecular conformation of the octasaccharide-antithrombin complex has been determined by NMR experiments and docking/energy minimization. The presence of the second 3-O-sulfated glucosamine in the octasaccharide induced more than one order of magnitude increase in affinity to antithrombin compared to the pentasaccharide AGA*IA.

Profiling glycol-split heparins by high-performance liquid chromatography/mass spectrometry analysis of their heparinase-generated oligosaccharides.

Glycol-split (gs) heparins, obtained by periodate oxidation/borohydride reduction of heparin currently used as an anticoagulant and antithrombotic drug, are arousing increasing interest in anticancer and anti-inflammation therapies. These new medical uses are favored by the loss of anticoagulant activity associated with glycol-splitting-induced inactivation of the antithrombin III (AT) binding site. The structure of gs heparins has not been studied yet in detail. In this work, ion pair reversed-phase high-performance liquid chromatography (IPRP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS) widely used for unmodified heparin has been adapted to the analysis of oligosaccharides generated by digestion with heparinases of gs heparins usually prepared from porcine mucosal heparin. The method was also found to be very effective in analyzing gs derivatives obtained from heparins of different animal and tissue origins. Besides the major 2-O-sulfated disaccharides, heparinase digests of gs heparins contain mainly tetra- and hexasaccharides incorporating one or two gs residues, with distribution patterns typical for individual gs heparins. A heptasulfated, mono-N-acetylated hexasaccharide with two gs residues was shown to be a marker of the gs-modified AT binding site within heparin chains.

The inhibition of the integrin VLA-4 in MV3 melanoma cell binding by non-anticoagulant heparin derivatives.

INTRODUCTION: The integrin VLA-4-mediated binding is important for the metastatic dissemination of melanoma cells. Recently we found that heparin possesses a binding capacity to VLA-4. This could contribute to the heparin function to attenuate metastasis in a selectin-dependent manner. Aiming to a purposive, anti-adhesive heparin application, structural requirements of heparin for VLA-4 recognition have to be elucidated. MATERIALS AND METHODS: A series of non-anticoagulant heparin derivatives were investigated concerning their inhibitory capacities for VLA-4 mediated binding of human melanoma MV3 cells to VCAM-1 under physiological flow conditions in vitro. A surface acoustic wave biosensor was applied to detect kinetic constants of selected derivatives binding to both, VLA-4 or P- and L-selectin. RESULTS: Experimental metastasis of MV3 cells in mice confirmed the relevance of VLA-4 for metastatic dissemination. LMWHs (enoxaparin, tinzaparin) efficiently blocked VLA-4 cell binding, dominantly via the integrin`s α-chain. Desulfation at 2-O-position, N-acetylation or a size smaller than tetradecasaccharide disfavoured VLA-4 inhibition. Glycol-splitting of heparin and thus higher chain flexibility is a tolerable parameter. A derivative with 50% 6-O-desulfation appeared promising and exceeded tinzaparin in VLA-4 inhibition, both compounds displayed binding affinities to VLA-4 in the low micromolar range. CONCLUSIONS: These findings provide structure-activity relationships for heparin VLA-4 binding, which partly differ from P- and L-selectin requirements. The data confirm that anti-coagulative and anti-adhesive function of heparin can be distinguished favouring applications of non-anticoagulant heparins in antimetastatic approaches without the risk of bleeding complications. The 50% 6-O-desulfated heparin-derivative appears promising to further evaluate the interference with selectin and VLA-4 binding functions in vivo.

SST0001, a chemically modified heparin, inhibits myeloma growth and angiogenesis via disruption of the heparanase/syndecan-1 axis.

PURPOSE: Heparanase promotes myeloma growth, dissemination, and angiogenesis through modulation of the tumor microenvironment, thus highlighting the potential of therapeutically targeting this enzyme. SST0001, a nonanticoagulant heparin with antiheparanase activity, was examined for its inhibition of myeloma tumor growth in vivo and for its mechanism of action. EXPERIMENTAL DESIGN: The ability of SST0001 to inhibit growth of myeloma tumors was assessed using multiple animal models and a diverse panel of human and murine myeloma cell lines. To investigate the mechanism of action of SST0001, pharmacodynamic markers of angiogenesis, heparanase activity, and pathways downstream of heparanase were monitored. The potential use of SST0001 as part of a combination therapy was also evaluated in vivo. RESULTS: SST0001 effectively inhibited myeloma growth in vivo, even when confronted with an aggressively growing tumor within human bone. In addition, SST0001 treatment causes changes within tumors consistent with the compound's ability to inhibit heparanase, including downregulation of HGF, VEGF, and MMP-9 expression and suppressed angiogenesis. SST0001 also diminishes heparanase-induced shedding of syndecan-1, a heparan sulfate proteoglycan known to be a potent promoter of myeloma growth. SST0001 inhibited the heparanase-mediated degradation of syndecan-1 heparan sulfate chains, thus confirming the antiheparanase activity of this compound. In combination with dexamethasone, SST0001 blocked tumor growth in vivo presumably through dual targeting of the tumor and its microenvironment. CONCLUSIONS: These results provide mechanistic insight into the antitumor action of SST0001 and validate its use as a novel therapeutic tool for treating multiple myeloma.

