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Fucosylated chondroitin sulfate (FCS) is a unique polysaccharide, first described nearly four decades ago, and found exclusively in sea cucumbers. It is a component of the extracellular matrix, possibly associated with peculiar properties of the invertebrate tissue. The carbohydrate features a chondroitin sulfate core with branches of sulfated α-Fuc linked to position 3 of the β-GlcA. FCSs from different species of sea cucumbers share a similar chondroitin sulfate core but the structure of the sulfated α-Fuc branches varies significantly. The predominant pattern consists of a single unit of sulfated α-Fuc, though some species exhibit branches with multiple α-Fuc units. This comprehensive review focuses on four major aspects of FCS. Firstly, we describe the initial approaches to elucidate the structure of FCS using classical methods of carbohydrate chemistry. Secondly, we highlight the impact of two-dimensional NMR methods in consolidating and revealing further details about the structure of FCS. These studies were conducted by various researchers across different countries and involving multiple species of sea cucumbers. Thirdly, we summarize the biological activities reported for FCS. Our survey identified 104 publications involving FCS from 42 species of sea cucumbers, reporting 10 types of biological activities. Most studies focused on anticoagulant and antithrombotic activities. Finally, we discuss future perspectives for studies related to FCS. These studies aim to clarify the evolutionary advantage for sea cucumbers in developing such a peculiar fucosylated glycosaminoglycan. Additionally, there is a need to identify the enzymes and genes involved in the metabolism of this unique carbohydrate., (© The Author(s) 2025. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Low-molecular-weight heparin represent a significant advancement in anticoagulant therapy with enoxaparin being a prominent example obtained exclusively through the fragmentation of porcine intestinal heparin. However, escalating demand and limited resources have raised concerns about enoxaparin supplementation. The current challenge involves exploring alternative heparin sources for large-scale enoxaparin production with bovine intestinal heparin emerging as a promising option. Our study demonstrates that enoxaparin derived from the available bovine heparin preparation differs significantly from the reference compound. Yet, the implementation of a straightforward purification step yields a preparation termed "high-anticoagulant bovine heparin". Fragmentation of this purified product through β-elimination produces enoxaparin akin to the standard from a porcine origin. To ensure physicochemical similarity, we employed various spectroscopic, enzymatic, and chromatographic tests to compare the new bovine-derived enoxaparin with the original porcine compound. Biological activity was confirmed through in vitro coagulation assays and assessments using an animal model of venous thrombosis. Our study affirms that the β-elimination reaction cleaves the bovine heparin chain without preferential breaks in regions with different sulfation patterns. Additionally, we scrutinized decasaccharides purified from enoxaparin preparations, providing a comprehensive demonstration of the similarity between products obtained from porcine and bovine heparin. In summary, our findings indicate that an enoxaparin equivalent to the original porcine-derived product can be derived from bovine heparin, given that the starting material undergoes a simple purification step., Competing Interests: The authors declare no competing financial interest., (© 2024 The Authors. Published by American Chemical Society.)

