New Anticoagulants

Within recent years, considerable interest has arisen in the new anticoagulant and non-anticoagulant actions of heparins and other glycosaminoglycans. In addition, oral direct factor (F) Xa inhibitors are being developed as an alternative treatment for thromboembolic disorders.

In this issue of Seminars in Thrombosis and Hemostasis, readers will find selected articles from a symposium organized by a group of German and Italian investigators dealing with glycosaminoglycans (GAGs), factor (F) Xa, and thrombin inhibitors. The meeting was held in the autumn of 2006 at Villa Vigoni in Loveno, Italy.

The first article in the issue is by Linhardt et al on the enzymatic synthesis of GAG heparin. Heparin and its low molecular weight heparin (LMWH) derivatives, widely used as clinical anticoagulants, are acidic polysaccharide members of a family of biomacromolecules called GAGs. Heparin and the related heparan sulfate (HS) are biosynthesized in the Golgi apparatus of eukaryotic cells. Heparin is a polycomponent drug that currently is prepared for clinical use by extraction from animal tissues. A heparin pentasaccharide, fondaparinux, has also been prepared through chemical synthesis for use as a homogenous anticoagulant drug. Recent enabling technologies suggest that it may now be possible to synthesize heparin and its derivatives enzymatically. Moreover, new technologies including advances in synthetic carbohydrate synthesis, enzyme-based GAG synthesis, micro- and nano-display of GAGs, rapid on-line structural analysis, and microarray/microfluidic technologies might be applied to the enzymatic synthesis of heparins with defined structures and exhibiting selected activities. The advent of these new technologies also makes it possible to consider the construction of an artificial Golgi to increase our understanding of the cellular control of GAG biosyntheses in this organelle.

Next, Vismara et al describe a structural modification of heparin induced by Felten-type depolarization processes. In detail, a LMWH obtained by a depolymerization process induced by a Fenton-type reagent was characterized in depth by nuclear magnetic resonance (NMR) spectroscopy. The depolymerization involves the cleavage of glycosidic bonds, leading to natural terminal reducing end residues, mainly represented by N-sulfated glucosamine. Natural uronic acids, especially the 2-O-sulfate iduronic acid, are also present as reducing residues. Unusual reaction results, such as the disappearance of the nonsulfated iduronic acid residues when followed by 6-O-nonsulfated glucosamine, and the decrease of the glucuronic acid when followed by the N-acetylglucosamine, were observed. Iduronic acid residues, followed by 6-O-sulfate glucosamine (A_Nx,6S), and the glucuronic acid residues, followed by A_NS residues, were not modified. A few minor internal chain modifications occur, possibly arising from oxidative breaking of the bond between C2-C3 of glucosamine and uronic acids, suggested by evidence of formation of new -COR groups. Finally, no change was observed in the content of the N-sulfated, 6-O-sulfated glucosamine bearing an extra sulfate on 3-O, which is considered the marker of the active site for antithrombin. With respect to the original heparin, this LMWH is characterized by a lower number of nonsulfated uronic acid residues, and as a consequence, by a lower degree of structural heterogeneity than LMWHs prepared with other procedures.

In the next article, Guerrini et al describe the structural differentiation of LMWHs by bidimensional NMR spectroscopy. Individual LMWHs exhibit distinct pharmacological and biochemical profiles because of manufacturing differences. Correlation of biological properties with particular structural motifs is a major challenge in the design of new LMWHs as well as in the development of generic versions of proprietary LMWHs. Two-dimensional NMR spectroscopy permits identification and quantification of structural peculiarities of LMWH preparations. In this article, heteronuclear single quantum coherence spectroscopy, previously used to determine variously substituted monosaccharide components of HS and HS-like GAG mimics, has been applied to the structural characterization of three commercially available LMWHs (enoxaparin, dalteparin, and tinzaparin). Relevant residues belonging to the parent heparin, as well as minor residues generated by each depolymerization procedure, have been characterized and quantified. The use of a high-sensitivity NMR spectrometer allowed the accurate quantification of residues with sensitivity better than 1 to 2%.

The article by Bisio et al analyzes the heparin species to cleavage by heparins using high-performance liquid chromatography (HPLC) and mass spectrometry (MS). Heparanase is an endo-β-d-glucuronidase that cleaves the heparan sulfate chains of HS proteoglycans and is implicated in angiogenesis and metastasis. With the aim of establishing a simple and reliable method for studying the susceptibility of heparin/HS oligosaccharides to be cleaved by heparanase, an on-line ion pair reversed-phase HPLC/electrospray ionization MS method was set up. The method works in the micromolar range of concentration and does not require derivatization of the substrate or of the products. It is based on mass identification of oligosaccharide fragments generated by heparanase and their quantification with reference to an internal heparin disaccharide standard. Substrates were (1) the synthetic pentasaccharides, and (2) two natural heparin octasaccharides. The present method could also be used for rapid screening of potential heparanase inhibitors.