Effects on molecular conformation and anticoagulant activities of 1,6-anhydrosugars at the reducing terminal of antithrombin-binding octasaccharides isolated from low-molecular-weight heparin enoxaparin.

Terminal 1,6-anhydro-aminosugars (1,6-anAS) are typical structural moieties of enoxaparin, a low-molecular-weight heparin (LMWH) widely used for prevention and treatment of thrombotic disorders. In the enoxaparin manufacturing process, these modified amino sugars are formed during the ß-eliminative cleavage of heparin. To investigate the effect of terminal anAS on antithrombin (AT) binding and on inhibition of factor Xa (FXa), two octasaccharides containing modified AT-binding pentasaccharide sequences were isolated from enoxaparin. The molecular conformation of the octasaccharides terminating with N-sulfo-1,6-anhydro-D-mannosamine and N-sulfo-1,6-anhydro-D-glucosamine, respectively, has been determined both in the absence and presence of AT by NMR experiments and docking simulations. Reduced overall contacts of the terminal anAS residues with the binding region of AT induce a decrease in affinity for AT as well as lower anti-FXa activity. The anti-FXa measured either in buffer or plasma milieu does not show any significant difference, suggesting that the inhibition of anti-FXa remains specific and biologically relevant.

Heparin-derived heparan sulfate mimics to modulate heparan sulfate-protein interaction in inflammation and cancer.

The heparan sulfate (HS) chains of heparan sulfate proteoglycans (HSPG) are "ubiquitous" components of the cell surface and the extracellular matrix (EC) and play important roles in the physiopathology of developmental and homeostatic processes. Most biological properties of HS are mediated by interactions with "heparin-binding proteins" and can be modulated by exogenous heparin species (unmodified heparin, low molecular weight heparins, shorter heparin oligosaccharides and various non-anticoagulant derivatives of different sizes). Heparin species can promote or inhibit HS activities to different extents depending, among other factors, on how closely their structure mimics the biologically active HS sequences. Heparin shares structural similarities with HS, but is richer in "fully sulfated" sequences (S domains) that are usually the strongest binders to heparin/HS-binding proteins. On the other hand, HS is usually richer in less sulfated, N-acetylated sequences (NA domains). Some of the functions of HS chains, such as that of activating proteins by favoring their dimerization, often require short S sequences separated by rather long NA sequences. The biological activities of these species cannot be simulated by heparin, unless this polysaccharide is appropriately chemically/enzymatically modified or biotechnologically engineered. This mini review covers some information and concepts concerning the interactions of HS chains with heparin-binding proteins and some of the approaches for modulating HS interactions relevant to inflammation and cancer. This is approached through a few illustrative examples, including the interaction of HS and heparin-derived species with the chemokine IL-8, the growth factors FGF1 and FGF2, and the modulation of the activity of the enzyme heparanase by these species. Progresses in sequencing HS chains and reproducing them either by chemical synthesis or semi-synthesis, and in the elucidation of the 3D structure of oligosaccharide-protein complexes, are paving the way for rational approaches to the development of HS-inspired drugs in the field of inflammation and cancer, as well in other therapeutic fields.

The tainted heparin story: an update.

Recently, certain batches of heparin have been associated with an acute, rapid onset of serious side-effects indicative of allergic-type reactions. These reports generated significant concern regarding the possible presence of a dangerous contaminant within heparin and highlighted the need to re-assess the purity criteria of heparin preparations for clinical use. Given the nature of the array of all possible contaminants, traditional screening tests cannot safely differentiate between contaminated and uncontaminated heparin preparations. Mono- and bi-dimensional NMR spectroscopy are powerful techniques that are able to detect and quantify a wide variety of potential sulfated polysaccharide contaminants. As such, these techniques are powerful tools for the analysis and assessment of heparin preparations.

Orthogonal analytical approaches to detect potential contaminants in heparin.