Metastasis is the leading cause of cancer-related deaths. Despite this relevance, there is no specific therapy targeting metastasis. The interaction of the tumor cell with platelets, forming microemboli is crucial for successful hematogenous dissemination. Heparin disrupts it by a P-selectin-mediated event. However, its clinical use for this purpose is hindered by the requirement of high doses, leading to anticoagulant-related side effects. In this study, we obtained a low-anticoagulant heparin through the fractionation of a pharmaceutical bovine heparin. This derivative was referred to as LA-hep and we investigated its efficacy in inhibiting metastases and explored its capacity of suppressing the interaction between tumor cells and platelets. Our data revealed that LA-hep is as efficient as porcine unfractionated heparin in attenuating lung metastases from melanoma and colon adenocarcinoma cells in an assay with a single intravenous administration. It also prevents platelet arrest shortly after cell injection in wild-type mice and suppresses melanoma-platelets interaction in vitro. Moreover, LA-hep blocks P-selectin's direct binding to tumor cells and platelet aggregation, providing further evidence for the role of P-selectin as a molecular target. Even in P-selectin-depleted mice which developed a reduced number of metastatic foci, both porcine heparin and LA-hep further inhibited metastasis burden. This suggests evidence of an additional mechanism of antimetastatic action. Therefore, our results indicate a dissociation between the heparin anticoagulant and antimetastatic effects. Considering the simple and highly reproducible methodology used to purify LA-hep along with the data presented here, LA-hep emerges as a promising drug for future use in preventing metastasis in cancer patients., Competing Interests: Declaration of Competing Interest The authors Juliana M. Motta, Kayene V. A. Micheli, Carlos Roberto-Fernandes, Paulo A. S. Mourão, and Mariana S. Pereira declare that they have a patent pending for the compound LA-hep entitled “Composição de heparina bovina de baixa atividade anticoagulante e seus usos para inibição do crescimento tumoral e metástase”, filed under number BR 10 2023 013 in the Instituto Nacional de Propriedade Intelectual – INPI (Brazilian Patent Office) by the company Heptech Biotecnologia LTDA (Brazil) and Universidade Federal do Rio de Janeiro (Brazil). The other authors have no financial conflicts of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Hypercoagulability, a major complication of metastatic cancers, has usually been treated with heparins from natural sources, or with their synthetic derivatives, which are under intense investigation in clinical oncology. However, the use of heparin has been challenging for patients with risk of severe bleeding. While the systemic administration of heparins, in preclinical models, has shown primarily attenuating effects on metastasis, their direct effect on established solid tumors has generated contradictory outcomes. We investigated the direct antitumoral properties of two sulfated fucans isolated from marine echinoderms, FucSulf1 and FucSulf2, which exhibit anticoagulant activity with mild hemorrhagic potential. Unlike heparin, sulfated fucans significantly inhibited tumor cell proliferation (by ~30-50%), and inhibited tumor migration and invasion in vitro. We found that FucSulf1 and FucSulf2 interacted with fibronectin as efficiently as heparin, leading to loss of prostate cancer and melanoma cell spreading. The sulfated fucans increased the endocytosis of β1 integrin and neuropilin-1 chains, two cell receptors implicated in fibronectin-dependent adhesion. The treatment of cancer cells with both sulfated fucans, but not with heparin, also triggered intracellular focal adhesion kinase (FAK) degradation, with a consequent overall decrease in activated focal adhesion kinase levels. Finally, only sulfated fucans inhibited the growth of B16-F10 melanoma cells implanted in the dermis of syngeneic C57/BL6 mice. FucSulf1 and FucSulf2 arise from this study as candidates for the design of possible alternatives to long-term treatments of cancer patients with heparins, with the advantage of also controlling local growth and invasion of malignant cells., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Ascidians are marine invertebrates that synthesize sulfated glycosaminoglycans (GAGs) within their viscera. Ascidian GAGs are considered analogues of mammalian GAGs and possess great potential as bioactive compounds, presenting antitumoral and anticoagulant activity. Due to its worldwide occurrence and, therefore, being a suitable organism for large-scale mariculture in many marine environments, our main objectives are to study Microcosmus exasperatus GAGs regarding composition, structure, and biological activity. We also aim to develop efficient protocols for sulfated polysaccharides extraction and purification for large-scale production and clinical applications. GAGs derived from M. exasperatus viscera were extracted by proteolytic digestion, purified by ion-exchange liquid chromatography, and characterized by agarose gel electrophoresis and enzymatic treatments. Anticoagulant activity was evaluated by APTT assays. Antitumoral activity was assessed in an in vitro model of tumor cell culture using MTT, clonogenic, and wound healing assays, respectively. Our results show that M. exasperatus presents three distinct polysaccharides; among them, two were identified: a dermatan sulfate and a fucosylated dermatan sulfate. Antitumoral activity was confirmed for the total polysaccharides (TP). While short-term incubation does not affect tumor cell viability at low concentrations, long-term TP incubation decreases LLC tumor cell growth/proliferation at different concentrations. In addition, TP decreased tumor cell migration at different concentrations. In conclusion, we state that M. exasperatus presents great potential as an alternative GAG source, producing compounds with antitumoral properties at low concentrations that do not possess anticoagulant activity and do not enhance other aspects of malignancy, such as tumor cell migration. Our perspectives are to apply these molecules in future preclinical studies for cancer treatment as antitumoral agents to be combined with current treatments to potentiate therapeutic efficacy.

Background  Intracerebral hemorrhage is the most serious complication of anticoagulant therapy but the effects of different types of oral anticoagulants on the expansion of these hemorrhages are still unclear. Clinical studies have revealed controversial results; more robust and long-term clinical evaluations are necessary to define their outcomes. An alternative is to test the effect of these drugs in experimental models of intracerebral bleeding induced in animals. Aims  To test new oral anticoagulants (dabigatran etexilate, rivaroxaban, and apixaban) in an experimental model of intracerebral hemorrhage induced by collagenase injection into the brain striatum of rats. Warfarin was used for comparison. Methods  Ex vivo anticoagulant assays and an experimental model of venous thrombosis were employed to determine the doses and periods of time required for the anticoagulants to achieve their maximum effects. Subsequently, volumes of brain hematoma were evaluated after administration of the anticoagulants, using these same parameters. Volumes of brain hematoma were evaluated by magnetic resonance imaging, H&E (hematoxylin and eosin) staining, and Evans blue extravasation. Neuromotor function was assessed by the elevated body swing test. Results and Conclusions  The new oral anticoagulants did not increase intracranial bleeding compared with control animals, while warfarin markedly favored expansion of the hematomas, as revealed by magnetic resonance imaging and H&E staining. Dabigatran etexilate caused a modest but statistically significant increase in Evans blue extravasation. We did not observe significant differences in elevated body swing tests among the experimental groups. The new oral anticoagulants may provide a better control over a brain hemorrhage than warfarin., Competing Interests: Conflicts of Interest None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. ( https://creativecommons.org/licenses/by/4.0/ ).)