The following article, by Bianchini et al, focuses on the variability of heparins and LMWHs. Chemical and physical characteristics, building blocks, constitutive disaccharides, sulfation degree, and biological activities of unfractionated heparins (UFHs) and LMWHs obtained by different depolymerization processes are examined comparatively in terms of structure characteristics, content of 1,6-anhydro rings, and other fingerprints. The heterogeneity of different LMWHs depends on different manufacturing processes and on particular specifications of pharmacopoeias. The reported examples prove that the variability among samples of LMWHs manufactured by the same process is quite limited. Most of the variability is derived from the parent UFH. In contrast, fingerprint groups and residues are specific to the depolymerization process and their extent can be roughly controlled through the process parameters.

The article by Harenberg et al describes the determination of antithrombin-dependent FXa inhibitors using the prothrombin-induced clotting time method. Prothrombinase-induced clotting time (PiCT) determines the anticoagulant effects of heparins, LMWHs, and direct thrombin inhibitors. At present, this is the only method that measures the effects of all of these inhibitors, in contrast to the prothrombin time, activated partial thromboplastin time (aPTT), Heptest, ecarin clotting time, and the chromogenic assays. The antithrombin-dependent direct FXa inhibitors fondaparinux and idraparinux were compared with the LMWH dalteparin on PiCT, aPTT, Heptest, and chromogenic anti-FXa assays in pooled human normal plasma samples. Fondaparinux and idraparinux prolonged the coagulation times in the PiCT, Heptest, and chromogenic FXa assays in a dose-dependent manner, in contrast to the aPTT. The authors conclude that PiCT is a suitable assay to determine the anticoagulant effects of these two new FXa inhibitors in patients receiving treatment with these compounds.

Nowak et al describe an assay to determine the thrombin-generating capacity of blood and plasma. Diagnostics of a hyper- or hypocoagulable state has been very difficult. The first attempt to solve this problem was the method of endogenous thrombin potential (ETP) by Hemker. In ETP, activators and a chromogenic substrate are added to diluted plasma samples and the thrombin generation is measured. By analysis of acquired data, three characteristics of ETP are seen: lag phase, peak thrombin, and velocity index. ETP is not suited for exact determination of maximum activated thrombin. Therefore, a new method was developed: the thrombin generation assay (THROGA). With the use of THROGA, the maximum generated thrombin in a blood or plasma sample can be measured easily. The background of the method is the addition of a certain amount of recombinant hirudin (r-hirudin) to the blood or plasma sample. After activation, the generated thrombin is bound quantitatively and neutralized by r-hirudin so that at the end of the activation phase the amount of generated thrombin can be determined easily and exactly by measurement of residual r-hirudin in the sample.

In the next article, Laux et al describe the preclinical and clinical characteristics of rivaroxaban, which is a new oral direct FXa inhibitor. There are several novel anticoagulants in development that target FXa-the pivotal point of the coagulation cascade. One promising agent is rivaroxaban (a highly selective, oral, direct FXa inhibitor), which is in advanced clinical development for the prevention and treatment of thromboembolic disorders. Oral rivaroxaban may be administered in fixed once-daily doses, with the potential for no coagulation monitoring. These properties, along with results from preclinical and clinical studies, suggest that rivaroxaban may have advantages over current treatments. Studies in arterial and venous animal models demonstrated that rivaroxaban has potent antithrombotic effects, without prolonging bleeding times. In healthy subjects, rivaroxaban was well tolerated, with a predictable pharmacological profile and a low propensity for clinically relevant drug-drug interactions. Phase II studies of rivaroxaban for the prevention of venous thromboembolism (VTE) after major orthopedic surgery support these findings. The results also suggested that a total daily dose range of 5 to 20 mg rivaroxaban had similar efficacy and safety to enoxaparin, and that 10 mg rivaroxaban once daily was the optimal dose. This review assesses the preclinical and clinical characteristics of rivaroxaban, and discusses phase II findings with rivaroxaban for the prevention of VTE after major orthopedic surgery.

An article by Mousa focuses on the emerging links between thrombosis, angiogenesis, and inflammation for heparin and heparin derivatives. The key reason behind the success of heparin in thrombosis and beyond is its polypharmacological sites of action for the prevention and treatment of multifactorial diseases that will only benefit slightly with single pharmacological mechanism-based agents. Thromboembolic disorders are driven by hypercoagulable, hyperactive platelet, proinflammatory, endothelial dysfunction, and proangiogenesis states. Heparin can effectively modulate all of those multifactorial components, as well as the interface among those components.