Heparin is a widely used anticoagulant and antithrombotic agent. Recently, a contaminant, oversulfated chondroitin sulfate (OSCS), was discovered within heparin preparations. The presence of OSCS within heparin likely led to clinical manifestations, most prevalently, hypotension and abdominal pain leading to the deaths of several dozens of patients. Given the biological effects of OSCS, one continuing item of concern is the ability for existing methods to identify other persulfonated polysaccharide compounds that would also have anticoagulant activity and would likely elicit a similar activation of the contact system. To complete a more extensive analysis of the ability for NMR and capillary electrophoresis (CE) to capture a broader array of potential contaminants within heparin, we completed a systematic study of NMR, both mono- and bidimensional, and CE to detect both various components of sidestream heparin and their persulfonated derivatives. We show that given the complexity of heparin samples, and the requirement to ensure their purity and safety, use of orthogonal analytical techniques is effective at detecting an array of potential contaminants that could be present.

Antithrombin-binding octasaccharides and role of extensions of the active pentasaccharide sequence in the specificity and strength of interaction. Evidence for very high affinity induced by an unusual glucuronic acid residue.

The antithrombotic activity of low molecular weight heparins (LMWHs) is largely associated with the antithrombin (AT)-binding pentasaccharide sequence AGA(*)IA (GlcN(NAc/NS,6S)-GlcA-GlcN(NS,3,6S)-IdoUA(2S)-GlcN(NS,6S)). The location of the AGA(*)IA sequences along the LMWH chains is also expected to influence binding to AT. This study was aimed at investigating the role of the structure and molecular conformation of different disaccharide extensions on both sides of the AGA(*)IA sequence in modulating the affinity for AT. Four high purity octasaccharides isolated by size exclusion chromatography, high pressure liquid chromatography, and AT-affinity chromatography from the LMWH enoxaparin were selected for the study. All the four octasaccharides terminate at their nonreducing end with 4,5-unsaturated uronic acid residues (DeltaU). In two octasaccharides, AGA(*)IA was elongated at the reducing end by units IdoUA(2S)-GlcN(NS,6S) (OCTA-1) or IdoUA-GlcN(NAc,6S) (OCTA-2). In the other two octasaccharides (OCTA-3 and OCTA-4), AGA(*)IA was elongated at the nonreducing side by units GlcN(NS,6S)-IdoUA and GlcN(NS,6S)-GlcA, respectively. Extensions increased the affinity for AT of octasaccharides with respect to pentasaccharide AGA(*)IA, as also confirmed by fluorescence titration. Two-dimensional NMR and docking studies clearly indicated that, although elongation of the AGA(*)IA sequence does not substantially modify the bound conformation of the AGA(*)IA segment, extensions promote additional contacts with the protein. It should be noted that, as not previously reported, the unusual GlcA residue that precedes the AGA(*)IA sequence in OCTA-4 induced an unexpected 1 order of magnitude increase in the affinity to AT with respect to its IdoUA-containing homolog OCTA-3. Such a residue was found to orientate its two hydroxyl groups at close distance to residues of the protein. Besides the well established ionic interactions, nonionic interactions may thus contribute to strengthen oligosaccharide-AT complexes.

Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events.

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked beta1-->3 to a beta-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here.

Minimum FGF2 binding structural requirements of heparin and heparan sulfate oligosaccharides as determined by NMR spectroscopy.

Heparin and heparan sulfate (HS) glycosaminoglycans (HSGAGs) are sulfated polysaccharidesthat play important roles in fundamental biological processes by binding to proteins. The prototypic exampleof HSGAG-protein interactions is that with the fibroblast growth factors (FGFs), specifically FGF1 andFGF2. Structural and biochemical studies have shown that the chain length, sulfation pattern, andconformation of HSGAGs play a critical role in FGF binding and activity. Previously, we showed that atetrasaccharide of the form ANS,6X-I2S-ANS,6X-I2S-OPr (where X is OH or O-sulfate and Pr is propyl) withat least one of the ANS,6X residues having a 6-O sulfate group was the minimum binding motif for FGF1[Guerrini, M., Agulles, T., Bisio, A., Hricovini, M., Lay, L., Naggi, A., Poletti, L., Sturiale, L., Torri, G.,and Casu, B. (2002) Biochem. Biophys. Res. Commun. 292, 222-230]. We report NMR structural analysisusing two-dimensional NOE spectroscopy (2D-NOESY) and transferred NOESY (trNOESY) on a non-6-O-sulfated synthetic tetrasaccharide TETRA (ANS-I2S-ANS-I2S-OPr) both in its free state and bound toFGF2. This tetrasaccharide comprises both the structural trisaccharide motif ANS-I2S-ANS that forms "kinks"in longer heparin chains induced by FGF binding [Raman, R., Venkataraman, G., Ernst, S., Sasisekharan,V., and Sasisekharan, R. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 2357-2362] and the common bindingmotif I2S-ANS-I2S present in octasaccharides that exhibited strong FGF2 binding [Kreuger, J., Salmivirta,M., Sturiale, L., Gimenez-Gallego, G., and Lindahl, U. (2001) J. Biol. Chem. 276, 30744-30752]. Thesedata suggest that TETRA could be the shortest HSGAG oligosaccharide that binds to FGF2. Furthermore,our study confirms that both the IdoA residues in TETRA adopt the chair 1C4 conformation upon FGF2binding to provide the best molecular fit in contrast to an analogous 6-O-sulfated tetrasaccharide motifobserved in the FGF2-HSGAG cocrystal structure where one of the IdoAs adopts skew-boat 2SOconformation. Thus, our study highlights the fact that the conformational plurality of IdoA is able toaccommodate the changes in the sulfation pattern to provide the necessary specificity for protein binding.