There is a strong scientific rationale to use nebulised unfractionated heparin (UFH) in treating patients with COVID-19. This pilot study investigated whether nebulised UFH was safe and had any impact on mortality, length of hospitalisation and clinical progression, in the treatment of hospitalised patients with COVID-19. This parallel group, open label, randomised trial included adult patients with confirmed SARS-CoV-2 infection admitted to two hospitals in Brazil. One hundred patients were planned to be randomised to either "standard of care" (SOC) or SOC plus nebulized UFH. The trial was stopped after randomisation of 75 patients due to falling COVID-19 hospitalisation rates. Significance tests were 1-sided test (10% significance level). The key analysis populations were intention to treat (ITT) and modified ITT (mITT) which excluded (from both arms) subjects admitted to ITU or who died within 24 h of randomisation. In the ITT population (n = 75), mortality was numerically lower for nebulised UFH (6 out of 38 patients; 15.8%) versus SOC (10 out of 37 patients; 27.0%), but not statistically significant; odds ratio (OR) 0.51, p = 0.24. However, in the mITT population, nebulised UFH reduced mortality (OR 0.2, p = 0.035). Length of hospital stay was similar between groups, but at day 29, there was a greater improvement in ordinal score following treatment with UFH in the ITT and mITT populations (p = 0.076 and p = 0.012 respectively), while mechanical ventilation rates were lower with UFH in the mITT population (OR 0.31; p = 0.08). Nebulised UFH did not cause any significant adverse events. In conclusion, nebulised UFH added to SOC in hospitalised patients with COVID-19 was well tolerated and showed clinical benefit, particularly in patients who received at least 6 doses of heparin. This trial was funded by The J.R. Moulton Charity Trust and registered under REBEC RBR-8r9hy8f (UTN code: U1111-1263-3136)., Competing Interests: Declaration of competing interest TW reports receiving fees and grant support from Synairgen, Bergenbio, AstraZeneca, Janssen, UCB and Valneva in the area of the submitted work, and from GSK, Boehringer-Ingelheim, AZ, and Roche outside the area of the submitted work. CP reports receiving personal fees and grant support from EpiEndo, Eurodrug, Recipharm and Glycosynnovation, and has equity in Verona Pharma, outside of the submitted work. AW reports receiving fees from Theravance in the area of work, and personal fees from Boehringer-Ingelheim, GSK and Novartis outside the submitted work. JS has equity in Ockham Biotech Ltd. who are developing inhaled heparin for the treatment of respiratory diseases. DS has received personal fees from Aerogen, AstraZeneca, Boehringer Ingelheim, Chiesi, Cipla, CSL Behring, Epiendo, Genentech, GlaxoSmithKline, Glenmark, Gossamerbio, Kinaset, Menarini, Novartis, Pulmatrix, Sanofi, Synairgen, Teva, Theravance and Verona., (Copyright © 2023. Published by Elsevier Ltd.)

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbβ3, a fundamental platelet glycoprotein, and αvβ3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1 H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of β-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC 50 95 nM), collagen (IC 50 57 nM), and thrombin (IC 50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbβ3 antagonist, capable of preventing arterial thrombosis., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Pharmaceutical heparins from different manufacturers may present heterogeneities due to particular extraction and purification procedures or even variations in the raw material manipulation. Heparins obtained from different tissues also differ in their structure and activity. Nevertheless, there is an increased demand for more accurate assessments to ensure the similarities of pharmaceutical heparins. We propose an approach to accurately assess the similarity of these pharmaceutical preparations based on well-defined criteria, which are verified with a variety of refined analytical methods. We evaluate six commercial batches from two different manufacturers which were formulated with Brazilian or Chinese active pharmaceutical ingredients. Biochemical and spectroscopic methods and analysis based on digestion with heparinases were employed to evaluate the purity and structure of the heparins. Specific assays were employed to evaluate the biological activity. We observed minor but significant differences between the constitutive units of the heparins from these two manufacturers, such as the content of N -acetylated α-glucosamine. They also have minor differences in their molecular masses. These physicochemical differences have no impact on the anticoagulant activity but can indicate particularities on their manufacturing processes. The protocol we propose here for analyzing the similarity of unfractionated heparins is analogous to those successfully employed to compare low-molecular-weight heparins.

