Tumour cell-activated platelets modulate the immunological activity of CD4+, CD8+, and NK cells, which is efficiently antagonized by heparin.

Platelets, key players in haemostasis, are progressively investigated with respect to their role in immunity and inflammation. Although the platelet support to haematogenous cancer cell metastasis has been the subject of multiple studies, their impact on anti-cancer immunity remains unaddressed. Here, we investigated the immunomodulatory potential of platelets upon their activation by MDA-MB-231 breast cancer cells in various in vitro approaches. We provide evidence that platelets as well as their tumour cell-induced releasates increased the ratio of regulatory T cells, shaping an immunosuppressive phenotype in isolated CD4+ cultures. The influence on CD8+ T cells was assessed by detecting the expression of activation markers CD25/CD69 and release of cytolytic and pro-inflammatory proteins. Notably, the platelet preparations differentially influenced CD8+ T cell activation, while platelets were found to inhibit the activation of CD8+ T cells, platelet releasates, in contrast, supported their activation. Furthermore, the NK cell cytolytic activity was attenuated by platelet releasates. Low molecular weight heparin (LMWH), the guideline-based anticoagulant for cancer-associated thrombotic events, is known to interfere with tumour cell-induced platelet activation. Thus, we aimed to investigate whether, unfractionated heparin, LMWH or novel synthetic heparin mimetics can also reverse the immunosuppressive platelet effects. The releasate-mediated alteration in immune cell activity was efficiently abrogated by heparin, while the synthetic heparin mimetics partly outperformed the commercial heparin derivatives. This is the first report on the effects of heparin on rebalancing immunosuppression in an oncological context emerging as a novel aspect in heparin anti-tumour activities.

The Control of Heparanase Through the Use of Small Molecules.

Despite the enormous progress made in recent years with antibodies, vaccines, antisense oligonucleotides, etc., the so-called "biological" approaches for tackling the control of various diseases, medicinal chemistry remains a bulwark to refer to for the development of new drugs. Also in the case of heparanase, medicinal chemistry has always been in the forefront to identify new inhibitors, through modification of natural macromolecules, e.g., sulfated polysaccharides like heparin, or of natural compounds isolated from bacteria or plants, or through rational design. In this chapter, the reader will find a detailed description of the most relevant small-molecule heparanase inhibitors reported so far in the scientific literature and in patent applications, with mention to the design strategy and to structure-activity relationships. Starting from heparanase inhibitors of natural origin and the attempts to improve their potency and selectivity, the reader will be guided through the major chemical classes of synthetic inhibitors, with representation of the structure of the most relevant compounds. The last paragraph is dedicated to a brief description of inhibitors that have reached clinical trials, highlighting their structure, mechanism, and improved derivatives.

Heparin mimetics with anticoagulant activity.

Heparin, a sulfated polysaccharide belonging to the glycosaminoglycan family, has been widely used as an anticoagulant drug for decades and remains the most commonly used parenteral anticoagulant in adults and children. However, heparin has important clinical limitations and is derived from animal sources which pose significant safety and supply problems. The ever growing shortage of the raw material for heparin manufacturing may become a very significant issue in the future. These global limitations have prompted much research, especially following the recent well-publicized contamination scandal, into the development of alternative anticoagulants derived from non-animal and/or totally synthetic sources that mimic the structural features and properties of heparin. Such compounds, termed heparin mimetics, are also needed as anticoagulant materials for use in biomedical applications (e.g., stents, grafts, implants etc.). This review encompasses the development of heparin mimetics of various structural classes, including synthetic polymers and non-carbohydrate small molecules as well as sulfated oligo- and polysaccharides, and fondaparinux derivatives and conjugates, with a focus on developments in the past 10 years.

Synthesis, Biological Evaluation and Reversal of Sulfonated Di- and Triblock Copolymers as Novel Parenteral Anticoagulants.

Despite targeting different coagulation cascade sites, all Food and Drug Administration-approved anticoagulants present an elevated risk of bleeding, including potentially life-threatening intracranial hemorrhage. Existing studies have not thoroughly investigated the efficacy and safety of sulfonate polymers in animal models and fully elucidate the precise mechanisms by which these polymers act. The activity and safety of sulfonated di- and triblock copolymers containing poly(sodium styrenesulfonate) (PSSS), poly(sodium 2-acrylamido-2-methylpropanesulfonate) (PAMPS), poly(ethylene glycol) (PEG), poly(sodium methacrylate) (PMAAS), poly(acrylic acid) (PAA), and poly(sodium 11-acrylamidoundecanoate) (PAaU) blocks are synthesized and assessed. PSSS-based copolymers exhibit greater anticoagulant activity than PAMPS-based ones. Their activity is mainly affected by the total concentration of sulfonate groups and molecular weight. PEG-containing copolymers demonstrate a better safety profile than PAA-containing ones. The selected copolymer PEG47-PSSS32 exhibits potent anticoagulant activity in rodents after subcutaneous and intravenous administration. Heparin Binding Copolymer (HBC) completely reverses the anticoagulant activity of polymer in rat and human plasma. No interaction with platelets is observed. Selected copolymer targets mainly factor XII and fibrinogen, and to a lesser extent factors X, IX, VIII, and II, suggesting potential application in blood-contacting biomaterials for anticoagulation purposes. Further studies are needed to explore its therapeutic applications fully.