Non-anticoagulant heparins and inhibition of cancer.

Low-molecular-weight heparins (LMWH) appear to prolong survival of patients with cancer. Such a beneficial effect is thought to be associated with interruption of molecular mechanisms involving the heparan sulfate (HS) chains of cell surface and extracellular matrix proteoglycans (HSPGs), growth factors and their receptors, heparanase, and selectins. The beneficial effects of heparin species could also be associated with their ability to release tissue factor pathway inhibitor from endothelium. The utility of heparin and LMWH as anticancer drugs is limited due to their anticoagulant properties. Non-anticoagulant heparins can be obtained either by removing chains containing the antithrombin-binding sequence, or by inactivating critical functional groups or units of this sequence. The non-anticoagulant heparins most extensively studied are regioselectively desulfated heparins and 'glycol-split' heparins. Some modified heparins of both types are potent inhibitors of heparanase. A number of them also attenuate metastasis in experimental models. With cancer cells overexpressing selectins, heparin-mediated inhibition of tumor cells-platelets aggregation and tumor cell interaction with the vascular endothelium appears to be the prevalent mechanism of attenuation of early stages of metastasis. The structural requirements for inhibition of growth factors, heparanase, and selectins by heparin derivatives are somewhat different for the different activities. An N-acetylated, glycol-split heparin provides an example of application of a non-anticoagulant heparin that inhibits cancer in animal models without unwanted side effects. Delivery of this compound to mice bearing established myeloma tumors dramatically blocked tumor growth and progression.

Heparanase, heparin and the coagulation system in cancer progression.

Heparanase is an endoglycosidase which cleaves heparan sulfate (HS) and hence participates in degradation and remodeling of the extracellular matrix (ECM). The enzyme also releases angiogenic factors from the ECM and thereby induces an angiogenic response in vivo. Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an accelerated growth and invasive phenotype in experimental animals. In contrast, heparanase gene silencing is associated with a marked inhibition of tumor progression. Heparanase upregulation correlates with increased tumor vascularity and poor postoperative survival of cancer patients. Studies on relationships between structure and the heparanase-inhibiting activity of nonanticogulant heparins systematically differing in their O-sulfation patterns, degrees of N-acetylation, and glycol-splitting of nonsulfated uronic acid residues, have permitted to select effective inhibitors of the enzymatic activity of heparanase. N-acetylated, glycol-split heparins emerged as highly effective and specific inhibitors of heparanase and tumor growth and metastasis. Several observations support the involvement of heparanase in haemostasis. A marked induction of tissue factor (TF) was noted in response to heparanase over-expression in tumor-derived cell lines and heparanase over-expressing transgenic mice. A direct correlation was also found between heparanase and TF expression levels in leukemia patients. TF induction was even more pronounced upon exogenous addition of heparanase to primary endothelial cells that do not normally express TF, and this induction was associated with enhanced coagulation. These and other results indicate that pro-heparanase is rapidly tethered on cell surfaces, partially depending on cell surface heparan sulfate, generating a local procoagulant effect. In addition, pro-heparanase can reverse the anti-coagulant effect of unfractionated heparin and the Factor Xa inhibitory activity of low molecular weight heparin (LMWH). These effects were also demonstrated in plasma derived from patients treated with LMWH. The pro-coagulant effects of pro-heparanase were also exerted by a peptide corresponding to its major functional heparin-binding domain. Heparanase pro-coagulant activities suggest its possible role as a natural regulator of heparinoid anti-coagulant activities, and point to a possible use of this molecule or its heparin binding domain as antidote for heparinoid therapies.