Scedosporium and Lomentospora are a group of filamentous fungi with some clinically relevant species causing either localized, invasive, or disseminated infections. Understanding how the host immune response is activated and how fungi interact with the host is crucial for a better management of the infection. In this context, an α-glucan has already been described in S. boydii , which plays a role in the inflammatory response. In the present study, an α-glucan has been characterized in L. prolificans and was shown to be exposed on the fungal surface. The α-glucan is recognized by peritoneal macrophages and induces oxidative burst in activated phagocytes. Its recognition by macrophages is mediated by receptors that include Dectin-1 and Mincle, but not TLR2 and TLR4. These results contribute to the understanding of how Scedosporium 's and Lomentospora 's physiopathologies are developed in patients suffering with scedosporiosis and lomentosporiosis.

The number of deaths associated with cardiovascular diseases (CVD) increases every year, leading to an intense search for new compounds that may be employed as anticoagulants. One of the classes of bioprospected molecules comprises sulfated polysaccharides (SP) from seaweed, as heparin displays many adverse effects associated with its use. The present study aimed to characterize and evaluate the anticoagulant potential of SP extracted from the green algae Halimeda opuntia. Four PS-rich fractions, F23, F44, F60 and F75, were obtained by proteolytic digestion in papain followed by ethanol precipitation. The presence of SP was confirmed by agarose gel electrophoresis, revealing different populations in each fraction. The F44 fraction is noteworthy compared to the other fractions, presenting a 5% yield compared to the initial algae weight and anticoagulant activity revealed by the activated partial thromboplastin time (APTT) assay (intrinsic/common coagulation pathway). Surprisingly, F44 purification (SP peak P1F44) resulted in prothrombin time (PT) activity (extrinsic coagulation pathway) at a 160 µg/mL, in addition to enhanced APTT activity. The P1F44 anticoagulant activity mechanism was shown to be dependent on two coagulations factors, IIa and Xa, more potent via IIa. Future assessments will be performed to assess this fraction in the medical clinic.

Heparin has already been found in a variety of animal tissues but only few of them became effective sources for production of pharmaceutical preparations. Here, we correlate physical-chemical features and anticoagulant activities of structurally similar heparins employed in the past (from bovine lung, HBL), in the present (from porcine intestine, HPI) and in development for future use (from ovine intestine, HOI). Although they indeed have similar composition, our physical-chemical analyses with different chromatography and spectrometric techniques show that both HOI and HBL have molecular size notably lower than HPI and that the proportions of some of their minor saccharide components can vary substantially. Measurements of anticoagulant activities with anti-FIIa and anti-FXa assays confirmed that HPI and HOI have potency similar each other but significantly higher than HBL. Such a lower activity of HBL has been attributed to its reduced molecular size. Considering that HOI also has reduced molecular size, we find that its increased anticoagulant potency might result from an improved affinity to antithrombin (three times higher than HBL) promoted by the high content of N ,3,6-trisulfated glucosamine units, which in turn are directly involved in the heparin-antithrombin binding. Therefore, the anticoagulant activity of different heparins is driven by a balance between different physical-chemical components, especially molecular size and fine-tuning composition. Although such minor but relevant chemical differences reinforce the concept that heparins from different animal sources should indeed be considered as distinct drugs, HOI could be approved for interchangeable use with the gold standard HPI and as a suitable start material for producing new LMWHs., Competing Interests: Conflict of Interest None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. ( https://creativecommons.org/licenses/by/4.0/ ).)

Heparin is a centennial anticoagulant drug broadly employed for treatment and prophylaxis of thromboembolic conditions. Although unfractionated heparin (UFH) has already been shown to have remarkable pharmacological potential for treating a variety of diseases unrelated with thromboembolism, including cancer, atherosclerosis, inflammation, and virus infections, its high anticoagulant potency makes the doses necessary to exert non-hemostatic effects unsafe due to an elevated bleeding risk. Our group recently developed a new low-anticoagulant bovine heparin (LABH) bearing the same disaccharide building blocks of the UFH gold standard sourced from porcine mucosa (HPI) but with anticoagulant potency approximately 85% lower (approximately 25 and 180 Heparin International Units [IU]/mg). In the present work, we investigated the pharmacokinetics profile, bleeding potential, and anticancer properties of LABH administered subcutaneous into mice. LABH showed pharmacokinetics profile similar to HPI but different from the low-molecular weight heparin (LMWH) enoxaparin and diminished bleeding potential, even at high doses. Subcutaneous treatment with LABH delays the early progression of Lewis lung carcinoma, improves survival, and brings beneficial health outcomes to the mice, without the advent of adverse effects (hemorrhage/mortality) seen in the animals treated with HPI. These results demonstrate that LABH is a promising candidate for prospecting new therapeutic uses for UFH., Competing Interests: Conflict of Interest None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution License, permitting unrestricted use, distribution, and reproduction so long as the original work is properly cited. ( https://creativecommons.org/licenses/by/4.0/ ).)