Heparin, Heparan Sulphate and Sepsis: Potential New Options for Treatment.

Sepsis is a life-threatening hyperreaction to infection in which excessive inflammatory and immune responses cause damage to host tissues and organs. The glycosaminoglycan heparan sulphate (HS) is a major component of the cell surface glycocalyx. Cell surface HS modulates several of the mechanisms involved in sepsis such as pathogen interactions with the host cell and neutrophil recruitment and is a target for the pro-inflammatory enzyme heparanase. Heparin, a close structural relative of HS, is used in medicine as a powerful anticoagulant and antithrombotic. Many studies have shown that heparin can influence the course of sepsis-related processes as a result of its structural similarity to HS, including its strong negative charge. The anticoagulant activity of heparin, however, limits its potential in treatment of inflammatory conditions by introducing the risk of bleeding and other adverse side-effects. As the anticoagulant potency of heparin is largely determined by a single well-defined structural feature, it has been possible to develop heparin derivatives and mimetic compounds with reduced anticoagulant activity. Such heparin mimetics may have potential for use as therapeutic agents in the context of sepsis.

Validation of Recombinant Heparan Sulphate Reagents for CNS Repair.

Therapies that target the multicellular pathology of central nervous system (CNS) disease/injury are urgently required. Modified non-anticoagulant heparins mimic the heparan sulphate (HS) glycan family and have been proposed as therapeutics for CNS repair since they are effective regulators of numerous cellular processes. Our in vitro studies have demonstrated that low-sulphated modified heparan sulphate mimetics (LS-mHeps) drive CNS repair. However, LS-mHeps are derived from pharmaceutical heparin purified from pig intestines, in a supply chain at risk of shortages and contamination. Alternatively, cellular synthesis of heparin and HS can be achieved using mammalian cell multiplex genome engineering, providing an alternative source of recombinant HS mimetics (rHS). TEGA Therapeutics (San Diego) have manufactured rHS reagents with varying degrees of sulphation and we have validated their ability to promote repair in vitro using models that mimic CNS injury, making comparisons to LS-mHep7, a previous lead compound. We have shown that like LS-mHep7, low-sulphated rHS compounds promote remyelination and reduce features of astrocytosis, and in contrast, highly sulphated rHS drive neurite outgrowth. Cellular production of heparin mimetics may, therefore, offer potential clinical benefits for CNS repair.

Heparin Mimetics and Their Impact on Extracellular Matrix Protein Assemblies.

Heparan sulfate is a crucial extracellular matrix component that organizes structural features and functional protein processes. This occurs through the formation of protein-heparan sulfate assemblies around cell surfaces, which allow for the deliberate local and temporal control of cellular signaling. As such, heparin-mimicking drugs can directly affect these processes by competing with naturally occurring heparan sulfate and heparin chains that then disturb protein assemblies and decrease regulatory capacities. The high number of heparan-sulfate-binding proteins that are present in the extracellular matrix can cause obscure pathological effects that should be considered and examined in more detail, especially when developing novel mimetics for clinical use. The objective of this article is to investigate recent studies that present heparan-sulfate-mediated protein assemblies and the impact of heparin mimetics on the assembly and function of these protein complexes.

Sulfonated non-saccharide molecules and human factor XIa: Enzyme inhibition and computational studies.

Human factor XIa (FXIa) is a serine protease in the intrinsic coagulation pathway. FXIa has been actively targeted to develop new anticoagulants that are associated with a reduced risk of bleeding. Thousands of FXIa inhibitors have been reported, yet none has reached the clinic thus far. We describe here a novel class of sulfonated molecules that allosterically inhibit FXIa with moderate potency. A library of 18 sulfonated molecules was evaluated for the inhibition of FXIa using a chromogenic substrate hydrolysis assay. Only six molecules inhibited FXIa with IC50 values of 4.6-29.5 µM. Michaelis-Menten kinetics indicated that sulfonated molecules are allosteric inhibitors of FXIa. Inhibition of FXIa by these molecules was reversed by protamine. The molecules also showed moderate anticoagulant effects in human plasma with preference to prolong activated partial thromboplastin time. Their binding to an allosteric site in the catalytic domain of FXIa was modeled to illustrate potential binding mode and potential important Arg/Lys residues. Particularly, inhibitor 16 (IC50  = 4.6 µM) demonstrated good selectivity over a panel of serine proteases including those in the coagulation process. Inhibitor 16 did not significantly compromise the viability of three cell lines. Overall, the reported sulfonated molecules serve as a new platform to design selective, potent, and allosteric inhibitors of FXIa for therapeutic applications.