In the following article, Simonis et al analyze the affinity and kinetics of different heparins to P- and L-selectin. Selectins are adhesion receptors that participate in inflammation and tumor cell metastasis. The anti-inflammatory and antimetastatic activities of heparins have been related partly to their ability to interact with P- and L-selectin. The recent findings that various heparins differ in antimetastatic activity were explained by differences in their P- and L-selectin binding ability. To obtain data to illustrate the binding characteristics, we detected for the first time the binding kinetics and affinity of the two LMWHs, enoxaparin and nadroparin, and of the unfractionated heparin Liquemin N to P- and L-selectin using a quartz crystal microbalance biosensor. The differences are caused by a higher association rate compared with that of the LMWHs. These data support recent findings of antimetastatic activities, but illustrate that the intrinsic selectin binding does not entirely reflect the antimetastatic activities in vivo.

Next, Borsig focuses on the antimetastatic activities of modified heparins. Heparin, which is traditionally used as an anticoagulant but has a variety of additional biological activities, was shown in several retrospective and prospective clinical trials to have an effect on cancer survival. Experimental evidence from animal models consistently demonstrates that heparin is an efficient inhibitor of metastasis. To clarify the mechanism of heparin antimetastatic activity, several biological effects are being investigated. Cancer progression and metastasis are associated with enhanced expression of heparanase, which is inhibited efficiently by heparin. Heparin is also a potent inhibitor of selectin-mediated interactions. P- and L-selectin were shown to contribute to the early stages of metastasis, which is associated with platelet-tumor cell thrombi formation. To delineate the biological activities of heparin contributing to metastasis inhibition, modified heparins with specific activities were evaluated. Low anticoagulant heparin preparations still inhibited metastasis efficiently, indicating that anticoagulation is not a necessary component for heparin attenuation of metastasis. Modified heparins characterized for heparanase inhibitory activity are also potential inhibitors of selectins. Selectin inhibition is a clear component of heparin inhibition of metastasis. The contribution of selectin or heparanase inhibition by heparin can provide evidence about its antimetastatic activity.

In the next contribution, Borgenström et al report results on O-sulfated bacterial polysaccharides with low anticoagulant activity on inhibition of metastases. Heparin-like polysaccharides possess the capacity to inhibit cancer cell proliferation, angiogenesis, heparanase-mediated cancer cell invasion, and cancer cell adhesion to vascular endothelia via adhesion receptors, such as selectins. The clinical applicability of the antitumor effect of such polysaccharides, however, is compromised by their anticoagulant activity. The authors compared the potential of chemically O-sulfated and N,O-sulfated bacterial polysaccharide (capsular polysaccharide from E. coli K5 [K5PS]) species to inhibit metastasis of mouse B16-BL6 melanoma cells and human MDA-MB-231 breast cancer cells in two in vivo models. We demonstrate that in both settings, O-sulfated K5PS was a potent inhibitor of metastasis. Reducing the molecular weight of the polysaccharide, however, resulted in lower antimetastatic capacity. Furthermore, we show that O-sulfated K5PS efficiently inhibited the invasion of B16-BL6 cells through Matrigel and also inhibited the in vitro activity of heparanase. Moreover, treatment with O-sulfated K5PS lowered the ability of B16-BL6 cells to adhere to endothelial cells, intercellular adhesion molecule-1, and P-selectin, but not to E-selectin. Importantly, O-sulfated K5PSs were largely devoid of anticoagulant activity. These findings indicate that O-sulfated K5PS polysaccharide should be considered as a potential antimetastatic agent.

The last article of this issue by Ferro et al deals with a new compound PI-88, and other novel heparin sulfate mimetics that inhibit angiogenesis. PI-88 is a promising inhibitor of tumor growth and metastasis expected to commence phase III clinical evaluation in 2007 as an adjuvant therapy for postresection hepatocellular carcinoma. Its anticancer properties are attributed to inhibition of angiogenesis via antagonism of the interactions of angiogenic growth factors and their receptors with HS. It is also a potent inhibitor of heparanase, an enzyme that plays a key role in both metastasis and angiogenesis. A series of PI-88 analogs have been prepared with enhanced chemical and biological properties. The new compounds consist of single, defined oligosaccharides with specific modifications designed to improve their pharmacokinetic properties. These analogs all inhibit heparanase and bind to the angiogenic fibroblast growth factor 1 (FGF-1), FGF-2, and vascular endothelial growth factor with similar affinity to PI-88. However, compared with PI-88, some of the newly designed compounds are more potent inhibitors of growth factor-induced endothelial cell proliferation and of endothelial tube formation on Matrigel. Representative compounds were also tested for antiangiogenic activity in vivo and were found to reduce significantly blood vessel formation. Moreover, the pharmacokinetic profile of several analogs was also improved, as evidenced primarily by lower clearance in comparison with PI-88. The current data support the development of HS mimetics as potent antiangiogenic anticancer agents.

I would like to thank all contributors for the excellent articles, and Professor Vertonneli and all his collaborators for the excellent organization and the congenial atmosphere at the German-Italian Cultural Centre at Villa Vigone.