Marine organisms are a source of active biomolecules with immense therapeutic and nutraceutical potential. Sulfated fucose-rich polysaccharides are present in large quantities in these organisms with important pharmacological effects in several biological systems. These polysaccharides include sulfated fucan (as fucoidan) and fucosylated chondroitin sulfate. The development of these polysaccharides as new drugs involves several important steps, among them, demonstration of the effectiveness of these compounds after oral administration. The oral route is the more practical, comfortable and preferred by patients for long-term treatments. In the past 20 years, reports of various pharmacological effects of these polysaccharides orally administered in several animal experimental models and some trials in humans have sparked the possibility for the development of drugs based on sulfated polysaccharides and/or the use of these marine organisms as functional food. This review focuses on the main pharmacological effects of sulfated fucose-rich polysaccharides, with an emphasis on the antidislipidemic, immunomodulatory, antitumor, hypoglycemic and hemostatic effects.

This study aimed to isolate, characterize chemical-structurally and evaluate the effects of polysaccharides from Caesalpinia (Libidibia) ferrea stem barks in the haemostatic system. The deproteinated-polysaccharide extract (PE-Cf) after being fractionated by ion exchange chromatography-DEAE-cellulose resulted in three fractions (FI, FII, FIII) containing total carbohydrates (14.3-38%), including uronic acid (5-16%), and polyphenols (0.94-1.7 mg/g GAE). The polysaccharide fractions presented polydisperse profile in polyacrylamide gel electrophoresis (detected by Stains-All) and molecular masses (9.5 × 10 4  Da-1.5 × 10 5  Da) identified by gel permeation chromatography. FT-IR showed absorption bands (1630 cm -1 , 1396-1331 cm -1 ), indicative of uronic acid, and a band at 1071 cm -1 , typical of COO - groups of galacturonic acid. The NMR spectra of C. ferrea polysaccharides revealed a central core composed mainly by 5-linked α-Araf and minority components as α-Rhap and α-GalAp. UV spectra of fractions revealed discrete shoulders at 269-275 nm, characteristic of polyphenolic compounds. In vitro, polysaccharides inhibited the intrinsic and/or common coagulation pathway (aPTT test) (2.0-3.7 fold) and the platelet aggregation induced by 3 μM adenosine diphosphate (25-48%) and 5 μg/mL collagen (24%), but not that induced by arachidonic acid. In vivo, the polysaccharides inhibited (36-69%) venous thrombosis induced by hypercoagulability and stasis, showing discrete hemorrhagic effect. In conclusion, the polysaccharides of C. ferrea barks, containing arabinose, galactose, rhamnose and uronic acid, possess anticoagulant, antiplatelet and antithrombotic properties of low hemorrhagic risk, suggesting potential applicability in thromboembolic disorders., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Marine ancestors of freshwater sponges had to undergo a series of physiological adaptations to colonize harsh and heterogeneous limnic environments. Besides reduced salinity, river-lake systems also have calcium concentrations far lower than seawater. Cell adhesion in sponges is mediated by calcium-dependent multivalent self-interactions of sulfated polysaccharide components of membrane-bound proteoglycans named aggregation factors. Cells of marine sponges require seawater average calcium concentration (10 mM) to sustain adhesion promoted by aggregation factors. We demonstrate here that the freshwater sponge Spongilla alba can thrive in a calcium-poor aquatic environment and that their cells are able to aggregate and form primmorphs with calcium concentrations 40-fold lower than that required by marine sponges cells. We also find that their gemmules need calcium and other micronutrients to hatch and generate new sponges. The sulfated polysaccharide purified from S. alba has sulfate content and molecular size notably lower than those from marine sponges. Nuclear magnetic resonance analyses indicated that it is composed of a central backbone of non- and 2-sulfated α- and β-glucose units decorated with branches of α-glucose. Assessments with atomic force microscopy/single-molecule force spectroscopy show that S. alba glucan requires 10-fold less calcium than sulfated polysaccharides from marine sponges to self-interact efficiently. Such an ability to retain multicellular morphology with low environmental calcium must have been a crucial evolutionary step for