Inhibition of Tumor-Host Cell Interactions Using Synthetic Heparin Mimetics.

Low-molecular-weight heparin (LMWH) is the guideline-based drug for antithrombotic treatment of cancer patients, while its direct antitumor effects are a matter of ongoing debate. Although therapeutically established for decades, LMWH has several drawbacks mainly associated with its origin from animal sources. Aiming to overcome these limitations, a library of synthetic heparin mimetic polymers consisting of homo- and copolymers of sulfonated and carboxylated noncarbohydrate monomers has recently been synthesized via reversible addition-fragmentation chain transfer polymerization. These heparin mimetics were investigated for their capacities to interfere with simulated steps of tumor cell metastasis. Among them, homo- and copolymers from sodium 4-styrenesulfonate (poly(SSS)) with acrylic acid (poly(SSS-co-AA)) with an MW between 5 and 50 kDa efficiently attenuated cancer cell-induced coagulation and thus platelet activation and degranulation similar to or even better than LMWH. Furthermore, independent of anticoagulant activities, these polymers affected other metastasis-relevant targets with impressive affinities. Hence, they blocked heparanase enzymatic activity outmatching commercial heparins or a glycosidic drug candidate. Furthermore, these polymers bind P-selectin and the integrin VLA-4 similar to or even better than heparin, indicated by a biosensor approach and thus efficiently blocked melanoma cell binding to endothelium under blood flow conditions. This is the first report on the prospects of synthetic heparin mimetics as promising nontoxic compounds in oncology to potentially substitute heparin as an anticoagulant and to better understand its role as an antimetastatic drug.

Antiviral Strategies Using Natural Source-Derived Sulfated Polysaccharides in the Light of the COVID-19 Pandemic and Major Human Pathogenic Viruses.

Only a mere fraction of the huge variety of human pathogenic viruses can be targeted by the currently available spectrum of antiviral drugs. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak has highlighted the urgent need for molecules that can be deployed quickly to treat novel, developing or re-emerging viral infections. Sulfated polysaccharides are found on the surfaces of both the susceptible host cells and the majority of human viruses, and thus can play an important role during viral infection. Such polysaccharides widely occurring in natural sources, specifically those converted into sulfated varieties, have already proved to possess a high level and sometimes also broad-spectrum antiviral activity. This antiviral potency can be determined through multifold molecular pathways, which in many cases have low profiles of cytotoxicity. Consequently, several new polysaccharide-derived drugs are currently being investigated in clinical settings. We reviewed the present status of research on sulfated polysaccharide-based antiviral agents, their structural characteristics, structure-activity relationships, and the potential of clinical application. Furthermore, the molecular mechanisms of sulfated polysaccharides involved in viral infection or in antiviral activity, respectively, are discussed, together with a focus on the emerging methodology contributing to polysaccharide-based drug development.

Sulfonated RAFT Copolymers as Heparin Mimetics: Synthesis, Reactivity Ratios, and Anticoagulant Activity.

The glycosaminoglycan heparin is a clinically important anticoagulant drug, primarily used to reduce the risk of blood clots (thrombosis) during surgery. Despite its importance in medicine and its continuous use over many decades, heparin suffers from several limitations associated with its heterogeneity and its extraction from animal tissues. In order to address these limitations, reversible addition-fragmentation chain transfer polymerization is utilized to prepare a library of heparin mimetic copolymers from the sulfonated monomers sodium 4-styrene sulfonate, potassium-3-sulfopropyl acrylate, potassium-3-sulfopropyl methacrylate, and sodium-2-acrylamido-2-methyl-1-propane sulfonate. Copolymers are prepared using combinations of two different monomers in various ratios. Monomer reactivity ratios are also determined for some representative monomer combinations, and all polymers are characterized by 1 H NMR spectroscopy and gel permeation chromatography. The anticoagulant activities of the copolymers are determined by activated partial thromboplastin time and thrombin clotting time assays and structure-activity relationships are explored.

Novel heparin mimetics reveal cooperativity between exosite 2 and sodium-binding site of thrombin.