freshwater sponges to successfully colonize inland waters., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Marine organisms have been proven to be a valuable source of bioactive compounds. Among them, we highlight the sulfated galactans (SGs) from seaweeds, which besides being massively exploited as industrial thickening and gelling agents (agarans and carrageenans), have also shown promising pharmacological properties. Investigations on the non-agaran/-carrageenan SG from the red algae Bothryocladia occidentalis (SGBo) have demonstrated clear correlations between physical-chemical features and biological activities. SGBo is composed of 2,3-disulfated (~33%) or 2-sulfated (33%) α-D-galactose linked to non- or 2-sulfated β-D-galactose repetitive disaccharide units. The notable serpin-dependent/-independent anticoagulant activity of SGBo (~130 international units [IU]/mg) is higher than those of other SGs containing less 2,3-disulfated α-D-galactose units and their low-molecular-weight derivatives, and thus is directly correlated to its high molecular mass (>200 kDa) and sulfation pattern. Although SGBo has antithrombotic efficacy equivalent to heparin and decreased bleeding potential at low-doses, high-doses substantially increase thrombus formation in animal models. Such an odd dose-dependent dual antithrombotic/prothrombotic activity has been attributed to the ability of SGBo to activate factor XII. In addition to anticoagulant properties, SGBo also exerts antimalarial, antileishmanial and antiophidic activities, and, therefore, has a remarkable potential for the research and development of novel drugs., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Marsypianthes chamaedrys (Lamiaceae) is a medicinal plant popularly used against envenomation by snakebite. Pharmacological studies have shown that extracts of M. chamaedrys have antiophidic, anti-inflammatory and anticoagulant properties, supporting the ethnopharmacological use. In this study, an aqueous extract of aerial parts of M. chamaedrys showed anticoagulant activity in the activated partial thromboplastin time assay (0.54 IU/mg). The bioassay-guided fractionation using ethanol precipitation and gel filtration chromatography on Sephadex G-50 and Sephadex G-25 resulted in a water-soluble fraction with increased anticoagulant activity (Fraction F2-A; 2.94 IU/mg). A positive correlation was found between the amount of uronic acids and the anticoagulant potential of the active samples. Chemical and spectroscopic analyses indicated that F2-A contained homogalacturonan, type I rhamnogalacturonan, type II arabinogalactan and α-glucan. UV and FT-IR spectra indicated the possible presence of ferulic acid. Pectic polysaccharides and type II arabinogalactans may be contributing to the anticoagulant activity of the aqueous extract of M. chamaedrys in the APTT assay., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Heparin or highly sulfated heparan sulfate (HS) has been described in different invertebrates. In ascidians (Chordata-Tunicata), these glycosaminoglycans occur in intracellular granules of oocyte accessory cells and circulating basophil-like cells, resembling mammalian mast cells and basophils, respectively. HS is also a component of the basement membrane of different ascidian organs. We have analyzed an HS isolated from the internal organs of the ascidian Phallusia nigra , using solution 1 H/ 13 C NMR spectroscopy, which allowed us to identify and quantify the monosaccharides found in this glycosaminoglycan. A variety of α-glucosamine units with distinct degrees of sulfation and N -acetylation were revealed. The hexuronic acid units occur both as α-iduronic acid and β-glucuronic acid, with variable sulfation at the 2-position. A peculiar structural aspect of the tunicate HS is the high content of 2-sulfated β-glucuronic acid, which accounts for one-third of the total hexuronic acid units. Another distinct aspect of this HS is the occurrence of high content of N -acetylated α-glucosamine units bearing a sulfate group at position 6. The unique ascidian HS is a potent inhibitor of the binding of human colon adenocarcinoma cells to immobilized P-selectin, being 11-fold more potent than mammalian heparin, but almost ineffective as an anticoagulant. Thus, the components of the HS structure required to inhibit coagulation and binding of tumor cells to P-selectin are distinct. Our results also suggest that the regulation of the pathway involved in the biosynthesis of glycosaminoglycans suffered variations during the evolution of chordates.