INTRODUCTION: Thrombin is a primary target of most anticoagulants. Yet, thrombin's dual and opposing role in pro- as well as anti- coagulant processes imposes considerable challenges in discovering finely tuned regulators that maintain homeostasis, rather than disproportionately changing the equilibrium to one side. In this connection, we have been studying exosite 2-mediated allosteric modulation of thrombin activity using synthetic agents called low molecular weight lignins (LMWLs). Although the aromatic scaffold of LMWLs is completely different from the polysaccharidic scaffold of heparin, the presence of multiple negatively charged groups on both ligands induces binding to exosite 2 of thrombin. This work characterizes the nature of interactions between LMWLs and thrombin to understand the energetic cooperativity between exosite 2 and active site of thrombin. MATERIALS AND METHODS: The thermodynamics of thrombin-LMWL complexes was studied using spectrofluorimetric titrations as a function of ionic strength and temperature of the buffer. The contributions of enthalpy and entropy to binding were evaluated using classic thermodynamic equations. Label-free surface plasmon resonance was used to assess the role of sodium ion in LMWL binding to thrombin at a fixed ionic strength. RESULTS AND CONCLUSIONS: Exosite 2-induced conformational change in thrombin's active site is strongly dependent on the structure of the ligand, which has consequences with respect to regulation of thrombin. The ionic and non-ionic contributions to binding affinity and the thermodynamic signature were highly ligand specific. Interestingly, LMWLs display preference for the sodium-bound form of thrombin, which supports the existence of an energetic coupling between exosite 2 and sodium-binding site of thrombin.

Heparin Mimetics: Their Therapeutic Potential.

Heparin mimetics are synthetic and semi-synthetic compounds that are highly sulfated, structurally distinct analogues of glycosaminoglycans. These mimetics are often rationally designed to increase potency and binding selectivity towards specific proteins involved in disease manifestations. Some of the major therapeutic arenas towards which heparin mimetics are targeted include: coagulation and thrombosis, cancers, and inflammatory diseases. Although Fondaparinux, a rationally designed heparin mimetic, is now approved for prophylaxis and treatment of venous thromboembolism, the search for novel anticoagulant heparin mimetics with increased affinity and fewer side effects remains a subject of research. However, increasingly, research is focusing on the non-anticoagulant activities of these molecules. Heparin mimetics have potential as anti-cancer agents due to their ability to: (1) inhibit heparanase, an endoglycosidase which facilitates the spread of tumor cells; and (2) inhibit angiogenesis by binding to growth factors. The heparin mimetic, PI-88 is in clinical trials for post-surgical hepatocellular carcinoma and advanced melanoma. The anti-inflammatory properties of heparin mimetics have primarily been attributed to their ability to interact with: complement system proteins, selectins and chemokines; each of which function differently to facilitate inflammation. The efficacy of low/non-anticoagulant heparin mimetics in animal models of different inflammatory diseases has been demonstrated. These findings, plus clinical data that indicates heparin has anti-inflammatory activity, will raise the momentum for developing heparin mimetics as a new class of therapeutic agent for inflammatory diseases.

Sulfated levan from Halomonas smyrnensis as a bioactive, heparin-mimetic glycan for cardiac tissue engineering applications.

Chemical derivatives of levan from Halomonas smyrnensis AAD6(T) with low, medium and high levels of sulfation were synthesized and characterized by FTIR and 2D-NMR. Sulfated levan samples were found to exhibit anticoagulation activity via the intrinsic pathway like heparin in a dose-dependent manner. Exceptionally high heparin equivalent activity of levan sulfate was shown to proceed via thrombin inhibition where decreased Factor Xa activity with increasing concentration was observed in antithrombin tests and above a certain concentration, levan sulfate showed a better inhibitor activity than heparin. In vitro experimental results were then verified in silico by docking studies using equilibrium structures obtained by molecular dynamic simulations and results suggested a sulfation dependent binding mechanism. With its high biocompatibility and heparin mimetic activity, levan sulfate can be considered as a suitable functional biomaterial to design biologically active, functionalized, thin films and engineered smart scaffolds for cardiac tissue engineering applications.

Incorporation of heparin into biomaterials.

This review provides an overview of the incorporation of heparin into biomaterials with a focus on drug delivery and the use of heparin-based biomaterials for self-assembly of polymer networks. Heparin conjugation to biomaterials was originally explored to reduce the thrombogenicity of materials in contact with blood. Many of the conjugation strategies that were developed for these applications are still popular today for other applications. More recently heparin has been conjugated to biomaterials for drug delivery applications. Many of the delivery approaches have taken advantage of the ability of heparin to bind to a wide variety of growth factors, protecting them from degradation and potentiating interactions with cell surface receptors. More recently, the use of heparin as a base polymer for scaffold fabrication has also been explored, often utilizing non-covalent binding of heparin with peptides or proteins to promote self-assembly of hydrogel networks. This review will highlight recent advances in each of these areas.