Colorectal cancer has an overexpression of galectin-3 that is related to cancer progression. A decreased risk of colon cancer can be related to consumption of dietary fibers, but the entire mechanism by which this protection occurs remains unclear. Pectin is a type of dietary fiber that possesses β-galactosides and can bind and inhibit galectin-3-mediated effects. Papaya fruit has a massive cell wall disassembling during ripening that naturally changes its pectin structure. Our work shows that different points in the ripening time of papaya fruit exhibit pectins (chelate-soluble fractions; CSF) that can or cannot inhibit galectin-3. The fraction that inhibits galectin-3 (3CSF) also diminishes the proliferation of colon cancer cell lines, and it is derived from an intermediate point of papaya ripening. Therefore, we related this to a papaya pectin structure-dependent effect, and the papaya fruit seems to have a pectin structure that is promising in decreasing the risk of colon cancer development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Unfractionated heparin (UFH) and their low-molecular-weight derivatives are sourced almost exclusively from porcine mucosa (HPI); however, a worldwide introduction of UFH from bovine mucosa (HBI) has been recommended to reinforce the currently unsteady supply chain of heparin products. Although HBI has different chemical composition and about half of the anticoagulant potency of HPI (∼100 and ∼180 international unit [IU]/mg, respectively), they have been employed as interchangeable UFHs in some countries since the 1990s. However, their use as a single drug provoked several bleeding incidents in Brazil, which precipitated the publication of the first monographs exclusive for HBI and HPI by the Brazilian Pharmacopoeia. Nevertheless, we succeed in producing with high-resolution anion-exchange chromatography a novel HBI derivative with anticoagulant potency (200 IU/mg), disaccharide composition (enriched in N ,6-disulfated α-glucosamine) and safety profile (bleeding and heparin-induced thrombocytopaenia potentials and protamine neutralization) similar to those seen in the gold standard HPI. Therefore, we show that it is possible to equalize the composition and pharmacological characteristics of these distinct UFHs by employing an easily implementable improvement in the HBI manufacturing., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

Most of the unfractionated heparin (UFH) consumed worldwide is manufactured using porcine mucosa as raw material (HPI); however, some countries also employ products sourced from bovine mucosa (HBI) as interchangeable versions of the gold standard HPI. Although accounted as a single UFH, HBI, and HPI have differing anticoagulant activities (~100 and 200 IU mg -1 , respectively) because of their compositional dissimilarities. The concomitant use of HBI and HPI in Brazil had already provoked serious bleeding incidents, which led to the withdrawal of HBI products in 2009. In 2010, the Brazilian Pharmacopeia (BP) formed a special committee to develop two complementary monographs approaching HBI and HPI separately, as distinct active pharmaceutical ingredients (APIs). The committee has rapidly agreed on requirements concerning the composition and presence of contaminants based on nuclear magnetic resonance and anion-exchange chromatography. On the other hand, consensus on the anticoagulant activity of HBI was the subject of long and intense discussions. Nevertheless, the committee has ultimately agreed to recommend minimum anti-FIIa activities of 100 IU mg -1 for HBI and 180 IU mg -1 for HPI. Upon the approval by the Brazilian Health Authority (ANVISA), the BP published the new monographs for HPI and HBI APIs in 2016 and 2017, respectively. These pioneer monographs represent a pivotal step toward the safest use of HBI and HPI as interchangeable anticoagulants and serve as a valuable template for the reformulation of pharmacopeias of other countries willing to introduce HBI.

Glycoconjugates extracted from Genipa americana leaves (PE-Ga) were separated into two fractions, denominated as PFI and PFII (total carbohydrate: 23-36%/uronic acid: 9-30%; protein:4-5%; polyphenols:0.776-0.812 mg/g), mainly composed by arabinose, galactose and uronic acid and presenting high (PFI) and low (PFII) molecular weight (based on polyacrylamide electrophoresis gel and gel permeation chromatography). Uronic acid was also detected by FT-IR (wavenumbers: 1410 and 1333 cm -1 ) and NMR (α-GalpA). Deproteinization of glycoconjugates showed reduced protein and polyphenol levels with loss of its biological effects. PE-Ga and PFII prolonged clotting time-aPTT (3.6 and 1.8x), while PE-Ga and PFI inhibited by 48% (100 μg/μL) the ADP-induced platelet aggregation. In vivo, these glycoconjugates at 1 mg/kg inhibited (37-53%) venous thrombus formation (4.7 ± 0.1 mg) and increased bleeding time (PE-Ga and PFI:3.0x; PFII:1.7x vs. PBS:906 ± 16.7 s). In conclusion, the arabinogalactan-rich glycoconjugate of G. americana leaves, containing uronic acid, present antiplatelet, anticoagulant (intrinsic/common pathway) and antithrombotic effects, with low hemorrhagic risk., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Essentials Bovine (HBI) and porcine (HPI) heparins differ in structure and anticoagulant activity. Protamine-neutralization was evaluated on a variety of physical-chemical methods. HBI requires more protamine than HPI to fully neutralize its anticoagulant activity. Protamine preferentially removes higher-sulfated chains of HBI while HPI is evenly precipitated., Summary: Background Protamine neutralization is an essential step for the safe use and inactivation of the unfractionated heparin (UFH) that is widely employed in surgical and non-surgical procedures involving extracorporeal circulation. Objective To compare protamine neutralization of different pharmaceutical-grade UFHs prepared from porcine or bovine intestine (HPI and HBI, respectively). HBI has approximately half the anticoagulant potency of HPI, mostly as consequence of its fraction enriched with N-sulfated α-glucosamine disaccharides. Methods Protamine neutralization of HPI and HBI was evaluated with in vitro, ex vivo and in vivo assays. We also performed in-depth assessments of the complexation of protamine with these distinct UFHs by using nuclear magnetic resonance and mass spectroscopy. Results HPI and HBI interact similarly with protamine on a mass/mass basis; however, HBI requires more protamine than HPI to have its anticoagulant activity fully neutralized, because of its lower potency, which entails the use of higher doses. Nuclear magnetic resonance spectra revealed that HPI precipitates homogeneously with protamine. On the other hand, the low-sulfated fraction of HBI, enriched with N-sulfated α-glucosamine, precipitates at higher concentrations of protamine than the fraction more like HPI, with a preponderance of N,6-disulfated α-glucosamine disaccharides. Finally, mass spectroscopy spectra showed that some of the different peptide components of protamine interact preferentially with the heparins, irrespective of their animal origin. Conclusion Our results have important medical implications, indicating that protamine neutralization of HBI, determined exclusively by point-of-care coagulation assessments, must fail because of its lower-sulfated fraction with reduced anticoagulant activity that could remain in the circulation after the neutralization procedure., (© 2018 International Society on Thrombosis and Haemostasis.)

Fucosylated chondroitin sulfates (FCSs) and sulfated fucans (SFs) are conspicuous components of the body wall of sea cucumbers (Holothuroidea). FCSs are composed of a central core of chondroitin sulfate (CS) decorated with branches of mono- or both mono- and disaccharides of α-fucose (FCS types I and II, respectively). FCSs type II have heterogeneous and irregularly distributed α-fucose branches; however, the novel FCS type II from Holothuria lentiginosa described herein via solution nuclear magnetic resonance has strikingly homogeneous α-fucose branches neatly distributed along its CS core. This FCS is built up of three distinct sequential units composed of the typical CS disaccharides of FCSs, rich in β-galactosamine-4,6diS, decorated with branches of α-Fucp-2,4diS, α-Fucp-3,4diS or α-Fucp[1→3]α-Fucp-4S[1→ linked to the position 3- of the β-glucuronic acid. Conformational analyses of these repetitive units revealed a fairly rigid structure despite of the high sulfate content of their α-fucose branches. We also determined the structure of the SF from H. lentiginosa as a repetitive tetrasaccharide sequence composed of →3]α-Fucp-2,4diS[1→3]α-Fucp[1→3]α-Fucp-2S[1→3]α-Fucp-2S[1→. Furthermore, we determined that the nonsulfated α-fucose units present in FCS type II did not interfere with their anticoagulant potencies and affinities to calcium. FCS is an autapomorphic molecular character of the class Holothuroidea and the composition of their α-fucose branches differs in a species-specific manner. Branches containing α-Fucp-2,4diS are the most common within the extant holothurians, being found in 90% of the FCSs characterized thus far.

Cationic dyes such as toluidin blue are commonly employed to visualize glycosaminoglycans (GAGs) on electrophoresis gels; however, the carbocyanine-based dye Stains-all have been increasingly used to stain the non-sulfated hyaluronic acid and other GAGs in submicrogram quantities. In this short communication, we demonstrate that Stains-all is able to stain the most common GAGs on polyacrylamide gels with distinct and contrasting colors in a reproducible manner. We also show that this staining method is useful to identify GAGs present both in mixtures and in submicrogram quantities. Therefore, Stains-all has shown to be useful in identifying GAGs on polyacrylamide gels with basis on their specific colors, at least on screening level., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Anticoagulant heparins are mostly obtained from porcine intestine. Occasionally they are also obtained from bovine intestine. Structural and functional analyses of pharmaceutical-grade heparins from these two sources using multiple methods such as NMR spectroscopy, in vitro and in vivo assays of the anticoagulant, antithrombotic and bleeding effects, complemented by fractionation on anion exchange chromatography, confirm they are different drugs. Although bovine heparin is more heterogeneous and less sulfated, heparins from both sources are overall made of a similar mixture of fractions, however with different proportions. Therefore, high-anticoagulant composites from bovine origin, similar to porcine counterparts, can be properly obtained., (Copyright © 2014 Elsevier Ltd. All rights reserved.)